Solano AN, Lou J, Scipioni L, Gratton E, Hinde E.
Brightness correlation spectroscopy tracks transcription factor diffusion as a function of oligomeric state within live cell nuclear architecture.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 302a, 1460-Plat.In the crowded environment of the cell nucleus, transcription factors form homo and hetero-oligomers that modulate the exploration volume available during DNA target search, as well as DNA binding affinity. To directly track this process in a living cell here we present brightness correlation spectroscopy as a method to extract oligomeric transcription factor dynamics within live-cell microscopy data. From correlation of brightness fluctuations that originate from a fluorescently tagged transcription factor this approach has the capacity to extract protein mobility as a function of oligomeric state, and spatiotemporally map the anisotropy of this parameter with respect to nuclear architecture within a rapid single-channel frame scan acquisition. Expanding this method, which can be applied to any oligomeric protein, to a dual-channel frame scan acquisition enables measurement of the spatiotemporal dynamics that underly hetero-oligomeric transcription factor complex formation. Application of one and two channel brightness correlation spectroscopy to the signal transducer and ... [truncated at 150 words]
Fazel M, Jazani S, LorenzoScipioni, Vallmitjana A, Gratton E, Digman MA, Pressé S.
High resolution fluorescence lifetime maps from minimal photon counts.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 141a, 688-Pos.Fluorescence lifetime imaging microscopy (FLIM) can be used to probe concentration profiles of key marco-molecules within sub-cellular environments. However, current analyses for FLIM require a large number of photons per pixel to provide such a quantitative picture of life. In order to acquire such large number of photons, we must either increase data acquisition time, which limits temporal resolution, or increase laser intensity, which causes greater photo damage to the sample, or both. Here, we propose to analyze FLIM data by leveraging tools from the Bayesian paradigm. Our framework also takes into account details of the experiment such as point spread function (PSF) and instrument response function (IRF) of arbitrary shapes. We show that our method is robust with respect to species lifetimes from below the IRF to exceeding inter-pulse times. Moreover, we achieve direct blind unmixing of lifetimes with sub-nanosecond resolution and sub-pixel spatial resolution using limited photon budgets. ... [truncated at 150 words]
Torrado B, Dvornikov A, Gratton E.
Using multi-channel detector for simultaneous hyperspectral and flim imaging in strongly scattering media.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 277a, 1342-Pos.We describe a method that allows imaging in scattering media, such as biological tissues, and perform multiplexed hyperspectral and FLIM analysis of these images. This method is based on a set of transmission filters placed in front of wide area 4-channel detector in the emission path of a fluorescence microscope. The special transmission filters with sin-cos or linear spectral profiles were used to resolve the emission wavelength in every pixel of an image. The set of filters are used to obtain the spectral phasor of the transmitted light, which is enough to perform spectral deconvolution over a broad wavelength range. With this method we can distinguish between free and protein-bound NADH, so it can be used in metabolic studies. In addition, a filter with transmission in the range of 400-500nm was used for simultaneous FLIM analysis of the NADH autofluorescence. The method can be applied to any type of imaging ... [truncated at 150 words]
-
Using phasors.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 411a, 1989-Pos.We have designed and built a fluorescence microscope that can simultaneously acquire and process spectral emission and fluorescence lifetime of a fluorophore in real time (Phasor S-FLIM). We also developed an analysis pipeline capable of unmixing up to 7 different organelle-directed dyes over time by using only the spectral information. In particular, we are able to visualize dyes that report on lysosomes, lipid droplets, tubulin, chromatin, mitochondria and Golgi apparatus. This kind of data can reveal not only the position of cellular structures inside the cells but also their dynamics and interplay in living cells, providing invaluable data for the that can be used to model cell functions at a level of detail that is missing today. Furthermore, part of what is learned from the data obtained at the cellular level can be scaled up to clinical samples, where the effect of drugs can be followed at the tissue level. ... [truncated at 150 words]
Hinde E, Lou J, Liang Z, Solano A, Scipioni L, Gratton E.
The phasor approach to histone FLIM-FRET microscopy maps nuclear wide heterochromatin structure at the level of nucleosome proximity in a living cell.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 412a, 1997-Pos.Inside the nucleus of an intact cell, DNA is folded around histone proteins into nucleosomes and compacted into a multi-layered three-dimensional chromatin network. The nanometre spacing between nucleosomes positioned throughout this structural framework is known to locally modulate local DNA template access and regulate genome function. However, given that this structural feature occurs on a spatial scale well below the diffraction limit, real time observation of nucleosome proximity in live cells by optical microscopy has proven technically difficult, despite recent advances in live cell super resolution imaging. A promising alternative solution is to measure and spatially map Förster resonance energy transfer (FRET) between fluorescently labelled histones - the core protein of a nucleosome. In recent work we established that the phasor approach to fluorescence lifetime imaging microscopy (FLIM) is a robust method for the detection of histone FRET which can quantify nuclear wide chromatin compaction at the level of nucleosome ... [truncated at 150 words]
Vallmitjana A, Vu TM, Gu J, La K, Flores J, Hedde PN, Zhao W, Gratton E.
Spectral and lifetime multiplexing with combinatorial labeling.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 139a, 681-Pos.It is currently of critical importance to enable technologies which permit the spatial analysis of multi-omics biomarkers, e.g. mRNA and proteins, by revealing their presence, counts, locations, dynamics, and interactions within each tissue and organ. Unfortunately, there is the lack of tools that can, in a single round of labelling and imaging, profile these biomarkers beyond 9 targets and scale from the molecular level to the tissue or organ level in large heterogeneous samples. Here, we introduce Multi-Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICATM), a new spatialomics technology that enables rapid, high-throughput, and highly multiplexed profiling of mRNA and protein targets while remaining cost-effective and easy to implement. MOSAICATM utilizes in situ labeling using combinatorial labeling of fluorescent species combined with spectral-lifetime microscopy and phasor-based image segmentation. The technology is highly scalable due to the orthogonality of the spectral and lifetime dimensions and the combinatorial approach to labelling. We ... [truncated at 150 words]
Scipioni L, Rossetta A, Tedeschi G, Anderson AJ, Gratton E.
Phasor S-FLIM for the physiological profiling of living cells.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 411a, 1990-Pos.Fluorescence Lifetime Imaging Microscopy (FLIM) and spectral imaging are two broadly applied methods for increasing dimensionality in microscopy. However, their combination is typically inefficient and slow in terms of both acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased Spectral FLIM (S-FLIM) system capable of extracting detailed and precise information about the photophysics of fluorescent specimens at optical resolution. The data are processed with the phasor approach to provide unbiased and fast analysis of S-FLIM data. We therefore named this system Phasor S-FLIM. Here, we exploit both spectral emission and fluorescence lifetime of environment-sensitive fluorescent dyes to report on multiple parameters related to cellular physiology. Environment-sensitive dyes are fluorophores that change their spectral emission and/or fluorescence lifetime according to changes in the nano-environment in which they localize. As an example, we can measure important physiological parameters such as mitochondrial membrane potential, lipid droplets composition ... [truncated at 150 words]
Jones B, Torrado B, Rosenberg A, Gratton E, Levi M, Ranjit S.
Determination of cardiac fibrosis using picrosirius red fluorescence.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 279a, 1349-Pos.Automatic measurement and quantification of collagen accumulation and fibrosis in the heart using multiphoton excitation methods is more difficult compared to other organs, as myosin and collagen both contribute to the second harmonic generation (SHG) signal, inhibiting any specificity of collagen only signals. Therefore, the most common fibrosis quantification using SHG often fails to quantify cardiac fibrosis. Picrosirius red (PSR) is a common dye that is used for the quantification of fibrosis using polarized light. In this work, we have used PSR stained rat heart samples, from the DOCA-salt (deoxycorticosterone acetate) model of chronic hypertension model, to identify and quantify the extent of fibrosis. The samples were excited with a 1040 nm excitation using a two photon excitation scheme and the red fluorescence emission was collected using a 575-645 nm filter. Phasor approach to fluorescence lifetime imaging (Phasor-FLIM) and spectra (spectral phasor) were used to identify the origin of this ... [truncated at 150 words]
Palomba F, Scipioni L, Truong T, Stukenberg TP, Jiang H, Gratton E, Digman MA.
Fluorescence methods for the biophysical characterization of protein phase separated condensates.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 280a, 1353-Pos.Protein condensates are self-assembled membrane-less organelles that can orchestrate complex functions through a controlled confinement or exclusion of biologically relevant molecules. The biophysical mechanisms on how condensates can orchestrate the activities is hampered by the lack of methods capable of assessing phenomena across large spatiotemporal scales. Here, we propose three novel fluorescence-based approaches for the characterization of protein phase separated condensates (PPSC) at optical resolution that can cover a spatiotemporal range from sub-nanoseconds to seconds and from nanometer to micrometers. 1) We developed an approach to characterize at nanoscale (10−12 s, 10−9 m) the water dynamics and environment polarity within the condensates by monitoring the changes of optical properties of polarity-sensitive probes (ACDAN and ACRYLODAN). We demonstrate that this method can be applied to assess the phase separation diagram of a model coacervate when the classical methods (FRAP, FCS) fail. 2) We found that the type of phase separation (liquid-like, ... [truncated at 150 words]
Zaccheo K, Benítez-Mata AB, Digman MA, Barbee KA.
Investigating the effects of alcohols and cholesterol manipulations on bovine aortic endothelial cell membrane properties using phasor analysis of laurdan fluorescence lifetime microscopy and spectral imaging.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 366a, 1777-Pos.Endothelial cell membranes are made up of a heterogenous mixture of lipids and proteins and include liquid ordered (Lo) domains called lipid rafts and caveolae. These compartments have higher concentrations of cholesterol and sphingolipids and are concentrated in signaling molecules. Perturbations in membrane fluidity, including changes in cholesterol content can lead to changes in the structure of the plasma membrane and impair signaling mechanisms. In order to study how changing membrane fluidity can lead to changes in cellular signaling, we must be able to characterize changes in fluidity. Here we utilize two different methods using the fluorescent dye Laurdan, which exhibits a red spectral shift in emission when in a more liquid disordered (Ld) domain, to examine the effects of alcohols and cholesterol manipulations on membrane fluidity and cholesterol content in bovine aortic endothelial cells (BAEC). Laurdan-labeled BAECs were treated with dodecanol and benzyl alcohol to fluidize the membrane, methyl-β-cyclodextrin ... [truncated at 150 words]
Benítez-Mata AB, Palomba F, Scipioni L, Digman MA.
Analysis of membrane fluidity disturbances by mutant huntingtin in different membranal compartments using phasor analysis of local ICS and spectral phasor analysis.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 138a, 676-Pos.Huntington's Disease (HD), caused by the expression of extended polyQ repeats in the protein huntingtin, is known to disturb cholesterol metabolism. The misfolded proteins alter biosynthesis and intracellular levels of cholesterol, leading to membranes deterioration. In this work, we aim to describe the effect of said disturbances in various membrane compartments caused by different polyQ expansions from the membrane fluidity point of view, an approach never applied before. To this end, we use the environment-sensitive fluorescent probe LAURDAN to study the membrane fluidity at different compartments of SHSY5Y cells modified to express the non-pathogenic (18QmCherry) and pathogenic (53QmCherry, 77QmCherry) protein. We focus on three categories of compartments: cellular plasma membrane, internal membranes, and lipid droplets, all known to be rich in cholesterol and other lipids. Our approach leverages LAURDAN's propensity to insert in hydrophobic regions, such as membranes, where its emission is the more blue-shifted the less water is accessible ... [truncated at 150 words]
Huang Y, Fang T, Wei J, Mena JEM, Lakey PSJ, Kleinman MT, Shiraiwa M, Digman MA.
Phasor approach FLIM as an indicator for NADPH oxidase during exposure to secondary organic aerosols.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 415a, 2012-Pos.Nicotinamide adenine dinucleotide phosphate oxidase (NOX) is a multi-subunit enzyme which is responsible for host defense and inflammation signaling against either bacterial infection or pollutants. During activation, free oxygen is turned into superoxide anions which further leads to cascade formation of reactive oxygen species (ROS). However, once accumulation of ROS exceeds cell capacity, it damages components including proteins, lipids, and DNA known as oxidative stress which contributes to various lethal impacts such as lipid peroxidation and cancer. Therefore, it gives rise to the importance of monitoring NOX activity to avoid ROS accumulation. While most measurements use fluorescent probes to detect ROS, it lacks the ability to differentiate readings between cellular and extracellular which does not directly relate to NOX activity only. Here we aim to detect the impact of secondary organic aerosols (SOAs) on macrophage metabolism using fluorescence lifetime imaging (FLIM) and correlate the changes in fluorescence lifetime of NAD(P)H ... [truncated at 150 words]
Tedeschi G, Scipioni L, Palomba F, Digman MA.
Multiparametric fluorescence imaging approaches to investigate metabolic signature, lipid droplets, and collagen remodeling in breast cancer spheroids.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 409a, 1980-Pos.Tumor spheroids are 3D cell cultures models to study the behavior of avascular solid tumors in a controlled environment. Cellular metabolism, lipid storage and their relationship with the extracellular matrix (ECM) have been shown to be important elements in the characterization of tumor cell proliferation and metastatic invasion, but their simultaneous assessment remains challenging. In order to obtain all the information simultaneously, we imaged tumor spheroid with the following three imaging approaches over a 24 hours period. The metabolic signature at the single cell level was obtained by analyzing NADH lifetime, using the Phasor Fluorescence Lifetime Imaging Microscopy (FLIM) approach. Differences in lipid droplets content were obtained by analyzing the emission signal of an environmental-sensitive (solvatochromic) dye, Nile Red, with Spectral Phasors. Finally, we evaluated the spheroids-ECM interaction with Second Harmonic Generation (SHG) with PLICS (Phasor analysis of Local Image Correlation Spectroscopy). The main advantage of the proposed techniques is ... [truncated at 150 words]
Zhu S, Scipioni L, Digman MA.
Phasor unmixing to reveal organelle organization and cellular response.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 278a, 1346-Pos.Organelles are a series of subcellular structures that perform various functions inside eukaryotic cells. The coordination between organelles play an important role in understanding the intricate biochemical processes within eukaryotic cells as organelles must work in concert to maintain cell function. Here, we perform simultaneous excitation of seven fluorophores and spectral phasor unmixing to achieve robust emission separation of all seven stained subcellular compartments and organelles including the Golgi apparatus, tubulin, lipid droplets, lysosomes, mitochondria, nuclear and mitochondrial DNA. Our imaging approach uses a single round of two-photon excitation at 780 nm coupled with multicolor organelle-directed staining to obtain multi-parametric physiological profiling of live MCF10A cells, a non-tumorigenic epithelial breast cancer cell line, under stress related treatments. We created an analysis pipeline to spectrally unmix the contribution of each organelle and obtain phenotypic signatures under each type of treatment. Ultimately, we characterized significant differences in organelle phenotypes upon application of ... [truncated at 150 words]
Lopez KL, Tedeschi G, Palomba F, Digman MA.
Metabolic fingerprinting of HUVEC cells during phenotypic shifts using fluorescence lifetime image microscopy (FLIM).
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 404a, 1954-Pos.Adult human vascular endothelial cells (ECs) change their phenotype in response to changes in vascular tumor microenvironments, oxygen concentration and during wound healing. During angiogenesis, the physiological process of creating new blood vessels, hundreds of genes are activated with one of the most important being VEGF (vascular endothelial growth factor). VEGF, the signaling protein, stimulates EC migration, proliferation, and vascularization. During angiogenesis, ECs undergo an ‘angiogenic switch’; they change from a resting (quiescent) phenotype to a more elongated and mobile (tip cells) or proliferative and mobile (stalk cells) phenotype. Differences between phenotypes are not easy to assess. Currently, the most common approaches include Seahorse assays, fluorescent labeling or transfections in an attempt to categorize the HUVEC phenotypes by their metabolic signature. Although these methods have shown the importance of metabolic profiling to separate the cells, they are invasive and may ultimately alter the cellular metabolism. Additionally, a non-invasive metabolic imaging ... [truncated at 150 words]
Brennan CK, Yao Z, Scipioni L, Chen H, Ng K, Tedeschi G, Digman MA, Prescher JA.
Multiplexed bioluminescence microscopy via phasor analysis.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 142a, 695-Pos.Imaging tools allow us to visualize biological processes, including single cell dynamics and metabolic fluxes. Tracking multiple cells or features over extended periods of time, though, remains challenging. Common approaches rely on fluorescent probes, which require intense excitation light that can damage cells and cause phototoxicity. Bioluminescence imaging (BLI) relies on light production from a chemical reaction, the luciferase-catalyzed oxidation of small molecule substrates (luciferins), and can offer a more bio-friendly alternative for monitoring cellular events in real time. Although luciferases and luciferins have been used for decades to image processes in complex tissues and preclinical models, microscopic BLI has been historically challenging due to a lack of detection methods and probes that can be easily resolved by their emission spectra. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. We applied the approach on a camera-based microscope by means ... [truncated at 150 words]
Palomba F, Digman MA, Luo X, Lee A.
NADH autofluorescence phasor FLIM for the metabolic characterization of T cell and leukemia cell in a droplet.
66th Annual Meeting of the Biophysical Society. San Francisco, California. February 19-23.
Biophys J. 2022; 121(3, Suppl 1): 359a, 1733-Pos.Immune cells are known to shift their metabolism when stimulated by the interaction with binding receptors. The study of metabolic activation can be very important for the development of immune therapies and for understanding the machinery of immune active cells. However, there are challenges for monitoring real-time cell to cell interactions. Here we report the combination of the autofluorescence NADH Phasor FLIM method with the metabolic fingerprint of Jurkat cells (T lymphocyte) co-encapsulated with K562 (leukemia cells) in a water-in-oil droplet microfluidic chamber. The NADH autofluorescence, produced by all cells, lifetime and percent fractional contribution is characterized by two states: i) the enzyme bound state,3.4 ns, representative of OXPHOS and ii) the free state, 0.4 ns, mainly attributed to Glycolysis. The cells are co-encapsulated in a 1:1 ratio in a water droplet. To measure the NADH autofluorescence FLIM fingerprints, we used two photon excitation at 740 nm, with a confocal ... [truncated at 150 words]
-
Metabolic imaging using the phasor approach to FLIM and tracking phenotypic change of mitochondria in cancer cells with Mitometer.
Single Molecule Spectroscopy and Superresolution Imaging XV. San Francisco, California. January 22-27, 2022.
The hallmark of metabolic alteration of increase glycolysis, i.e. Warburg effect, in cancer cells together with atypical extracellular matrix structure may be responsible for tumor cell aggressiveness and drug resistance. Here we apply the phasor approach technique in fluorescence lifetime imaging microscopy (FLIM) as a novel method to measure metabolic alteration as a function of ECM mechanics. We imaged and compared triple-negative breast cancer (TNBC) cells to non-cancerous cells on various ECM stiffness. Dysregulation of mitochondrial motion may contribute to the fueling of bioenergy demands in metastatic cancer. To measure mitochondria motion and analyze their fusion and fission events, we developed a new algorithm called “mitometer” that is unbiased, and allows for automated segmentation and tracking of mitochondria in live cell 2D and 3D time-lapse images.
Vorontsova I, Vallmitjana A, Nakazawa Y, Torrado B, Schilling TF, Hall JE, Gratton E, Malacrida L.
Aquaporin 0a is required for water homeostasis in the zebrafish lens in vivo.
6th International Conference on the Lens. Kailua Kona, Hawaii. December 8-13, 2019. 2019; ICL 2019 Meeting Program: 23.Purpose: Aquaporin 0 (AQP0) is essential for lens development and transparency. Mammalian AQP0 has multiple cellular functions including water transport and adhesion, which have been impossible to study individually. An ancestral teleost genome duplication gave rise to two AQP0 orthologs in zebrafish, aqp0a and aqp0b, which apparently have functionally diverged. In vitro, both Aqp0s permeate water. We have shown that CRISPR-Cas9 mediated null aqp0a-/- mutants form cataract, while aqp0b-/- lenses look like wild-type, consistent with distinct functions. In this study, we test adhesive properties, and the requirements of Aqp0a and Aqp0b for water homeostasis in zebrafish lenses in vivo.
Methods: Heterologous expression in adhesion-deficient mouse fibroblast L-cells was used to test adhesive properties of Aqp0s. To study lens water homeostasis in vivo, we have employed a permeable, non-toxic solvatochromic fluorescence probe, ACDAN (6-acetyl-2-dimethylaminonaphthalene). Spectral phasor analysis of the ACDAN signal allows mapping of dipolar relaxation (DR) onto lens images, which we ... [truncated at 150 words]
-
3D orbital tracking under STED microscopy.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 440a, 2174-Pos.Orbital tracking is a well-established particle tracking technique in which the scanner of a conventional fluorescence scanning microscope is controlled to scan orbits around an object instead of forming a raster image. By recording the fluorescence along the orbits, a movement of the object inside the orbit is detected as an increase in the intensity measured at a particular point in the orbit which is in turn used to re-center the subsequent orbits in the appropriate position. Movements along the z direction can be detected by performing two orbits, one above and one below, using a piezo objective. This allows following the object in time and obtaining a 3D trajectory from which we can extract information about the diffusion and the structure of the biological system in which the object is moving. Moreover, the fluorescence intensity signal acquired at each orbit during a tracking experiment provides a space-time image of ... [truncated at 150 words]
Gratton E, Rossetta A, Chen H.
A new faster Flimbox with higher harmonic content and multi detector input.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 441a, 2179-Pos.The FLIMbox is an FPGA chip programed to perform frequency-domain FLIM acquisition. The original FLIMbox setup (Colyer et al.) was designed around Xilinx Spartan3 FPGA chip. The original design had a maximum internal frequency of operation of 320 MHz, which in our design, limits the spectral bandwidth to the first harmonic of a laser source at 80 MHz, typically used for FLIM images in 2-photon setups. The new Xilinx Kintex-7 FPGA KC705 Evaluation Kit has much higher internal operational frequencies allowing the acquisition of harmonic content in excess of 1GHz. Also this new FPGA chip can be used with a USB3 interface with a large increase in transmission speed. More importantly, the Kintex 7 board can operate on many inputs simultaneously allowing data acquisition of photomultiplier arrays (8x8). In this work we show the larger harmonic content obtained with this chip as well as the capability for multi detector acquisition. ... [truncated at 150 words]
Tedeschi G, Scipioni L, Trinh A, Lee KA, Malacrida LS, Digman MA, Gratton E.
Characterization of the metabolic state and molecular crowding in breast cancer spheroids.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 421a, 2080-Pos.Spheroids are 3D cultures of cells specifically grown in order to create a single spherical cell mass, which can be used as a model for studying the behavior of tumors in a controlled environment. Despite being a simplistic model, it allows for the rapid creation of the samples and displays a more complex physiology when compared to 2D cultures [1].
The spheroids grown for this study are derived from two different breast adenocarcinoma cell lines, MCF7 and MDA-MB231, which display less and more invasive behavior, respectively. From 20,000 cells, our protocol provides spheres with a diameter of approximately 400 μm. The spheres were then transferred to a collagen Type-1 gel matrix in order to mimic the extracellular matrix.
Once obtained and transferred in the collagen, the spheres were observed at different time points over 24 hours. We measured their single cell metabolic signature by analyzing the NADH lifetime with the Phasor-FLIM approach ... [truncated at 150 words]
-
Adaptive optics in strongly scattering samples.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 441a, 2181-Pos.One of the challenges in deep tissue microscopy is the sharp decline in image quality as depth is increased. To improve image quality, we are developing an adaptive optical system in our multiphoton microscope, the DIVER (Deep Imaging Via Enhanced photon Recovery). The transmission geometry used in the DIVER allows us to ramp up laser power as needed to counteract the effects of scattering and absorption without blinding the detector with out of focus surface fluorescence, which is a constraint in epi-detection schemes. Although the photons contributing to the multiphoton excitation are mostly spatially and temporally coherent, phase delays introduced by the spatial variations in refractive index throughout the tissue distort the focal volume. We use a segmented hexagonal deformable mirror from Boston Micromachines Corporation with 111 actuators to cancel out the low spatial frequency aberrations introduced by the tissue as well as those inherent in the imaging system. To ... [truncated at 150 words]
Malacrida LS, Scipioni L, Gratton E.
In vivo chromatin compaction changes as detected by water dipolar relaxations: the molecular crowding role revealed by the ACDAN fluorescence.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 70a, 355-Pos.Chromatin occupies territories in the nucleus in regards to its different status, hetero and euchromatin. Chromatin dynamics responds to chemical and physical inputs, producing changes in the molecular crowding and thermodynamic parameters. Using ACDAN photophysics we demonstrate the correlation between the water dipolar relaxation and the chromatin status(Malacrida-2017). ACDAN was synthesized to sense polarity and dipolar relaxation in proteins(Weber-1979). Inside the cell one of the main dipolar relaxation active molecules is water. Using spectral phasors we analyzed the ACDAN spectral shift due to modification of chromatin compaction using chemical and physical stimuli. First, we produce double-strand DNA break (DSB) by irradiating a small ROI (780nm ∼25mW, 10x10pixels, ∼150nm/pixel) inside the nucleus. Using Trichostatin-A (TSA, a histone deacetylase inhibitor) we produced chromatin decompaction by the unfolding of the heterochromatin driven by the inhibition of removal of acetyl group from histones. To induce chromatin compaction we used Okadaic acid (OA, a protein ... [truncated at 150 words]
Hedde PN, Staaf E, Singh SB, Johansson S, Gratton E.
Dynamics of activating and inhibitory receptors in Murine Natural Killer cells revealed by 2D pair correlation function analysis.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 280a, 1380-Pos.Natural Killer (NK) cells are innate immune cells that mediate anti-viral immunity and protect against cancer. Their killing threshold is determined by a balance between inhibitory and activating membrane receptors. By expressing self MHC class I specific inhibitory receptors they become educated, i.e., they learn to recognize self from non-self. Educated NK cells are more efficient in killing aberrant cells. However, the mechanism for this functional difference between educated and uneducated NK cells is still unclear. In a previous study, we found that murine educated NK cells exhibited faster movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A by means of image mean square displacement analysis. Yet, these values represent a spatial average over a region of interest at the cell membrane. Here, we utilized the 2D pair correlation function approach in order to study the spatial distribution of obstacles to NK cell receptors at the ... [truncated at 150 words]
-
Hyperspectral imaging in scattering media using two filters.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 441a, 2178-Pos.Hyperspectral imaging is a common technique in fluorescence microscopy to obtain the emission spectrum at each pixel of an image. However, methods to obtain spectral resolution based on diffraction gratings or integrated prisms work poorly when the sample is strongly scattering. We developed a microscope named the DIVER that collects the fluorescence emission over a very large angle and sample area. We have shown that this microscope, depending on the tissue thickness, can capture several orders of magnitude more light than conventional microscopes using epi detection, which in turn significantly increases imaging depth. Since the fluorescence light after passing through the multiple scattering sample is not collimated, the use of grating or prisms strongly limits the amount of light that can be used with available hyperspectral devices. Here we show that two filters that accept uncollimated light over a large aperture are sufficient to calculate the spectral phasor rather than ... [truncated at 150 words]
Begarani F, D'Autilia F, Signore G, Gratton E, Beltram F, Cardarelli F.
Capturing metabolism-dependent solvent polarity fluctuations in a trafficking Lysosome.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 307a, 1516-Plat.The eukaryotic cell compartmentalization acts as a master regulator of metabolism by arranging specific sets of molecular components in spatially confined, membrane-enclosed, intracellular structures (e.g. organelles, endosomes, vesicles). Here, peculiar physicochemical properties of the local environment do emerge and participate to the regulation of molecular processes. In spite of the huge amount of available environmental probes, experiments on sub-cellular structures are severely challenged by their restless 3D-movement. Classical optical microscopy tools fail to subtract the 3D evolution of the system while preserving the temporal resolution required to probe the physicochemical properties of the local environment. This bottleneck is tackled by focusing an excitation light-beam in a periodic orbit around the structure of interest. The recorded signal is used as feedback to localize the structure position with unprecedented temporal resolution: microseconds along the orbit, milliseconds between orbits. The lysosome is proposed as paradigmatic intracellular target. 6-acetyl-2-dimethylaminonaphthalene (ACDAN) is used as a ... [truncated at 150 words]
Scipioni L, Lanzanò L, Diaspro A, Gratton E.
Airyscan CCA provides structural and dynamics fingerprinting of subcellular compartments in living cells.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 282a, 1389-Pos.In this work we will present our current advances in the development of the CCA (Comprehensive Correlation Analysis) technique using the Zeiss Airyscan detector. This detector consists of 32 GaAsP PMT arranged in a hexagonal pattern, featuring a fast temporal sampling (down to 1.28 μs per frame). It can also be used in a super-resolution configuration as per the ISM [1] principle, while the arrangement of the detectors allows for the implementation of advanced state-of-the-art spatiotemporal correlation techniques. We will present a novel application of the spot-variation FCS technique [2] in super-resolution, as well as the implementation of the 2D-pCF [3], iMSD [4] and number and brightness [5] analysis using this fast detector array. This simultaneous analysis of the same dataset provides biophysical information regarding the fluorescent probe and the surrounding environment, such as diffusion coefficient, concentration, diffusion modality, environment organization, direction and anisotropy of molecular flows, diffusion connectivity and ... [truncated at 150 words]
Ranjit S, Malacrida LS, Gratton E.
The fluorescence lifetime of bound NADH: clues from the phasor plots.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 565a, 2804-Pos.Changes in metabolism in cells are often studied by the ratio of NAD+ to NADH. This ratio is correlated to free and enzyme bound NADH, which can be used as a measurement of metabolic states in cells using fluorescence lifetime imaging (FLIM). Binding of NADH to the enzyme causes separation between nicotinamide and adenine moieties of Nicotinamide adenine dinucleotide (NADH) from their collapsed structure in solution and results in increase of fluorescence quantum yield and lifetime. The extent of increase in fluorescence lifetime is dependent on the apoenzyme and presence of auxiliary ions. Reports from past studies show distinctive discrepancies in calculation of the bound NADH lifetime, often related to complications in sample preparation and limitations in data acquisition. In this work, we show that in presence of oxalic ion, proper preparation of lactate dehydrogenase (LDH) bound NADH has a lifetime of 3.4 ns and is positioned on the universal ... [truncated at 150 words]
Chen H, Ma N, Kagawa K, Kawahito S, Digman MA, Gratton E.
Widefield multi-frequency fluorescence lifetime imaging using a two-tap CMOS camera with lateral electric field charge modulators.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 567a, 2812-Pos.Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) measures fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD-FLIM system based on a CMOS camera equipped with two-tap true correlated double sampling (CDS) lock-in pixels and lateral electric field charge modulators. Due to the fast intrinsic response and modulation of the camera, our system allows parallel multi-frequency FLIM in one measurement via Fast Fourier Transform. We demonstrate that at a fundamental frequency of 20MHz, 31-harmonics (620MHz) can be measured with 64 phase images per laser repetition period. As a proof of principle, we analyzed cells transfected with Cerulean and with a construct of Cerulean-Venus that shows Förster resonance energy transfer at different modulation frequencies. We also tracked the temperature change of living cells via the fluorescence lifetime of Rhodamine B at different frequencies. Furthermore, we can rapidly distinguish the pre-implantation embryo development stages at the ... [truncated at 150 words]
Cinco R, Hedde PN, Malacrida L, Digman MA, Gratton E.
Multi-modal fluorescence characterization of cell cycle progression and cytokinesis.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 24a, 125-Plat.Cell division is central to both the creation of life and the propagation of diseases such as cancer and neurodegenerative disorders. While many of the genes and enzymes responsible for regulating cell cycle progression have been extensively characterized, much of the cellular bioenergetics responsible for fueling cell cycle progression and biophysics of cell division is poorly defined. Here, we characterize cell cycle progression in human HeLa cells using Fluorescent Ubiquitination-based Cell Cycle Indicator (FUCCI) with a variety of advanced fluorescence optical approaches. Application of NADH phasor Fluorescence Lifetime Imaging Microscopy (FLIM) identified significant changes both in NADH concentration and free/bound NADH between several stages of the cell cycle defined by FUCCI. We also present detailed timelapse images of human HeLa cells undergoing cytokinesis using NADH Phasor FLIM, Laurdan Phasor FLIM, and timelapse 740nm 2-Photon spectral phasor to spatially characterize and quantify metabolism, membrane fluidity, and unlabeled fluorescence emission during the ... [truncated at 150 words]
Digman MA, Lefebvre AEYT, Adame FA, Fong E.
The phasor FLIM analysis monitors metabolic changes at the leading edge in response to Rac photo-activation and mitochondrial transport in MDA MB231 cells.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 442a, 2185-Pos.Subcellular enrichments can promote metabolic shifts in cancer cells that may in turn increase invasiveness. There is a direct relationship between mitochondrial transport at the leading edge and increased invasion within various cancer types including prostate, breast, lung and glia. This recruitment is increased when PI3K inhibitors are added as cancer treatment but instead of reducing cancer invasion, it stimulates the reactivation of Akt2 or mTOR that causes the contradictory increase in tumor cell invasion. Given that mTOR controls actin cytoskeleton dynamics through Rac GTPase activation, we reasoned that activating Rac may increase mitochondria recruitment. We investigated mitochondrial recruitment by photo-activating the Rac GTPAse photoactivatable protein in living cells and monitored local energy metabolism at the leading edges of the cell membrane using the phasor approach to Fluorescence Lifetime imaging Microscopy (FLIM). We also hypothesis that matrix density may regulate intracellular tension at cell's leading edge. This tension may trigger ... [truncated at 150 words]
Lefebvre AEYT, Adame FA, Digman MA.
A non-invasive metabolic investigation of breast cancer invasion.
63rd Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 2–6, 2019.
Biophys J. 2019; 116(3, Suppl 1): 550a, 2727-Pos.Over 90% of cancer related deaths are the result of complications arising due to cancer metastasis. The reconfiguration of an invasive cancer cell's metabolic signature due to its mechanosensing ability is well acknowledged yet remains poorly understood. By probing the metabolic response of cells of differing invasivity using non-invasive imaging techniques, novel insights into the mechanisms of metastatic disease may be elicited.In this work, we used fluorescence lifetime imaging microscopy (FLIM) together with phasor analysis, to determine the free to bound NADH ratio present within the different cells. This free to bound NADH ratio analysis can determine whether a cell is relying relatively more on glycolytic metabolism or on oxidative phosphorylation. Additionally, we used spectral imaging with phasor analysis on LAURDAN stained cells, allowing us to determine the cell's membrane fluidity under varying conditions. We probed the metabolic and fluidity profiles of three breast cells of varying invasivity. These cells ... [truncated at 150 words]
Tenner A, Chu SH, Wetsel R, Cinco R, Gratton E, Fonseca M.
Complement proteins associated with synapse proteins in an Alzheimer's disease mouse model lacking C5aR1.
27th International Complement Workshop (ICW). Santa Fe, New Mexico. September 16–20, 2018.
Mol Immunol. 2018; 102: 218.Activation of the complement pathway by the fibrillar Aß plaques in Alzheimer's disease (AD) brains and AD mouse models contributes to glia1activation and inflammation, and is associated with cognitive loss. Complement is also necessary for synapse pruning in AD and other neurological disorders. Complement activation generates the proinflammatory cleavage fragment C5a. Previous work in our lab has shown that pharmacological inhibition or genetic lack of C5a receptor 1 (C5aR1) has beneficial effects in suppressing pathology and cognitive loss in mouse models of AD. Using superresolution microscopy, the influence of C5a/C5aR1 signaling on synapses in the Arctic mouse model of AD was investigated. Wildtype (WT), Arctic, C5aR1KO and ArcticC5aR1KO mice at different ages were immunostained with presynaptic synaptophysin (SYN) or Vglut1 and postsynaptic PSD95 or Homer 1, or with C1q and a presynaptic marker. Individual and colocalized puncta as well as C1q-tagged synapses in the CA1 stratum radiatum (SR) and the ... [truncated at 150 words]
Hadraba D, Lopot F, Paesen R, Ameloot M, Jelen K, Digman MA.
Mechanical testing of connective tissue from nano to macro.
19th Annual UC Systemwide Bioengineering Symposium. Riverside, CA. June 21-23, 2018.
UCR BIEN Symposium Complete ABSTRACT Book. 2018; 1: 108.Introduction: Connective tissue is an inhomogeneous composite structure with high variability in mechanical properties. The mechanical properties originate in a hierarchical organization of proteins from which type I collagen plays the most evident role. This role seems to differ at each hierarchical level. For example, type I collagen triple helix returns the highest Young’s modulus but only fibers are aligned into a zig-zag wavy pattern, termed the crimp pattern. The mechanical properties are also not stationary and alter with age or disease, and therefore, conducting an experiment on the crimp pattern behavior under mechanical load simultaneously with the measurement on the helical pitch angle of type I collagen molecule can provide a new insight into the clinical classification of connective tissue.
Materials and Methods: The rabbit tendons were gripped in an in-house built tensile instrument that was placed at the microscope stage. The tendons were imaged using polarized second harmonic generation ... [truncated at 150 words]
Trinh AL, Chen H, Chen Y, Hu Y, Li Z, Siegel ER, Linskey ME, Wang PH, Zhou YH, Digman MA.
Distinguishing stem-like tumor-initiating cells in malignant glioma by phasor fluorescence lifetime imaging.
19th Annual UC Systemwide Bioengineering Symposium. Riverside, CA. June 21-23, 2018.
UCR BIEN Symposium Complete ABSTRACT Book. 2018; 1: 113.Introduction: Intra-tumoral heterogeneity is associated with therapeutic resistance of cancer. In particular, stem-like tumor initiating cell (STIC) subpopulations within malignant gliomas tumors have been shown to be responsible for treatment resistance and tumor recurrence. Therefore, there exists a need to monitor functional tumor subpopulations during treatment and cancer progression. Reduced nicotinamide adenine dinucleotide (NADH) is a metabolic coenzyme essential in cellular respiration. Fluorescence lifetime imaging microscopy (FLIM) of NADH has been demonstrated to be a powerful label-free indicator for inferring metabolic states of living cells. In this study, FLIM of NADH was applied to distinguish between malignant glioma subpopulations of STIC and tumor mass-forming cells (TMC). In addition, changes in the fluorescence lifetimes of NADH were compared to cellular respiration (oxidative phosphorylation and glycolysis), EGFR expression, and cell-growth rates.
Materials and Methods: Human glioblastoma-derived cell line U251 were enriched for STIC and TMC subpopulations by established cell culture conditions. U251 STIC ... [truncated at 150 words]
Rahim MK, Gratton E, Haun JB.
Transforming FLIM into a high-content molecular analysis platform.
19th Annual UC Systemwide Bioengineering Symposium. Riverside, CA. June 21-23, 2018.
UCR BIEN Symposium Complete ABSTRACT Book. 2018; 1: 69.Introduction: Tumors are complex and heterogeneous, and therefore diagnostic techniques will have to comprehensively assess molecular features across diverse cell types and functional states. Current methods that can provide both spatially-resolved and quantitative molecular interpretation of tissues are limited in multiplexing capacity or are complex and time-consuming. To address these limitations, we have developed a multiplexed imaging platform that utilizes fluorescence lifetime imaging microscopy (FLIM). Fluorescence lifetime refers to the duration of fluorescence light emission after excitation, and it can be leveraged to resolve fluorescent probes that have the same color. Moreover, the phasor approach to FLIM greatly simplifies lifetime analysis. Instead of fitting complicated exponential functions, the phasor approach provides a graphical representation that enables the unmixing of more than one lifetime using simple geometrical considerations. In this study, we are developing new methodologies to quantitatively detect 4 molecular probes within the same spectral window using FLIM and the ... [truncated at 150 words]
Leemans SW, Dvornikov A, Gratton E.
Adaptive optics in deep tissue microscopy.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 533a, 2638-Pos.Multiphoton fluorescence microscopy is a powerful technique to visualize the form and function of the cells in tissue at sub-micron scales. To diminish fluorescence emission losses due to light scattering in the sample, our lab has developed a transmission fluorescence microscope to recover more of the emitted fluorescence from biological samples. Multiphoton fluorescence is sensitive to the peak power of the light, so the efficiency of the process improves when pulse duration is decreased and the focal volume is minimized. The DIVER (Deep Imaging Via Emission Recovery) microscope uses a pulse compressor that pre-compensates for dispersion in the excitation path to minimize pulse duration. Spatial variations in the index of refraction in tissue distort the focal volume, resulting in decreased resolution as imaging depth is increased. We compensate for spatial variations in index by conjugating the wavefront of the excitation light to the optical aberrations that are introduced by the ... [truncated at 150 words]
Scipioni L, Diaspro A, Lanzanò L, Gratton E.
AiryScan comprehensive superresolution correlation analysis.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 533a, 2641-Pos.AiryScan is a detector array developed by Zeiss that consists in 32 GaAs PMT detectors arranged in a hexagonal pattern. The development of single point detector arrays is recently increasing also thanks to the possibility of achieving super-resolution without signal loss by means of Image Scanning Microscopy (ISM, Sheppard et al. 2013). Furthermore, super-resolution techniques applied to fluorescence correlation techniques have shown to be effective in retrieving information that is not accessible with diffraction-limited methods, giving new valuable tools to biological research. Being essentially a high-speed nano-camera, AiryScan allows, in principle, for the implementation of most of the state-of-the-art correlation techniques at super-resolution in a single analysis. In this work we will present the parallel implementation of fluorescence correlation spectroscopy (FCS, Digman et al. 2011), pair-correlation function (pCF, Hinde et al. 2010) and connectivity analysis, image-derived mean square displacement (iMSD, Di Rienzo et al. 2014), number and brightness (N&B, Digman ... [truncated at 150 words]
Malacrida LS, Hedde PN, Ranjit S, Cardarelli F, Gratton E.
Elucidating invisible barriers and obstacles to molecular diffusion in live cells by the spatial pair-correlation function: a connectivity view of the cell.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 166a, 835-Pos.Despite recent advances in optical super-resolution, we lack of methods to visualize the path followed by diffusing molecules in the cell. Fluorescence correlation spectroscopy (FCS) provides molecular dynamics at the single molecule level; however, the classical single point FCS lacks spatial resolution. Methods based on image analysis like raster-scanning and spatiotemporal image correlation still needs spatial averaging over relatively large areas; hence the spatial resolution is compromised. Here, we develop a new method based on the spatial pair-cross-correlation in two dimensions (2D-pCF) to obtain relatively high-resolution images of molecular diffusion dynamics and transport in live cells. The 2D-pCF method measures the time for a particle to go from one location to another by cross-correlating the intensity fluctuations at specific points in an image. Hence, a visual map of connectivity for the average path followed by molecules is created. To test our approach we use a nanometer channel simulation and a ... [truncated at 150 words]
Torrado B, Malacrida L, Badano JL, Irigoín F, Gratton E.
Intracellular transport characterization of the transcription factor Gli2 by fluorescence correlation spectroscopy approaches.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 630a, 3123-Pos.Gli2 is the principal transcriptional activator of the Hedgehog signalingin mammals. Upon pathway activation, Gli2 moves into the cilium before reaching the nucleus. While two conserved classical nuclear localization signals (cNLS) mediate Gli2 nuclear localization via importin (Imp)-α/β1, these sequences are not required for Gli2 ciliary import (Torrado et al, 2016. PLoSONE ;11(8)). However, Importin-β2 is involved in Gli2 ciliary entry, and might also collaborate in Gli2 nuclear entry (Torrado et al, 2016. PLoSONE ;11(8)). Several questions about Gli2 intracellular traffic still remain opened. To analyze the nucleo-cytoplasmic transport of Gli2 we used fluorescence correlation spectroscopy methods, in particular pair correlation function and Number & Brightness approaches. We compared Gli2 nuclear transport when the pathway is on and off. Gli2 ciliary transport was analyzed by fluorescence correlation spectroscopy approaches combined with 3D-particle tracking so we could avoid the out of focus produced by the movement of the cilium. We applied ... [truncated at 150 words]
Ranjit S, Dvornikov A, Levi M, Gratton E.
Multicomponent analysis of phasor plot to decipher changes in metabolic trajectory of biological systems.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 167a, 843-Pos.Recent developments of phasor approach to Fluorescence Lifetime Imaging (FLIM) in cells undergoing oxidative stress or cultured in oleic acid enriched media and in mice liver sections having high fat diet, have shown presence of a third autofluorescent component indicative of lipid droplets besides the phasor components from free and enzyme bound NADH in the 400-450 nm emission wavelength. This third component changes the position and shape of the phasor distribution away from the metabolic trajectory, defined by the line joining the phasor position of free and enzyme bound NADH, and makes the study of changes in metabolism using the ratio of free to bound NADH difficult. However, the phasor rule of addition applies to any number of components and this was exploited to create a multicomponent analysis. A change in phasor distribution towards free or bound NADH can then be separated and analyzed independent of the third individual. Law ... [truncated at 150 words]
Digman MA, Palomba F, Kong X, Yokomori K, Gratton E.
Pair correlation analysis of Ku dynamics upon DNA damage.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 351a, 1740-Pos.We used the pair correlation function (pCF) analysis developed for fast frame acquisition to measure the changes in molecular connectivity of the Ku protein following double strand DNA damage. Ku protein binds to the ends of double-strand breaks and is required in DNA-repair for non-homologous end joining (NHEJ). Before damage, Ku diffuses freely with low anisotropy in the entire nucleus. After damage, we observe the formation of barriers of Ku at the damage site. In the rest of the nucleus the diffusion coefficient of Ku increases that persists four hours post damage. The diffusion anisotropy tensor of Ku in the nucleus was measured and an increase in anisotropy compared to the control cells without damage was observed. Given that DNA-PKcs play a critical role in NHEJ, loss of their activity can lead to a reduced repair rate and would alter Ku motility at the damage sites. To test this, we ... [truncated at 150 words]
Hedde PN, Malacrida L, Ahrar S, Siryaporn A, Gratton E.
SideSPIM - a flexible multipurpose platform for light sheet microscopy.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 187a, 945-Plat.We devised a flexible multipurpose platform for light sheet microscopy. Based on a regular inverted microscope, a custom designed chamber is used to allow selective plane illumination from the side (sideSPIM). This eliminates the need of dipping into the sample container, hence small sample volumes (~100 µl) can be realized and objective lenses can be interchanged quickly. We demonstrate that this flexible multipurpose platform is suitable for a large variety of applications involving samples of substantially different sizes and geometries. For example, we captured the microcirculation of erythrocytes in a zebrafish embryo at 5 stacks/s (40 planes), studied the growth of bacteria biofilms under flow using a fluidic device, and probed the diffusion of EGFP tagged proteins in mammalian cells by acquiring fast time series (200 fps) and subjecting them to fluorescence fluctuation based image mean square displacement (iMSD) and two-dimensional pair cross-correlation function (2D-pCF) analysis. We further evaluated the ... [truncated at 150 words]
Cinco R, Hedde PN, Digman MA, Gratton E.
Spatial characterization of NADH concentration and diffusion in cells and tissue.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 335a, 1662-Pos.Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH play a central role in bioenergetics as an enzymatic cofactor and reducing equivalent in cellular metabolic and redox reactions. The NADH/NAD+ ratio has significant impact on energy production, cell survival, proliferation, longevity and aging. Lifetime measurements of free/bound NADH autofluorescence has been demonstrated to approximate the NADH/NAD+ ratio and has been established as a reliable means for estimating the mode of metabolism used by cells and tissues. NADH microenvironment concentrations, diffusion speed, and potential sequestration areas may dictate NADH availability for biochemical reactions and thereby modulate cell metabolism and signaling. To approximate the spatial distribution and diffusion of NADH in cells, we excited NADH at 740 nm 2-Photon excitation and performed raster image correlation spectroscopy (RICS) and number and brightness (N&B). Application of RICS to determine NADH diffusion did not yield enough photons for a correlation to be detected, since the ... [truncated at 150 words]
-
Spectral resolution in fluorescence microscopy in strongly scattering media.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 166a, 838-Pos.Spectral phasors were introduced as alternative to spectral demixing, for the determination of FRET and for the measurement of dipolar relaxation in membranes. The basic concept in the spectral phasor approach is that the entire spectrum is not needed for some analysis, but only some of the elements of the spectral distribution are sufficient. In the case in which the spectrum is dominated by few spectral bands, this approach is very efficient and provides fast and valid alternative to the analysis of the full spectrum. One example of a system that could benefit from the phasor analysis is in tissue spectroscopy in which the light to be collected has undergone multiple scattering so that it cannot be easily focused on the entrance slit of a dispersive element or transformed in a parallel beam for use with hyperspectral cameras. In a typical application of thick tissue spectroscopy, the light, after being ... [truncated at 150 words]
Murata MM, Kong X, Yokomori K, Digman MA.
Fluorescence lifetime of NADH reveals PARP-dependent increase of oxidative phosphorylation critical for cell survival.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 83a, 425-Pos.The DNA damage response (DDR) pathway triggers a chain of reactions that are critical for the maintenance of cell survival and genome integrity. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor that catabolizes nicotinamide adenine dinucleotide (NAD+) to form poly(ADP-ribose) on target proteins for initial chromatin organization and DNA repair pathway choice at damage sites. It has been reported that this depletion of NAD+ leads to a loss of a ATP and subsequent metabolic collapse ultimately leading to cellular death in DNA damaged cells. However, it is not clear whether or not regulating metabolism can overcome this PARP-dependent cell death pathway as a cell survival mechanism. We are investigating if inhibition of oxidative phosphorylation increases cellular damage sensitivity and if that correlates with cell survival. This may be dependent on PARP activity and the effect can last longer than the transient depletion of the total NADH level. Here we ... [truncated at 150 words]
Mah EJ, Yee AF, Digman MA.
Metabolism modulation of cancer cells on varying substrate stiffnesses.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 19a, 101-Plat.ancer cells sense their microenviroment and respond to biophysical cues that activate signaling networks through ion channels and integrin proteins. This allows cells to adapt by adjusting their cellular shape and tension that remodels the extracellular matrix (ECM). New evidence shows that these ECM signaling cascades may increase glucose uptake leading to altered metabolic states. The hallmark of metabolic alteration of increase glycolysis, i.e. Warburg effect, in cancer cells together with atypical ECM structure may be responsible for tumor cell aggressiveness and drug resistance. While it is known that tumor cells stiffen the ECM as the tumor progression occurs, a direct relationship between ECM stiffness and altered metabolism has not been explicitly measured. Here we apply the phasor approach to fluorescence lifetime imaging microscopy (FLIM) as a method to measure metabolic as a function of ECM mechanics. We imaged two breast cancer cell lines of high and low aggressiveness (MDA-MB231 ... [truncated at 150 words]
-
Laurdan imaging and spectral phasor analysis reveals increased membrane fluidity in Huntington disease.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 177a, 891-Pos.Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by the expansion of the CAG trinucleotide repeats in exon 1 of the gene encoding polyglutamine (polyQ). This is an incurable neurodegenerative disorder with complex pathogenies including protein aggregation and dysregulation of lipid homeostasis. Lipids are vital for brain function. Altered lipid metabolism has been linked to dysfunction in Huntington disease. In particular, cholesterol metabolism appears to be disturbed in patients with HD and in animal models. Here we used a biophysical approach to examine the effect of HD in the cell plasma membrane fluidity influenced by the pathogenic protein (97QmRuby) compared to non-pathogenic protein (25QmRuby) and non-labeled cells. To characterize membrane fluidity and hydration in HD, we used the fluorescent membrane probe LAURDAN that is sensitive to physical states of phospholipids. Using LAURDAN emission we characterized membrane fluidity. We further validated our results using secondary fluorescent membrane probe Nile ... [truncated at 150 words]
Cinco R, Digman MA, Gratton E, Luderer U.
Spatial characterization of bioenergetics and metabolism of primordial to preovulatory follicles in whole ex vivo murine ovary.
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
Previous work characterizing ovarian bioenergetics has defined follicular metabolism by measuring metabolic by- products in culture media. However, culture conditions perturb the native state of the follicle, and these methods do not distinguish between metabolism occurring within oocytes or granulosa cells. We applied the phasor approach to fluorescence lifetime imaging microscopy (phasor FLIM) at 740-nm two-photon excitation to examine the spatial distri- bution of free and protein-bound nicotinamide adenine dinucleotide hydride (NADH) during primordial through preovulatory stages of follicular development in fresh ex vivo murine neonatal and gonadotropin stimulated prepubertal ovaries. We obtained subcellular resolution phasor FLIM images of primordial through primary follicles and quantified the free/bound NADH ratio (relative NADH/NAD+) sepa- rately for oocyte nucleus and oocyte cytoplasm. We found that dynamic changes in oocyte nucleus free/bound NADH paralleled the developmental maturation of primordial to primary follicles. Immunohistochemistry of NAD+-dependent deacetylase SIRTUIN 1 (SIRT1) in neonatal ovary revealed that ... [truncated at 150 words]
Cinco R, Digman MA, Gratton E, Luderer U.
Characterization of NADH/NAD+ ratio during early stages of ovarian follicular activation in live murine neonatal ovaries.
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
Follicular bioenergetics is a critical determinant of oocyte quality and ovarian aging. Ovarian bioenergetics has largely focused on defining follicular metabolism by measuring metabolic byproducts in cultured media. However, culture conditions required for these data compromise the native biological cell state. Furthermore, there are few data characterizing early follicular metabolism before the primary stage, likely due to limitations in small follicle isolation.
Nicotinamide adenine dinucleotide (NAD) and its reduced form NADH play a central role in bioenergetics as one of the main energy currencies of the cell. The NADH/NAD+ ratio has significant impact on energy production, proliferation, cell survival, and aging. NADH is naturally fluorescent and its free and bound states can be measured by fluorescence lifetime imaging microscopy using 740nm 2-photon excitation. The development of the phasor approach to fluorescence lifetime imaging microscopy (Phasor FLIM) offers a straightforward approach to interpretation of lifetime differences at the pixel level. Phasor FLIM ... [truncated at 150 words]
-
Label-free fluorescence lifetime imaging microscopy (FLIM) to study metabolism and oxidative stress in biological systems.
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
Study of cellular metabolism and its influence on physiological functions and pathology along with investigation of oxidative stress in pathogenesis are essential for fundamental biology as well as biomedical research. Optical imaging offers the opportunity to assess these indices non-invasively. In this work we apply two photon fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores for label-free metabolic and oxidative stress imaging in a wide range of biological samples. Analysis of FLIM data was performed by applying the ‘fitfree’ phasor approach where each pixel of the image is transformed to its corresponding phasor on the phasor plot. Biological systems are a rich resource of autofluorescent biomolecules. Their fluorescence lifetimes are sensitive to alteration of normal physiology, making them attractive endogenous probes. We discovered one such endogenous fluorophore with characteristic long fluorescent lifetime. We hypothesized these long lifetime species (LLS) to be fluorescent products of lipid oxidation by reactive oxygen species ... [truncated at 150 words]
-
Intrinsic biomarker for oxidative stress by fluorescence lifetime imaging microscopy (FLIM).
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
Reactive oxygen species (ROS), when present above normal physiological levels, causes oxidative stress (OS), leading to damage of proteins and DNA and lipid peroxidation. OS has been associated with myriad of diseases like inflammation, diabetes mellitus, cardiovascular diseases, and cancer. Hence an OS biomarker would have immense clinical importance. There is a dearth of nondestructive techniques to measure OS in vivo. This work shows non-invasive detection of OS by fluorescence lifetime imaging microscopy (FLIM) of an intrinsic probe. Recognition of this endogenous biomarker enables label-free OS imaging. We identified this as fluorescent product of lipid oxidation by ROS. Using FLIM-phasor analysis, we can identify the presence and spatial location these species with a characteristic long lifetime (LLS). We could correlate the LLS to lipid droplets in the oleic acid treated HeLa cells using third harmonic generation (THG) imaging and coherent anti-Stokes Raman scattering (CARS) microscopy. For chemical analysis, we performed ... [truncated at 150 words]
-
Imaging techniques to measure spatio-temporal dynamics and metabolic alterations of p53 upon DNA damage.
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
For biological reaction-diffusion systems, live single cell spatiotemporal analysis of protein dynamics provides a mean to observe stochastic biochemical signaling which may lead to better understanding of cancer cell invasion, stem cell differentiation and other fundamental biological processes. This talk will describe methodologies used to investigate p53 activity and alteration of the metabolic pathway upon DNA damage. p53 is a tumor suppressor protein that regulates target genes involved in DNA damage migration and repair. If cells become stressed due to DNA damage, p53 will form tetramers in specific chromatin sites and active genes that trigger cell cycle arrest and/or apoptosis. We used the Number and Molecular Brightness (N&B) method to map aggregation processes in the entire cell upon DNA damage with cisplatin (a chemotherapy agent); thus revealing the spatial distribution of events, the site of tetramer formation and the time sequence of the aggregation events with quantitative information about the ... [truncated at 150 words]
Digman MA, Rottschäfer L, Gratton E, Hedde PN.
Fluorescence diffusion tensor maps p53 activation acquired with SPIM.
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
The transcription factor, p53, can activate the DNA repair or apoptotic pathway in response to cellular stress. Specific binding sites must be accessible to active either pathway. Thus, specific chromatin location as well as accessibility are required for stable tetramers of p53 to active either response pathway. To gain information regarding p53 fast dynamic binding interaction and diffusion to localized nuclear regions, we used single plane illumination microscopy (SPIM) to acquire high speed frame rate images with a time resolution below 1 ms. Cells were treated with either anisomyocin or cisplatin to induce cellular stress. To obtain maps of protein diffusion and directional local motion the fluorescence Diffusion Tensor Imaging (fDTI) method, inspired by the Diffusion Tensor imaging in the MRI field, was used. This powerful technique provides maps of molecular diffusivity and protein interactions in the context of their endogenous environments. The fDTI is based on spatial pair correlations ... [truncated at 150 words]
Mah EJ, Yee AF, Digman MA.
Focal adhesion protein reorganization and tension on nanotopography.
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
Surfaces with nanostructures have been shown to modulate cell contact points (e.g. focal adhesions, FAs) and have the potential to create selective adhering surfaces for implants. Previouse studies have shown that cells on nanolines form FAs and migrate parallel to the structures. In addition, FAs on nanostructures are seen to form on the edges of the features due to increased tension in these regions. However, the architecture of the FA complex on nanostructures and how this a ects cell tension have yet to be studied. Here, we utilized the z-phasor analysis to measure the distance between FA proteins, integrin and paxillin, within breast cancer cell line MDA-MB-231. Tension at the FAs were measured done with a vinculin and talin FRET tension sensor (VinTS and TalTS, respectively). Insight on the mechanosensing signals which occur in these cells will help further the understanding of environmental signals that trigger metastasis.
-
Correlation of molecular diffusion in live cells.
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
The coordination of cell functions requires that molecules move in the cell interior to find their partners. In the cell, the mechanisms for molecular motion are poorly understood. While in an isotropic fluid, diffusion is the default mechanism of motion, in the cell interior diffusion is hindered by barriers and by transient binding. Also molecules can move by active transport. One universal transport processes which is still debated is the shuttling of molecules between the cell membrane and other locations where the molecules will deliver a signal. Although we have made important progresses in understanding directed motion, less is understood in regard to paths for diffusion and in general the connectivity of the cell interior. In this presentation I will discuss the development of tools that could help us in measuring the path that molecules follow in the cell to reach their target. Among the tools needed for measuring diffusion ... [truncated at 150 words]
-
Selective plane illumination microscopy in the conventional inverted microscope geometry.
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
During the last decade, selective plane illumination microscopy (SPIM) has proven to be one of the most suitable techniques for three-dimensional time lapse imaging. By confining the excitation light to a sheet, SPIM combines axial sectioning capability with minimal light exposure and fast, camera-based image acquisition. However, the typical arrangement of two objective lenses perpendicular to each other provides a number of challenges in terms of instrument design and sample geometry, especially if the use of high numerical aperture (NA) lenses is desired. A popular approach is to dip into the sample container from the top, both lenses at a 45 degree angle with respect to the sample plane. Instead, our new design is based on a regular inverted microscope where the sample is illuminated from the side via an accessory. A custom designed chamber is used to allow side illumination. This way, all microscope ports remain available for other ... [truncated at 150 words]
Hedde PN, Ranjit S, Gratton E.
Fluorescence anisotropy imaging in 3D with single plane illumination microscopy.
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
Anisotropy imaging is a powerful tool to study the organization, composition and dynamics of molecules in biological systems. Changes in the organization and conformation of biomolecules within cells and tissues can be visualized at the single pixel level. Fluorescence anisotropy is typically probed with laser scanning and epifluorescence-based techniques. However, those techniques are limited in either axial resolution, image acquisition speed, or, by photobleaching. In the last decade, selective plane illumination microscopy (SPIM) has emerged as the best choice for three-dimensional time lapse imaging since it combines axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure [1,2]. We demonstrate how SPIM can be used for three-dimensional fluorescence anisotropy imaging of live cells [3]. To verify the accuracy of our SPIM system, we first measured and compared the anisotropy of Rhodamine110 solutions with different water/glycerol ratios with results obtained using a spectrofluorimeter. Then, we visualized the aggregation states ... [truncated at 150 words]
Malacrida LS, Ranjit S, Gratton E.
Water dipolar relaxation inside nucleus: taking advantage of ACDAN spectroscopic properties and spectral phasors.
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
Hetero and euchromatin are described at cell interphase as two different supramolecular organizations of the DNA and histones. Heterochromatin was described by electron transmission microscopy as a high electron density structure when compared to euchromatin. The information on the amount of global hydration and molecular crowding in hetero and euchromatin are still missing. These properties are relevant to understand biophysical and biochemical functions of the cell. In this study, we measure the water dynamic in hetero and euchromatin using the solvatochromic properties of ACDAN (6-acetyl-2-dimethylaminonaphthalene). The spectral phasor analysis of the ACDAN fluorescence at pixel level resolution revealed the water dynamic in the nucleus of NIH-3T3 cells. Furthermore, by using the 2D pair-Correlation-Function (so-called connectivity maps) analysis of the ACDAN fluorescence fluctuation, we were able to measure diffusion of A! CDAN in the nucleus at the interphase. To correlate the regions of different dipolar relaxation in the nucleus, we used ... [truncated at 150 words]
Malacrida LS, Ranjit S, Gratton E.
Water activity inside the nucleus: Some clues using ACDAN fluorescence and its implications in the chromatin supramolecular organization.
10th International Weber Symposium. Búzios, Brazil. May 28-June 2, 2017.
Hetero and euchromatin are described at cell interphase as two different supramolecular organizations of the DNA and histones. Heterochromatin was described by electron transmission microscopy as a high electron density structure when compared to euchromatin. The information on the amount of global hydration and molecular crowding in hetero and euchromatin are still missing. These properties are relevant to understand biophysical and biochemical functions of the cell. In this study, we measure the water dynamic in hetero and euchromatin using the solvatochromic properties of ACDAN (6-acetyl-2-dimethylaminonaphthalene). The spectral phasor analysis of the ACDAN fluorescence at pixel level resolution revealed the water dynamic in the nucleus of NIH-3T3 cells. Furthermore, by using the 2D pair-Correlation-Function (so-called connectivity maps) analysis of the ACDAN fluorescence fluctuation, we were able to measure diffusion of ACDAN in the nucleus at the interphase. To correlate the regions of different dipolar relaxation in the nucleus, we used a ... [truncated at 150 words]
Hinde E, Pandzic E, Yang Z, Ng I, Bogoyevitch M, Jans D, Gratton E, Gaus K.
Tracking oligomeric transcription factor dynamics by pair correlation of molecular brightness (pCOMB).
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 20a.Oligomerisation of transcription factors controls their translocation into the nucleus and DNA binding activity. Here we present a fluorescence microscopy method termed pCOMB (pair correlation of molecular brightness) that tracks the mobility of different oligomeric species within live cell nuclear architecture.1 pCOMB amplifies the signal from the brightest species present and filters the dynamics of the extracted oligomeric population based on arrival time between two locations. Here we use this method to demonstrate a dependence of signal transducer and activator of transcription 3 (STAT3) mobility on oligomeric state. We find that upon entering the nucleus STAT3 dimers must first bind DNA to form STAT3 tetramers, which are also DNA-bound but exhibit a different mobility signature. Examining the dimer-to-tetramer transition by a cross pair correlation analysis (cpCOMB) reveals chromatin accessibility to modulate STAT3 tetramer formation. Thus the pCOMB approach is suitable for mapping the impact oligomerisation has on transcription factor dynamics.
Lanzanò L, di Bona M, Scipioni L, Sarmento MJ, Gratton E, Vicidomini G, Diaspro A.
Heterogeneity of the nuclear environment investigated by superresolution microscopy and fluorescence correlation spectroscopy.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 142a.Genomes are more than one-dimensional entities purely defined by their linear DNA sequences [Misteli T, Cell (2007)]. A long standing challenge in biology is to decipher the principles of organization of what can be considered, by analogy with human-created libraries, the cell's primary unit of information storage and retrieval. In this respect, the development of super-resolved fluorescence microscopy has provided a new toolbox to peer into the nucleus. For instance, super-resolution microscopy has been recently applied to the investigation of nanoscale chromatin architecture, revealing nucleosome higher-order organization into heterogeneous ‘clutches’ and epigenetically dependent folding motives [Ricci M et al, Cell, (2015); Boettiger A et al, Nature (2016)].
However, dynamic properties of proteins in the nucleus are also critical for their function [Misteli T, Cell (2007)]. Fluorescence Correlation Spectroscopy (FCS) has been used to map the dynamics of several proteins in the nucleus with diffraction-limited spatial resolution. Here we combine a novel ... [truncated at 150 words]
Hedde PN, Malacrida L, Gratton E.
Selective plane illumination microscopy in the conventional inverted microscope geometry.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 145a.During the last decade, selective plane illumination microscopy (SPIM) has proven to be one of the most suitable techniques for three-dimensional time lapse imaging. By confining the excitation light to a sheet, SPIM combines axial sectioning capability with minimal light exposure and fast, camera-based image acquisition [1]. However, the typical arrangement of two objective lenses perpendicular to each other provides a number of challenges in terms of instrument design and sample geometry, especially if the use of high numerical aperture (NA) lenses is desired. A popular approach is to dip into the sample container from the top, both lenses at a 45 degree angle with respect to the sample plane [2,3]. Instead, our new design is based on a regular inverted microscope where the sample is illuminated from the side via an accessory. A custom designed chamber is used to allow side illumination. This way, all microscope ports remain available for ... [truncated at 150 words]
Digman MA, Rottschäfer L, Gratton E, Hedde PN.
Fluorescence diffusion tensor maps of p53 activation acquired with SPIM.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 148a.The transcription factor, p53, can activate the DNA repair or apoptotic pathway in response to cellular stress. Specific binding sites must be accessible to active either pathway. Thus, specific chromatin location as well as accessibility are required for stable tetramers of p53 to activate either response pathway. To gain information regarding p53 fast dynamic binding interaction and diffusion to localized nuclear regions, we used single plane illumination microscopy (SPIM) to acquire high speed frame rate images with a time resolution below 1 ms. Cells were treated with either anisomyocin or cisplatin to induce cellular stress. To obtain maps of protein diffusion and directional local motion the fluorescence Diffusion Tensor Imaging (fDTI) method, inspired by the Diffusion Tensor imaging in the MRI field, was used. This powerful technique provides maps of molecular diffusivity and protein interactions in the context of their endogenous environments. The fDTI is based on spatial pair correlations ... [truncated at 150 words]
Ranjit S, Aguilar-Arnal L, Stringari C, Sassone-Corsi P, Gratton E.
Spatial dynamics of SIRT1 relates to metabolic transitions in the cell nucleus.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 149a.SIRT1 is a NAD+ -dependent deacetylase functioning as metabolic sensor of cellular energy and it adapts different biochemical pathways to the changes in the environment. SIRT1 substrates include histones and proteins related to enhancement of mitochondrial and antioxidant protection. Fluctuations in intracellular NAD+ levels regulate SIRT1 activity, yet the exact pathway SIRT1 enzymatic activity impacts NAD+ levels and its intracellular distribution remains unclear. Here, we demonstrate that SIRT1 determines the nuclear organization of protein bound NADH. Using multiphoton microscopy in live cells, we show that free and bound NADH are compartmentalized inside of the nucleus, and its subnuclear distribution depends on SIRT1. Importantly, SIRT6, a chromatin-bound deacetylase of the same class does not influence NADH nuclear localization. In addition, using fluorescence fluctuation spectroscopy, especially phasorFCS in single living cells, we reveal that NAD+ metabolism in the nucleus is linked to subnuclear dynamics of active SIRT1. SIRT1 diffuses faster on the ... [truncated at 150 words]
Cardarelli F, Gratton E, Beltram F, di Rienzo C.
Spatiotemporal fluctuation analysis: a powerful tool for the future nanoscopy of dynamic molecular processes.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 149a.A major challenge of present and future biophysics is to quantitatively study how biomolecules dynamically fulfill their physiological role in living cells, tissues, or entire organisms. In the last few decades, new experimental methodologies were introduced that are able to unveil details on a length scale that is a tiny fraction of the wavelength of light, thus moving spatial resolution far beyond the diffraction limit set by Ernst Abbe's equation. Still, the enormous wealth of information available today from optical microscopy measurements on living samples is often underexploited. We argue that spatiotemporal fluorescence correlation spectroscopy (spFCS) can enhance the performances of current nanoscopy methods and provide further insight into dynamic molecular processes of high biological relevance (Di Rienzo, C. et al. Biophys J, 111, 679-685, 2016). We present exemplary biological applications of spFCS to measure dynamic molecular parameters well below the diffraction limit in a standard optical setup, including the ... [truncated at 150 words]
Datta R, Bhattacharjee A, Hochbaum A, Gratton E.
FLIM phasor fingerprint of bacterial metabolic state.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 186a.While a large number of bacteria are deemed beneficial, there is an increasing number of strains associated with pathology, including life-threatening diseases. Moreover, during the last decade, a large number of new infectious strains have been discovered. Treatment is often hindered by emergence multi-drug resistant bacterial strains. Bacteria adapt to adverse environmental conditions by changing their metabolic activity. Further, there also exists metabolic diversity within the same population. Non-invasive assessment of bacterial metabolism could shed light on their physiological status. In this work we employ two-photon fluorescence lifetime imaging microscopy (FLIM) of live, clinically relevant bacteria. FLIM of autofluorescent metabolic coenzyme reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, has been extensively exploited for label-free metabolic imaging of mammalian cells to study cancer and other diseases. However, it has not been explored to similar extents in bacteria. We apply phasor analysis to FLIM and create FLIM-phasor fingerprints of bacteria species Escherichia coli, ... [truncated at 150 words]
Malacrida LS, Ranjit S, Gratton E.
Water activity inside the nucleus: some clues using ACDAN fluorescence and its implications in the chromatin supramolecular organization.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 218a.Hetero and euchromatin are described at cell interphase as two different supramolecular organizations of the DNA and histones. Heterochromatin was described by electron transmission microscopy as a high electron density structure when compared to euchromatin. The information on the amount of global hydration and molecular crowding in hetero and euchromatin are still missing. These properties are relevant to understand biophysical and biochemical functions of the cell. In this study, we measure the water dynamic in hetero and euchromatin using the solvatochromic properties of ACDAN (6-acetyl-2-dimethylaminonaphthalene). The spectral phasor analysis of the ACDAN fluorescence at pixel level resolution revealed the water dynamic in the nucleus of NIH-3T3 cells. Furthermore, by using the 2D pair-Correlation-Function (so-called connectivity maps) analysis of the ACDAN fluorescence fluctuation, we were able to measure diffusion of ACDAN in the nucleus at the interphase. To correlate the regions of different dipolar relaxation in the nucleus, we used a ... [truncated at 150 words]
Bouzin M, Heylman C, Zhao W, Gratton E.
Particle counting by confocal microscopy: increasing the dynamic range via multiple-slits fluorescence detection.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 295a.Fast clinical diagnostics and the early-stage identification of bloodstream infections require culture-free real-time detection of pathogens at low concentrations, ranging from 105 down to a few particles per milliliter of blood. Without a priori knowledge of target concentration, the possibility of accessing the five-orders-of-magnitude clinically relevant concentration range using only one data-acquisition run is desirable. We demonstrate here that such a high dynamic range is achievable with a portable, low-cost, horizontal-geometry confocal microscope. A nanoliter-size excitation volume, key to enabling short (min) sample scanning times, is positioned inside a cylindrical cuvette; slow (mm/s) translation and fast (10-1000 rpm) rotation of the cuvette allow transporting fluorescent particles across the excitation volume thus generating fluctuations in intensity time trace, which is analyzed by a pattern-recognition algorithm. Conventional detection through a single pinhole limits to concentrations below 50000 particles/ml, when multiple particles in the observation volume hamper the detection of one particle at ... [truncated at 150 words]
Digiacomo L, Marchini C, Digman MA, Gratton E, Caracciolo G.
Intracellular dynamics of nanoparticles probed by an image-derived mean square displacement approach.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 296a.The interactions among living matter and nanoparticle-based drug delivery systems highly regulate their efficiency and strongly affect their intracellular trafficking, which can be explored by manifold fluorescence spectroscopy techniques. However, none of these experimental tools has been specifically developed to take into account a spatial distribution of directed motions, commonly arising from the active transport of nanoparticles along cytoskeletal networks. To fulfill this gap, we show how a two-dimensional motion driven by Brownian diffusion and flow terms that are uniformly distributed in an angular range can be fully characterized by exploiting general concepts of the spatiotemporal image correlation analysis. The proposed approach can be regarded as an extension of the image-derived mean square displacement method and recovers dynamic and geometric features, which are commonly achieved through single particle analyses. Starting from a time series of the collected images, a spatiotemporal correlation function is computed and studied over the entire domain ... [truncated at 150 words]
Gratton E, Ma N, Digman MA, Malacrida L.
Of absolute concentrations of NADH in cells using the Phasor FLIM method.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 581a.One of the properties of fluorescence lifetime is its independence on concentration. While this is considered to be an advantage, when analyzing images of cell and tissues we often need to also measure the absolute concentration of a fluorophore. The concentration value is commonly obtained by comparison of the intensity of the fluorophore in the tissue and in a solution of known concentration. The underlying hypothesis is that the quantum yield of the fluorophore is the same in the sample and in the solution used for calibration. In cells and tissue this assumption is not always valid especially for a fluorophore like NADH that changes the quantum yield by a large factor when free in solution with respect to when bound to enzymes. We propose a graphical method using the phasor representation of the fluorescence decay to derive the absolute concentration of NADH in cells. The method requires the measurement ... [truncated at 150 words]
-
NADH auto fluorescence reveals new metabolic signatures in yeast and mammalian cells.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 282a.Yeast cells and their extracts were reported to show glycolytic fluctuations with a frequency of several minutes with addition of supplements. There is a distinct phase relation of NADH and other oscillating metabolites and intermediates which can be studied observing the auto fluorescence. New fast and efficient fluorescence lifetime techniques emerged in last two decades accelerated the progress of revealing the true nature of these fluctuations. We address these dynamic changes happening in live yeast systems by imaging them in-vivo time lapse lifetime imaging and correlate it with carbon metabolism reported in mammalian and cancer cells. The similarity between cancer cell metabolic flux and rapidly proliferating yeast cells have recently been of scientific interest to elucidate cancer cell pathways. Our findings shed new light into these metabolic responses with help of auto fluorescence lifetime measurements and live flux measurements. In addition, we aid these observations with the rigorous validation of ... [truncated at 150 words]
-
Metabolic Shifts in Huntington Disease Revealed by Fluorescence Lifetime Imaging Microscopy.
61st Annual Meeting of the Biophysical Society. New Orleans, Louisiana. February 11–15, 2017.
Biophys J. 2017; 112(3, Suppl 1): 584a.Huntington disease (HD) is a progressive autosomal neurodegenerative disorder due to the expanded CAG or polyglutamine (polyQ HTT) repeats in the HD gene mapped into the 4th chromosome. One of the early sign and hallmark of the individual carrying mutated gene of HD is metabolic dysfunction. HD patients suffer from excessive weight loss correlated with progression of the disease despite of adequate calorie intake. However, this effect is not fully understood. In this work, we use fluorescence lifetime imaging microscopy (FLIM) coupled with phasor analysis to measure metabolic alteration of HD in living cells and in the Drosophila eye discs model. We used endogenous NADH autofluorescence as an intrinsic biomarker and cofactor of metabolism. We are able to get pixel maps of metabolic alteration in HEK293 cells and in the eye discs of transgenic Drosophila expressing expanded polyQ HTT. Our results show significant shift toward larger free to bound ratio ... [truncated at 150 words]
Scipioni L, di Bona M, D'Amora M, Gratton E, Diaspro A, Lanzanò L.
An organelle sizer based on local image correlation spectroscopy detects changes in subcellular morphology.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 485a, 2388-Pos.Organelles represent the scale of organization immediately below that of the cell itself, and their composition, size and number are tailored to their functions. The most fundamental geometrical feature of any organelle is its size. However, it can be difficult to make measurements on morphologically complex structures [1].
With this thought in mind, we developed an algorithm based on the principles of Image Correlation Spectroscopy [2] able to perform very fast size measurements on either a single image or a time sequence. For every pixel, the algorithm computes the autocorrelation function of the pixel’s surroundings, obtaining a decreasing correlation function the decay rate of which is linked to the size of the structures; then, the map of autocorrelation functions is analyzed by phasor analysis [3]. The resulting image shows, pixel by pixel, a map of the apparent sizes of the structures present in the image.
The algorithm has been validated on simulated ... [truncated at 150 words]
Dvornikov A, Ranjit S, Gratton E.
Application of the DIVER detection method to multiphoton microscopy and FLIM.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 482a, 2373-Pos.In two-photon microscopy of turbid media, such as biological tissues, achievable imaging depth depends on ability of detection system to detect weak emission signals induced in deep tissue layers. In the conventional epi-detection method photons are collected from a relatively narrow sample area and acceptance angle, so most of emitted photons remain undetected. The DIVER (Deep Imaging Via Emission Recovery) method utilizes wide photocathode area detector and matching refractive index in the optical path from the sample surface to photocathode; that allows efficient collection of photons from wide area of a turbid sample at almost any angle. Using this method, the imaging depth was increased by factor of 4-6 compared to conventional epi-detection. In addition, the DIVER method offers superior sensitivity in SHG imaging and allows detection of short wavelength emission, up to 320nm. This, in turn, allows THG and three-photon imaging using conventional Ti:Sa laser excitation wavelengths. Coupling with ... [truncated at 150 words]
Gratton E, di Rienzo C, Cardarelli F, Hedde PN.
Connectivity map of the cell interior.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 15a, 86-Plat.We are developing an imaging platform that has the appropriate spatial and time resolution to investigate fundamental biological process in regard to signaling events in cells. The technology is based on fluctuation spectroscopy and the concept of spatial cross-correlation of fluctuations at different locations (the pair correlation function approach) originally developed in our lab. This technology is a radical departure from conventional fluctuation spectroscopy methods commonly done at single points in a cell or in small regions, yet providing the same information about diffusion and binding but with additional crucial advantages. The main advantage of the pair-cross-correlation approach is that it provides the full diffusion tensor at every point in the cell, which is crucial for the determination of the flow of molecules in the cell interior. The pair-cross-correlation function is zero if the two points are not in molecular communication while is different from zero if a molecule can ... [truncated at 150 words]
-
Fluorescence anisotropy imaging in 3D with single plane illumination microscopy.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 482a, 2374-Pos.Anisotropy imaging is a powerful tool to study the organization, composition and dynamics of molecules in biological systems. Changes in the organization and conformation of biomolecules within cells and tissues can be visualized at the single pixel level. Fluorescence anisotropy is typically probed with laser scanning and epifluorescence-based techniques. However, those techniques are limited in either axial resolution, image acquisition speed, or, by photobleaching. In the last decade, selective plane illumination microscopy (SPIM) has emerged as the best choice for three-dimensional time lapse imaging since it combines axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure [1,2]. We demonstrate how SPIM can be used for three-dimensional fluorescence anisotropy imaging of live cells [3]. To verify the accuracy of our SPIM system, we first measured and compared the anisotropy of Rhodamine110 solutions with different water/glycerol ratios with results obtained using a spectrofluorimeter. Then, we visualized the aggregation states ... [truncated at 150 words]
-
Intrinsic biomarker for oxidative stress by FLIM.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 165a, 825-Pos.Reactive oxygen species (ROS), when present above normal physiological levels, causes oxidative stress (OS), leading to damage of proteins and DNA and lipid peroxidation. OS has been associated with myriad of diseases like inflammation, diabetes mellitus, cardiovascular diseases, and cancer. Hence an OS biomarker would have immense clinical importance. There is a dearth of nondestructive techniques to measure OS in vivo. This work shows non-invasive detection of OS by fluorescence lifetime imaging microscopy (FLIM) of an intrinsic probe. Recognition of this endogenous biomarker enables label-free OS imaging. We identified this as fluorescent product of lipid oxidation by ROS. Using FLIM-phasor analysis, we can identify the presence and spatial location these species with a characteristic long lifetime (LLS). We could correlate the LLS to lipid droplets in the oleic acid treated HeLa cells using third harmonic generation (THG) imaging and coherent anti-Stokes Raman scattering (CARS) microscopy. For chemical analysis, we performed ... [truncated at 150 words]
Malacrida LS, Jameson DM, Gratton E.
Linear combination between lifetime and spectral phasor plots: a new approach to study membrane organization with Laurdan.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 492a, 2428-Pos.The application of lifetime and spectral phasor to the study fluorescence emission has become a useful tool to analyze and interpret fluorescence results. Jameson et al(1) using the Weber(2) notation for a fluorescence lifetime emission, previously showed the possibility to plot the vector M (demodulation) with an angle @ (delta phase) to the x axial, given the called the phasor plot (PP). The coordinates in the x and y, are the real (Glf=M.cos@) and imaginary (Slf=M.sin@) part of the demodulation. In a similar fashion Fereidouni et al(3), Fourier transformed the spectrum of a fluorescent molecule in a complex number (Gss, Sss), the scatter plot of the data was called the spectral phasor plot (SP). Malacrida et al(4) prior showed the goodness of the lifetime (PP) and spectral phasor (SP) plots to study the emission of LAURDAN in membranes with diferent physical states.
At this work we are introducing a new approach, ... [truncated at 150 words]
Chen H, Gratton E, Digman MA.
Self-assisted optothermal trapping of gold nanorods under two-photon excitation.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 505a–506a, 2493-Pos.We report self-assisted optothermal trapping and patterning of gold nanorods (GNRs) on glass surfaces with a femtosecond laser. We show that GNRs are not only the trapping targets, but also can induce convective flows that drive more particles toward the trap. The trapping phenomenon is the net result of thermophoresis and convective flow caused by localized heating, which is due to the conversion of absorbed photons into heat at GNR’s longitudinal surface plasmon resonance wavelength. We investigated the optothermal trapping of GNRs at the glass surface which can be obtained with laser power as low as 0.5 mW at 840 nm. The attraction of particles toward the central hot spot can be larger than the typical field of view, e.g. attraction toward the trap was observed from a range of 210 µm × 210 µm. By moving the laser focus away from the glass surface, at certain distances, ring patterns ... [truncated at 150 words]
Ranjit S, Aguilar-Arnal L, Stringari C, Sassone-Corsi P, Gratton E.
Spatial dynamics of SIRT1 dictate metabolic transitions in the cell nucleus.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 237a-238a, 1180-Pos.Sirtuins are a highly conserved family of NAD+-dependent deacylases that function as metabolic sensors by detecting the cellular energy state to modulate biochemical pathways to allow the cell to adapt to the environment. SIRT1, one of the seven mammalian sirtuins, has been widely studied. Previous reports have implicated that SIRT1 activity can be regulated by fluctuations in intracellular NAD+ concentrations. However the direct impact of SIRT1 on the regulation of the metabolism of its own cofactor, NAD+, remains unclear. Here, we demonstrate the nuclear organization of NAD+/NADH states are determined by SIRT1. Phasor approach to two photon autofluorescence lifetime microscopy in live cells shows that the redox state is compartmentalized inside of the nucleus, and that the subnuclear distribution as well as the redox ratio itself depends on SIRT1. Moreover, this effect seems specific for SIRT1, as the other nuclear sirtuin SIRT6, doesn't play a major role. Fluorescence fluctuation spectroscopy ... [truncated at 150 words]
Ma N, Digman MA, Donovan P.
Non-invasive fluorescence lifetime imaging (FLIM) of stem cell signature metabolic states.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 143a, 721-Pos.Development of the early post-implantation gastrulating human embryo has largely been inaccessible to comprehensive molecular study for obvious moral and ethical reasons. During gastrulation, a flat sheet of pluripotent cells called the epiblast undergoes ordered differentiation, giving rise to three primary germ layers that form fetal tissues. These events, triggered in part by specific growth factors (GFs), involve changes in cell shape, cell migration, chromatin architecture, cell cycle, etc., all processes requiring metabolic resources. Human embryonic stem cells (hESCs), thought to be equivalent to human epiblast cells, could provide an important new window into development and allow us to better understand how this stage is controlled at the molecular level and risks to early human development. Here we explore the metabolic changes that occur in Human embryonic Stem Cells (hESCs) exposed to growth factors such as WNT3 and BMP4. We take advantage of the Non-invasive phasor approach to fluorescence lifetime ... [truncated at 150 words]
Murata MM, Kong X, Yokomori K, Digman MA.
Fluorescence lifetime mapping of NADH reveals DNA repair activity in live cells.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 64a-65a, 339-Pos.Reduced nicotinamide adenine dinucleotide (NADH) is an important coenzyme that catalyzes many cellular functions including energy production and DNA damage repair. The ratio of free and bound NADH has recently emerged as a regulator of transcriptional activity and age-associated diseases. The DNA damage response, which requires NADH to gain full functionality, senses and repairs any breaks and errors in the genome. Uncovering the role of this ubiquitous molecule in response to DNA damage will further our understanding of how DNA protects itself and provide insight on how cancer cells evade these processes. In laser-induced damage experiments, HeLa cells were damaged with a femto-second pulsed two-photon laser at a wavelength of 780nm. Fluorescence lifetime imaging microscopy (FLIM) data was acquired and processed using the FLIM-phasor method. Induction of DNA damage and initiation of DNA repair were confirmed via immunostaining for γH2AX and PAR. The lifetime of NADH species in each pixel ... [truncated at 150 words]
Mah EJ, Liang EI, Yee AF, Digman MA.
Focal adhesion formation and reorganization on nanopatterned surfaces.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 133a, 673-Pos.Understanding cell to substrate or cell to cell interactions can contribute to improving biocompatibility of implants, wound healing, tissue engineering, and cancer metastasis. Cell signaling is regulated by the interactions of membrane bound protein with the extracellular matrix (ECM) that control changes during cell migration, adhesion, and proliferation. These changes are often regulated by focal adhesion (FA) complexes. These FAs are anchoring points of the cell to the substrate and are important mechanotranducers for a variety of fundamental cell functions. It is known that cells on nanolined surfaces form their FA parallel to the nanostructures and affects cell behavior. There has been little study of the spatial organization of the individual FA proteins with respect to these structures and how mechanosensing machinery is affected. In this study, we used the z-phasor super-resolution analysis technique to look at the architecture of the FA complex. We have found that FA proteins are ... [truncated at 150 words]
Trinh A, Zhou YH, Digman MA.
Identification of glioblastoma subpopulations by FLIM.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 651a, 3213-Pos.Glioblastoma multiforme (GBM) is one of the most aggressive forms of brain cancer. The tumor is known to be composed of heterogeneous subpopulations of cells, potentially playing a role in therapeutic resistance. Therefore, an understanding of the tumor heterogeneity is essential for the development of effective therapies. To further study the tumor heterogeneity, a culture method was developed to isolate distinct subpopulations from an established glioma cell line. These subpopulations include neural stem-like cells and tumor mass cells. Previous studies have demonstrated that these subpopulations have different phenotypes, responses to radiotherapy, and levels of oxidative stress. In this study, fluorescence lifetime imaging microscopy (FLIM) on the metabolic coenzyme, reduced nicotinamide adenine dinucleotide (NADH) was performed. NADH is an essential cofactor for oxidative phosphorylation and glycolysis, therefore the NADH lifetime can be used as an indicator for metabolic states, with longer lifetimes correlating to oxidative phosphorylation and shorter lifetimes correlating to ... [truncated at 150 words]
Chacko JV, Gopalakrishna-Pillai S, Digman MA.
Metabolic profiling in metastatic cancer cells using frequency domain fluorescence lifetime microscopy.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 167a, 833-Pos.NADH (Nicotinamide adenine dinucleotide) is a cofactor for many enzymes and enzymatic activities in living cells. NADH is auto-fluorescent in its free and bound states with different fluorescent lifetimes. This intracellular NADH distribution can be imaged using fluorescence lifetime imaging microscopy (FLIM), either frequency domain or time domain based hardware. Many pioneering studies place NADH as a natural sensor for based oxidation-reduction states, confluency, glucose levels, proliferation, etc. Frequency domain FLIM imaging aided with phasor representation allows one to profile the distribution of NADH in real time and study any changes in terms of spatial coordinates. We investigate two parallel studies based on fingerprints from healthy, cancerous and stromal cells: 1) in-vitro cellular environments and following migrating cells in a 3D extracellular matrix 2) in-vivo autofluorescence study of tissues. We use different methods such as spectral phasors (two dimensional phasor plots for spectral images), optical redox ratio (Flavin Adenine Dinucleotide/NADH) ... [truncated at 150 words]
-
Spatio-temporal dynamics and metabolic alterations of P53 upon DNA damage.
60th Annual Meeting of the Biophysical Society. Los Angeles, California. February 27-March 2, 2016.
Biophys J. 2016; 110(3, Suppl 1): 5a, 35-Subg.For biological reaction-diffusion systems, live single cell spatio-temporal analysis of protein dynamics provides a mean to observe stochastic biochemical signaling which may lead to better understanding of cancer cell invasion, stem cell differentiation and other fundamental biological processes. We used the Number and Molecular Brightness (N&B) method to map aggregation processes of the p53 protein in the live cells upon DNA damage with cisplatin (a chemotherapy agent); thus revealing the spatial distribution of events, the site of tetramer formation and the time sequence of the aggregation events with quantitative information about the distribution and size of any intermediate aggregates that are formed. p53 is a tumor suppressor protein that regulates target genes involved in DNA damage migration and repair. Upon cell stress, p53 will form tetramers in specific chromatin sites and activate genes that trigger cell cycle arrest and/or apoptosis. To gain information regarding fast dynamic processes we use line ... [truncated at 150 words]
-
Phasor florescent lifetime imaging approach to understand and analyze cellular mechanism disturbed by Huntington disease.
Annual Meeting of the American Society for Cell Biology 2015. San Diego, CA, December 12-16, 2015.
ASCB2015 Abstract Book. 2015; , P47.Huntington disease is an autosomal dominant inherited neurodegenerative disease characterized by movement, cognitive and emotional disorders. Despite its gene discovery on 1993, there is still no treatment to delay the onset or slow down the progression of the disease. This disorder is characterized by expansion of cytosine adenine guanine, CAG, triplet repeats due to a mistaken copy of Huntington gene on the forth chromosome[1‐3].Protein aggregation and inclusion is the hallmark of the Huntington disease. PolyGlutamine, PolyQ, in particular shows to be the major component of proteins deposit in patients’ brain[4, 5]. Yang and colleagues also investigated the polyQ aggregates, and found it to be highly toxic when directed to the cell nucleus[6]. It is essential to detect, study, and characterize such aggregates when aiming to study toxicity mechanism in live cells. For this purpose, we have used the phasor approach to Fluorescence Lifetime Imaging Microscopy (FLIM) of Nicotinamide adenine dinucleotide ... [truncated at 150 words]
Mah EJ, Liang EI, Wu SC, Dickson MN, Yee AF, Digman MA.
Aggregation states of focal adhesion proteins on nanopatterned surfaces.
Annual Meeting of the American Society for Cell Biology 2015. San Diego, CA, December 12-16, 2015.
ASCB2015 Abstract Book. 2015; , P586.Selective cell adhesion has great importance in designing biomedical implants to control growth and proliferation. Cell migration and adhesion is regulated through a complex mechanosensing network consisting of focal adhesions, composed of protein complexes, that link to the actin cytoskeleton. Focal adhesions act as the anchoring points of the cell to the substrate and the size of the adhesions play a prominent role in migration properties of the cells. It has been long recognized that cells on nanopillared surfaces are weakly adhered than those on flat or nanolined surfaces. How these associations occur are not well characterized. Insights into protein behavior and regulation during cell migration are primarily derived from biochemical assays that lack information about when and where proteins interact. Most recently, advances in fluctuation correlation microscopy have opened the possibility of measuring spatial‐temporal protein interactions in living cells. Here, we used the Numbers and Brightness (N&B) analysis to ... [truncated at 150 words]
Trinh AL, Zhou Y, Digman MA.
Glioblastoma heterogeneity by FLIM analysis.
Annual Meeting of the American Society for Cell Biology 2015. San Diego, CA, December 12-16, 2015.
ASCB2015 Abstract Book. 2015; , P1148.Glioblastoma multiforme (GBM) is one of the most aggressive forms of brain cancer. The tumor is known to be composed of heterogeneous subpopulations of cells, potentially playing a role in therapeutic resistance. Therefore, an understanding of the tumor heterogeneity is essential for the development of effective therapies. To further study the tumor heterogeneity, a culture method was developed to isolate distinct subpopulations from an established glioma cell line. These subpopulations include neural stem‐like cells and tumor mass cells. Previous studies have demonstrated that these subpopulations have different phenotypes, responses to radiotherapy, and levels of oxidative stress. In this study, fluorescence lifetime imaging microscopy (FLIM) on the metabolic coenzyme, reduced nicotinamide adenine dinucleotide (NADH) was performed. NADH is an essential cofactor for oxidative phosphorylation and glycolysis, therefore the NADH lifetime can be used as an indicator for metabolic states, with longer lifetimes correlating to oxidative phosphorylation and shorter lifetimes correlating to ... [truncated at 150 words]
-
Phaso-FLIM analysis of metabolic indexes in differing breast cancer cell lines with treatment of caffeine.
Annual Meeting of the American Society for Cell Biology 2015. San Diego, CA, December 12-16, 2015.
ASCB2015 Abstract Book. 2015; , P1895.This experiment was performed to illustrate the metabolic signature of varying breast cancer cell lines after treatment with differing concentrations of caffeine. The objective was to determine whether caffeine can alter cancerous phenotypes in a concentration‐dependent manner and to show that metabolic analysis using fluorescence lifetime imaging microscopy (FLIM) is a viable, noninvasive method of determining cancer cell metabolism. Using the phasor approach to FLIM and autofluorescence of Nicotinamide adenine dinucleotide (NADH) as a biomarker for metabolic changes, we defined significant patterns in metabolic indexes. By identifying the ratio of free to protein bound NADH, we differentiated two forms of cellular metabolism , glycolysis and oxidative phosphorylation. The MB231 cell line, not treated with caffeine, expectedly showed a highly glycolytic profile. With 2 mM treatment, there was a moderate shift towards oxidative phosphorylation, indicating a less cancerous phenotype. Finally, with 10 mM treatment, a drastic jump occurred towards oxidative phosphorylation, ... [truncated at 150 words]
-
Chromatin structure and dynamics.
Italian National Conference on Condensed Matter Physics. Palermo, Italy. September 28-October 2, 2015.
Chromatin structure, compaction and remodeling at the micro and nanometer scale have fundamental roles in many biological events. Chromatin compaction produces heterogeneity of the cell nucleus which results in structural and transport properties which have been only partially studied. Although the nucleosome structure has been in part deciphered, the topology of chromatin structure at the micron scale remains unresolved. In this work, we studied chromatin organization using the orbital 3D tracking technique. This method provides insight of local structure and transport properties at the nano scale by following the trajectories of molecules during gene expression.
-
Chromatin structure and dynamics.
23rd International Union for Biochemistry and Molecular Biology (IUBMB) Congress and 44th Annual Meeting of the Brazilian Society for Biochemistry and Molecular Biology (SBBq). August 24–28, 2015. Foz do Iguaçu, Brazil..
Chromatin structure, compaction and remodeling at the micro and nanometer scale have fundamental roles in many biological events. Chromatin compaction produces heterogeneity of the cell nucleus which results in structural and transport properties which have been only partially studied. Although the nucleosome structure has been in part deciphered, the topology of chromatin structure at the micron scale remains unresolved. In this work, we studied chromatin organization using the orbital 3D tracking technique. This method provides insight of local structure and transport properties at the nano scale by following the trajectories of molecules during gene expression.
Borrego S, Fahrmann J, Stringari C, Gratton E, Fiehn O, Kaiser P.
Metabolite changes associated with methionine stress sensitivity of cancer.
11th Annual international conference of the metabolomics Society. San Francisco, CA. June 29-July 2, 2015.
Abstract Book. 2015; Online.Altered cellular metabolism has gained larger recognition as a hallmark of cancer in recent years. While mechanistic insights into the metabolic changes in cancer are limited, the importance of methionine metabolism in cancer cell proliferation has been known and studied for over 30 years. These early studies describe a “methionine-dependence” or “methionine stress sensitivity” of cancer as a phenomenon where the majority of cancer cells cannot proliferate in growth media in which methionine is replaced by its metabolic precursor homocysteine. Interestingly, non-transformed cells are unaffected by this metabolite replacement. Previously, we identified a cell cycle arrest in cancer cells as a response to methionine stress and a global metabolic shift toward a more oxidative, or quiescent, cell state. Using the breast cancer cell line MDA-MB468, we investigate the metabolic response to methionine stress by mass spectroscopy. As a control, we have derived rare clones from MDA-MB468 cells (referred to as ... [truncated at 150 words]
-
TU-EF-303-03: Single cell measurements of transcription heterogeneity of highly mobile identical genes.
57th Annual Meeting of the American Association of Physicists in Medicine. Anaheim, CA. July 12-16, 2015.
Med Phys. 2015; 42(6): 3619.Many medical physics diagnostic and therapeutic developments have significant overlap with biophysical approaches to measuring and modeling molecular and cellular systems. Although these methods may differ dramatically in terms of temporal and spatial scales, many of the underlying concepts are identical, suggesting potential synergies across the scales. For example, while clinical imaging is typically in the (sub)millimeter and second regime, biophysical imaging technologies reach sub-nanometer and sub-picosecond scales. Despite these scale differences, the use of molecular labeling, resulting large datasets, and need for quantitative analysis methods are similar, and solutions developed for one application are easily translated to others. Similarly, mathematical models used to understand clinical responses are often based on molecular and cellular networks that have been elucidated by biophysical studies. In turn, such models are directly useful for guiding on-going biophysical and biomedical research. Relevant biophysical applications are found in the cancer, neuroscience, and cardiac fields, which are ... [truncated at 150 words]
Santoro Y, van de Giessen M, Zarandi SM, Pigazzi A, Cerussi A, Tromberg B, Gratton E.
Diffuse optical spectroscopy probe for therapy monitoring in colorectal cancer: In-vivo optical properties of healthy colon tissue from Yorkshire pigs.
2015 Pathways to Cures: Clinical Translational Research Day at UCI.
Poster Session Abstracts. 2015; : 9, 12.The purpose of this study is to measure and obtain for the first time the optical properties of normal swine colon tissue in vivo in the near infrared range using a diffuse optical spectroscopy (DOS) instrument and a novel probe designed for endoscopic use. Optical parameters of colon tissue were determined using the diffusion approximation of the radiative transport theory of light propagation. The need of a noninvasive technique arises from the importance of monitoring colorectal cancer response to therapy. Diffuse optical spectroscopy imaging (DOSI) has previously been successful in the prediction of neoadjuvant chemotherapy response in breast cancer, as early as one day after the beginning of the therapy. Rectal cancer is characterized by a low 5-year survival rate. To increase survival, patients with rectal cancer receive similar neoadjuvant chemotherapy and radiation treatments of those with breast cancer. However, very often (up to 30%) such therapies do not reach ... [truncated at 150 words]
Stortz M, Bruno L, Annibale P, Gratton E, Pecci A, Levi V.
Intranuclear dynamics of the glucocorticoid receptor and its co-regulator TIF2/GRIP1.
3rd South American Symposium in Signal Transduction and Molecular Medicine (Sistam 2015). Bailoche, Argentina. April 2-6, 2015.
The glucocorticoid receptor (GR) is a ligand-activated transcription factor and plays a relevant role in physiology, with a great variety of effects. GR can be directly recruited to specific response elements or can also interact with other transcription factors finally inducing or repressing gene expression. The activity of GR is modulated by different co-regulators, e.g. TIF2/GRIP1. GR and TIF2 do not distribute homogeneously within the nucleus but accumulate in distinctive clusters.The functional role of this particular intranuclear organization is still unknown thus we decided to study it using advanced fluorescence microscopy techniques. We observed that TIF2 forms few large clusters in the nucleus that redistribute in the presence of GR activated by dexamethasone (DEX) to many small TIF2 clusters that co-localize with GR foci. In order to study the dynamics of these two proteins in the nucleus and the relevance of their spatial distribution, we performed fluorescence correlation spectroscopy (FCS) ... [truncated at 150 words]
Dobrinskikh E, Ranjit S, Montford J, Dvornikov A, Lehman A, Nemenoff R, Gratton E, Levi M, Furgeson S.
Characterizing fibrosis in mouse kidney using fluorescence lifetime and second harmonic generation imaging microscopy in unilateral ureteral obstruction model.
FASEB J. 2015; 29(1 Supplement), 719.19.Renal tubulointerstitial fibrosis is considered to be the final common pathway for most forms of chronic kidney disease (CKD). Fibrosis has been traditionally characterized by histological studies such as Picro-Sirius Red (PSR) staining, Masson Trichrome staining or collagen immunohistochemistry. Such methods are limited due to variability in staining and pathologist scoring. The goal of this study is to compare histologic measures of renal fibrosis to Fluorescence Lifetime Imaging (FLIM) and Second Harmonic Generation (SHG) techniques in our deep tissue imaging microscope called DIVER. Male C57Bl6 mice were subjected to unilateral ureteral obstruction (UUO), a well-characterized model of fibrosis. At 21 days, both kidneys were collected. Serial sections of both kidneys were analyzed by PSR staining or FLIM with SHG. Quantification of a whole kidney section from the PSR staining showed 34.32±0.99% area of fibrosis in the injured kidney compared to 5.55±1.07% in the control kidney. Using the Phasor approach to ... [truncated at 150 words]
Kang DK, Ali M, Digman M, Gratton E, Peterson E, Zhang K, Zhao W.
Rapid detection of single bacteria in unprocessed blood using the integrated comprehensive droplet digital detection (IC 3D) system.
4th Annual Conference of the Society for Laboratory Automation and Screening (SLAS). Washington, DC. February 9-11, 2015.
Scientific Program Abstract Compendium. 2015; : 103.Blood stream infections or sepsis is a major health problem worldwide with extremely high mortality that is partly due to the inability to rapidly detect and identify bacteria in the early stages of infection. Here we present a new technology termed “Integrated Comprehensive Droplet Digital Detection (IC 3D)” that is able to selectively detect bacteria directly from milliliters of whole blood at single-cell sensitivity in a one-step, culture- and amplification-free process within 1-3 hours which is currently not possible. The IC 3D integrates real-time bacterium-sensing DNAzyme sensors, droplet microencapsulation and a high throughput 3D particle counter system. Using E. coli as a target, we demonstrate that the IC 3D can provide absolute quantification of both stock isolates of E. coli and clinical isolates in spiked blood within a broad range of extremely low concentration from 1 to 10,000 bacteria/mL with exceptional robustness and limit of detection in the single digit ... [truncated at 150 words]
-
Mapping P53 alterations in metabolism upon DNA damage using the phasor/FLIM and number & molecular brightness analysis.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 478a, 2413-Pos.p53 is a tumor suppressor protein that regulates target genes involved in DNA damage migation and repair. If cells become stressed due to DNA damage, p53 will induce genes that trigger cell cycle arrest and/or apoptosis. p53 has a dual role in promoting oxidative phosphorylation (oxsphos) and glycolysis upon cellular stress. However, the metabolic function of p53 has not been fully explored. p53 metabolic activities may play an important role in normal growth, development, and tumor suppression, it might also be misused to help promote, rather than hinder, tumor development. To further assess p53 activity, we investigated the effect of metabolic changes under the same conditions and tested if the concentration of p53 influences the balance between apoptosis and DNA repair. We used the fluorescent lifetime imaging microscopy (FLIM) phasor approach to detect changes in oxsphos and glycolysis. Overall our data show the formation of p53 protein near the nucleolus ... [truncated at 150 words]
Lanzanò L, Hernandez IC, Castello M, Gratton E, Diaspro A, Vicidomini G.
Background-free super-resolution microscopy of subcellular structures by lifetime tuning and photons separation.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 359a.The visualization at the nanoscale level inside cells is a fundamental need in molecular biology. The challenge of increasing the spatial resolution of an optical microscope beyond the diffraction limit can be reduced to a spectroscopy task by proper manipulation of the molecular states. The nanoscale spatial distribution of the molecules inside the detection volume of the microscope can be encoded within the fluorescence dynamics and can be decoded by resolving the signal into its dynamics components [1]. We present here a robust and general method, based on the phasor analysis [2], to spatially sort the fluorescent photons on the basis of the associated molecular dynamics and without making use of any fitting procedure. In a specific implementation of this method, we generate spatially controlled gradients in the fluorescence lifetime by stimulated emission [3]. The separation of the time-resolved fluorescence components sorts photons according to their spatial positions. Spatial resolution ... [truncated at 150 words]
Levi M, Dobrinskikh E, Ranjit S, Montford J, Dvornikov A, Lehman A, Furgeson S, Nemenoff R, Gratton E.
Characterizing fibrosis in mouse kidney using auto fluorescence FLIM and SHG in UUO model.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 478a, 2410-Pos.Chronic kidney disease (CKD) arises from a diverse list of renal injuries. Renal fibrosis is considered to be the final common pathway for most forms of CKD and involves glomerular sclerosis and/or interstitial fibrosis. Because of that there is great interest in identifying renal fibrosis in the early stages of CKD to prevent progression. Unilateral ureteral obstruction (UUO) is a well-described model of CKD and renal fibrosis. Kidney injury and fibrosis usually are assessed by histology. Histological methods include Picro-Sirius Red staining, Masson's Trichrome staining or immunohistochemistry for collagen isoforms. Quantitation of fibrosis using histologic techniques may be difficult due to variability in staining and pathologist scoring. The goal of this study is to compare histologic measures of renal fibrosis to Fluorescence Lifetime Imaging (FLIM) and Second Harmonic Generation (SHG) techniques in our deep tissue imaging microscope called DIVER. Male C57Bl6 mice were subjected to UUO of right kidney. At ... [truncated at 150 words]
Heylman CM, Datta R, Conklin BR, George SC, Gratton E.
Classifying the electrophysiological effects of chronotropic drugs on human induced pluripotent stem cell-derived cardiomyocytes using voltage sensitive dyes and supervised machine learning.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 110a, 553-Pos.The emergence of human induced pluripotent stem (hiPS) cell technology has expanded the possibilities for sourcing human cardiomyocytes (hiPS-CMs). Novel microscopy and analysis methods serve to accelerate development and validation of in vitro hiPS-CM models for drug screening. Voltage sensitive dyes (VSD) allow non-invasive, non-destructive, and longitudinal assessment of hiPS-CM electrophysiology at the sub-cellular membrane scale. In this study, we successfully use 2-photon microscopy to capture VSD signal at the cellular membrane scale generated from actively beating hiPS-CMs exposed to the chronotropic drugs, propranolol (10−5 M) and isoproterenol (10−7 M). We use SimFCS software, developed at the Laboratory for Fluorescence Dynamics at the University of California, Irvine, to remove motion artifact and assess the resultant signal over time. We are able to generate a waveform of VSD fluorescence that is representative of the changing membrane potential (i.e. the depolarization of an action potential). A number of characteristics of these waveforms ... [truncated at 150 words]
Bachir AI, Zareno J, Kubow KE, Han S, Moissoglu K, Danuser G, Gratton E, Plow E, Horwitz AR.
Correlative traction force microscopy and fluorescence fluctuation analysis of molecular aggregation and complex formation in cell adhesions in distinct microenvironments.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 493a-494a, 2492-Plat.The mechanical properties of the cellular microenvironment regulate processes that include migration, proliferation, and differentiation. These interactions occur at adhesions, which serve as both traction points and signaling hubs and mediate bi-directional sensing and responses to specific features of the surrounding extracellular matrix (ECM). Adhesions execute these activities through an intricate network of putative molecular interactions that largely remain to be demonstrated and characterized functionally in living cells. The challenge is to capture the highly localized and transient associations that characterize these activities in adhesions and determine how they respond to different microenvironments. In this study, we use high-resolution fluorescence fluctuation microscopy to map the formation and stoichiometry of integrin-associated complexes in the adhesions that populate the leading edge of migrating cells. We focus on putative integrin activating (kindlin and talin) and actin-linking (talin, vinculin and a-actinin) molecules and show that all molecules are present in adhesions as soon as ... [truncated at 150 words]
-
Fluorescence fluctuation microscopy techniques to study mRNA synthesis and dynamics.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 324a-325a, 1629-Pos.Over the last ten years fluorescence microscopy methods have provided significant insights in the study of both the kinetics of mRNA synthesis at an actively transcribing gene (or gene array) and its dynamic in the nucleoplasm of living cells.
The availability of a system such as the MS2-GFP fusion protein, which directly labels the mRNA, has allowed obtaining an estimation of the RNA polymerase elongation speed in living cells. There is however a large heterogeneity observed in RNA Polymerase II (PolII) elongation rates measured from fluorescence assays.
To shed further light on the source of this heterogeneity we introduce and discuss here a novel method based on the phasor analysis of steady state MS2-mRNA fluorescence trajectories. When applied to the study of PolII kinetics, we demonstrate that this approach allows resolving PolII elongation rates in a range from a few to hundreds of basepairs per seconds.
In order to couple this information to ... [truncated at 150 words]
Ranjit S, Gratton E, Lanzanò L.
Mapping diffusion in a living cell using the phasor approach.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 324a, 1628-Pos.The diffusion of a fluorescent protein within the cell has been measured by using either the fluctuation based techniques (FCS, RICS) or through particle tracking or through FRAP. However none of these methods enable us to measure the diffusion of the fluorescent particle at each pixel of the entire image and create a detailed diffusion map of the cell. Measurement using the conventional single point FCS at every individual pixel results in the long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation we have developed new modes of scanning. In this new method of modified raster scanning, the laser scans each individual line multiple times before moving to the next line. The difference from the RICS approach is in RICS the data is acquired by scanning each frame once and then scanning the image multiple times. The other mode resembles single ... [truncated at 150 words]
Serebryannyy L, Parilla M, Annibale P, Cruz C, Kudryashov D, Gratton E, Gottardi CJ, de Lanerolle P.
Nuclear actin dynamics regulate nuclear organization and transcription.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 536a, 2716-Pos.Actin is an important and elegant mechanotransducer that transfers messages from the extracellular environment into the nucleus, thus conferring changes in both the physical properties and the genetic program of the cell. Although actin in the nucleus has been established in a growing number of functions, the form of nuclear actin remains poorly understood as there are no apparent actin filaments in the nucleus. Therefore, we tested how disrupting the form of nuclear actin impacts function and what effects this may have on the nucleus. To do so, we formed stable nuclear actin filaments using a variety of methods, including the nuclear enrichment of actin binding proteins supervillin and α-E-catenin and the expression of a mutant form of skeletal α-actin. Using fluorescence recovery after photobleaching (FRAP), we found that stabilization of nuclear actin filaments significantly impairs actin dynamics within the nucleus. The formation of nuclear actin filaments coincides with striking ... [truncated at 150 words]
di Rienzo C, Gratton E, Beltram F, Cardarelli F.
Probing short-range protein brownian motion in the cytoplasm of living cells.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 324a, 1627-Pos.The translational motion of small molecules in cells appears to be suppressed compared to what is observed in dilute solutions. Although, the rotation of small proteins is almost unhindered, pointing out a local aqueous environment. Different theoretical models provide explanations for this apparent discrepancy but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive experimental test of the nature of the translational motion in cells is still missing owing to the lack of techniques capable of probing protein motion with the required temporal and spatial resolution. We show that fluorescence-fluctuation analysis of raster scans at variable time scales can provide this information. By using GFP, we measure protein translational motion at the unprecedented time-scale of 1 microsecond, unveiling unobstructed Brownian motion from 25 to 100 nanometers, and partially-suppressed diffusion above 100 nm. Experiments on in vitro model systems attribute this effect to the ... [truncated at 150 words]
Hedde PN, Stakic M, Gratton E.
Rapid measurement of molecular transport and interaction inside living cells with single plane illumination microscopy.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 165a, 827-Pos.Fundamental physiological processes within cells and tissues including cell adhesion, signaling, movement, division or metabolism are based on the transport and interaction of biomolecules. Biomolecular dynamics is usually measured by applying single particle tracking (SPT) analysis to camera images or by using fluorescence fluctuation spectroscopy (FFS) methods based on single point detection. We show that image mean square displacement (iMSD) analysis, applied to single plane illumination microscopy (SPIM) data, is a faster and more efficient way of unravelling rapid, three-dimensional molecular transport and interaction within solutions and in living cells. From a stack of camera images recorded in a few seconds only, the type of dynamics such as free diffusion, flow or binding can be identified and quantified without suffering from the limitation of current camera frame rates. Also, the SPIM-iMSD method does not require calibration of the microscope point spread function (PSF) and light exposure levels are very low. ... [truncated at 150 words]
Wright BK, Jones MR, Digman MA, Gratton E.
Real-time analysis of endogenous nuclear NADH in differentiating cells using the spectral phasor approach.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 73a, 369-Pos.NADH is an endogenous autofluorescent regulatory metabolite detected in the nuclear regions of live cells which when analysed for the bound and free form can aid in determining a cell's metabolic status and molecular activity. Detecting the spectral differences of free and bound NADH in live cells is currently limited due to the very small differences in emission. The Spectral Phasor technique enables not only examination of small shifts in spectral emissions but also provides the spatial location of spectrally different components in live cells without any prior knowledge of the species. The phasor representation enables direct comparison of either optical sections (i.e. different focal planes) of one cell or multiple cells for global analysis.
Here we describe the use of Spectral Phasors to spatially map NADH's spectral emission in the nucleus of live cells under normal culture conditions and those stimulated into the early stages of differentiation. A comparison of ... [truncated at 150 words]
Anzalone A, Perez-Lopez A, Raffatellu M, Gratton E.
The lifetime NAD(P)H fingerprint of Salmonella infection.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 150a, 746-Pos.NAD(P)H is and endogenous fluorescent coenzyme that has been used as a metabolic biomarker in recent studies using the FLIM technique. Here we describe the NAD(P)H FLIM technique that provides a label-free imaging method to monitor Salmonella enterica infection inside differentiated macrophages. We use FLIM NAD(P)H signature as a marker of bacterial metabolism. NAD(P)H in Salmonella shows a different lifetime fingerprint when the bacteria is grown in a soft agar and when the bacteria infects differentiated macrophages. Specifically, we observed that the free/bound ratio of NAD(P)H in Salmonella changes from a more bound NAD(P)H to a more free state when the bacteria is inside the host cell. During the bacterial infection we also monitored the changes of NAD(P)H surrounding the bacteria inside the macrophage. The NAD(P)H signal around the bacterium when inside the host is interpreted as a marker of the NADPH oxidase enzyme. In contrast to related organisms like ... [truncated at 150 words]
-
Transcription kinetics heterogeneity of highly mobile identical genes revealed by simultaneous measurement at the single cell level.
59th Annual Meeting of the Biophysical Society. Baltimore, Maryland. February 7-11, 2015.
Biophys J. 2015; 108(2, Suppl 1): 507a, 2560-Plat.Fluorescence microscopy was used in combination with molecular constructs to measure in real time and in living cells the transcriptional activity of individual genes and their displacement within the nucleus. The question whether local mobility of individual chromatin regions is correlated to their transcriptional output is still the subject of active research. There is a consensus that the local chromatin environment and physical accessibility may play a role in modulating transcription, possibly determining the large heterogeneity observed in RNA Polymerase II (PolII) elongation rates measured from fluorescence assays. Employing high-speed fluorescence nanoimaging we measure here at ms-temporal resolution PolII elongation and we are able to resolve the minute displacements of identical active genes surrounding a labeled transgene array. We observe a correlation between the transcriptional activity and sub-micrometer movements of the active genes that is evidence of an active molecular mechanism determining displacements of the active genes following transcriptional bursts. ... [truncated at 150 words]
-
Mapping p53 aggregation and changes in metabolism upon DNA damage using the Phasor/FLIM and Number & Molecular Brightness analysis.
CFCCC Scientific Retreat. September 26-27, 2014. Palm Springs, California.
Scientific-Retreat-Program. 2014; : 19.p53 is a tumor suppressor protein that regulates target genes involved in DNA damage mitigation and repair. If cells become stressed due to DNA damage, p53 will induce genes that trigger cell cycle arrest and/or apoptosis. p53 has a dual role in promoting oxidative phosphorylation (oxsphos) and glycolysis upon cellular stress. However, the met abolic function of p53 has not been fully explored. p53 metabolic activities may play an important role in normal growth, development, and tumor suppression, it might also be misused to help promote, rather than hinder, tumor development. To further assess p53 activity, we investigated the effect of metabolic changes under the same conditions and tested if the concentration of p53 influences the balance between apoptosis and DNA repair. We used the fluorescent lifetime imaging microscopy (FLIM) phasor approach to detect changes in oxsphos and glycolysis. Overall our data show the formation of p53 protein near the ... [truncated at 150 words]
Moens PDJ, Digman MA, Gratton E.
Modes of diffusion of Cholera toxin bound to GM1 on live cell membrane by image mean square displacement (iMSD) analysis.
International Biophysics Congress 2014. Brisbane, Australia. August 3-7, 2014.
IUPAB2014 Abstracts. 2014; , #514.The image-Mean Square Displacement technique (iMSD) applies the calculation of the mean square displacement (MSD) used in single molecule tracking to images without resolving single particles. The iMSD plot obtained is similar to the MSD plot obtained using the single-particle tracking technique and is used to reconstruct the protein diffusion law. From these plots, we can determine whether the labeled molecules are undergoing pure isotropic, restricted, corralled, transiently confined or directed diffusion. In our study total internal reflection fluorescence (TIRF) microscopy images were taken of Cholera toxin subunit B (CtxB) membrane labeled cells. We mapped the modes of CTxB diffusion when bound to GM1 in live cell membranes and found different types of diffusion across the cell including isotropic, confined and transiently confined. However, no directed diffusion could be observed. We show that repeated illumination of the same area of the cell leads to a transition of diffusion type from ... [truncated at 150 words]
Hinde E, Pandzic E, Gratton E, Gaus K.
Imaging the diffusive route of transcription factor activity in live cells by fluctuation analysis.
International Biophysics Congress 2014. Brisbane, Australia. August 3-7, 2014.
IUPAB2014 Abstracts. 2014; , #14.Dynamic changes in higher order chromatin structure modulate the accessibility of DNA toward transcription, DNA repair and replication. The spatiotemporal scale upon which these dynamics occur however, render them ‘invisible’ to live cell imaging. Here we present a novel method to measure the real time accessibility of chromatin that is based on pair correlation analysis of EGFP (an inert tracer) molecular flow throughout the chromatin network1. From detected changes in the arrival time of EGFP molecules into and out of the chromatin, this method detects millisecond changes in protein access to condensed chromatin with sub-micron resolution. Using this method we have probed the naturally occurring changes in chromatin compaction during the cell cycle and in combination with laser micro-irradiation, monitored for the first time the in vivo structural rearrangements of chromatin during repair2,3. The pair correlation method thus offers a unique opportunity to probe the directionality of intra-nuclear traffic, by ... [truncated at 150 words]
Digman MA, Gratton E, Chiu CL.
Nano-scale spatial-temporal dynamics of Paxillin and actin in 3D collagen matrices.
International Biophysics Congress 2014. Brisbane, Australia. August 3-7, 2014.
IUPAB2014 Abstracts. 2014; , #273.Organization and dynamics of focal adhesion proteins have been well characterized in cells grown on two-dimensional (2D) cell culture surfaces which are debatably not a suitable representation of the anatomy and tissue microenvironment. Limited imaging technologies capable of measuring protein interactions in real time and space for cells grown in 3D is a major impediment in understanding how proteins function under different environmental cues. In this study, we applied the nano-scale precise imaging by rapid beam oscillation (nSPIRO) technique and combined the fluorescence correlation spectroscopy (FCS) and the Number and Brightness (N&B) methods to investigate paxillin and actin dynamics at focal adhesions in the 3D microenviroment. Both MDA-MB-231 cells and U2OS cells produce elongated protrusions with high intensity regions of paxillin in cell grown in 3D collagen matrices. Using FCS we found higher percentage of slow diffusing proteins at these focal spots, suggesting assembling/disassembling processes. In ... [truncated at 150 words]
-
Chromatin structure and dynamics.
International Biophysics Congress 2014. Brisbane, Australia. August 3-7, 2014.
IUPAB2014 Abstracts. 2014; , #17.Chromatin structure, compaction and remodeling at the micro and nanometer scale have fundamental roles in many biological events. Chromatin compaction produces heterogeneity of the cell nucleus which results in structural and transport properties which have been only partially studied. Although the nucleosome structure has been in part deciphered, the topology of chromatin structure at the micron scale remains unresolved. In this work, we studied chromatin organization using the orbital 3D tracking technique. This method provides insight of local structure and transport properties at the nano scale by following the trajectories of gold nanoparticle that are trapped in the chromatin. Using two-photon excitation, the fluorescence of fluorophores that are in the nanometer proximity to the gold particle is strongly enhanced. We have recently shown that metallic NPs do not bleach or blink upon continuous illumination, are extremely stable, very bright and their luminescence spans over the visible spectrum. These characteristics allow ... [truncated at 150 words]
Jahid S, Dvornikov AS, Gratton E.
3D tissue reconstruction by using deep tissue fluorescence imaging system and FLIM.
15th Annual UC Systemwide Bioengineering Symposium. University of California, Irvine. June 18-20, 2014.
We have demonstrated a new detection method used in two-photon fluorescence microscope called the DIVER (Deep Imaging via Enhanced-Photon Recovery) with enhanced capabilities for deep tissue imaging. The microscope uses a large area photo-detector to collect scattered emission photons directly from the wide area of a specimen. This detection scheme allows for increasing the imaging depth in tissues by about 6 folds due to a unique detector design and its increased photons collection efficiency compared to conventional methods. The DIVER system is also capable of performing Fluorescence Lifetime Imaging (FLIM), a very powerful tool to segregate various features in cells and tissues. The combination of deep tissue imaging and fluorescence lifetime provides contrasted images based on physiological parameters at depths that are not achievable by conventional microscopes. The DIVER potentially can be useful for imaging of various tissue samples, in particular for human skin cancer diagnostics, providing 3D cellular resolved ... [truncated at 150 words]
Datta R, Heylman C, Tran D, George SC, Gratton E.
Non-invasive optical metabolic imaging of three-dimensional cardiac tissue model in a polydimethylsiloxane microfluidic device.
15th Annual UC Systemwide Bioengineering Symposium. University of California, Irvine. June 18-20, 2014.
There is a growing importance of development of microfludic device based, microphysiological tissue systems which simulates structure and function of human organs. Such tissue chips find application in drug toxicity screening, point-of-care diagnostic devices, targeted drug delivery and so on. Optical imaging proves to be a powerful, non- invasive tool to monitor development of such tissue systems. In this work, we show label-free metabolic imaging of induced pluripotent stem cell derived cardiomyocyte (iPS-CM) 3D spheroids co-cultured with endothelial cells (to form vessel network) and fibroblast in tissue microchamber of a polydimethylsiloxane (PDMS) microfluidic device. We performed fluorescence lifetime imaging microscopy (FLIM) of endogenous fluorophore, reduced nicotinamide adenine dinucleotide (NADH), a metabolic coenzyme which plays myriad of roles in cellular oxidation and reduction reactions. The phasor approach to FLIM was applied to map free to protein bound NADH ratio distribution in the iPS-CM spheroid and surrounding vessel network, indicative of the ... [truncated at 150 words]
Kang DK, Ali MM, Digman MA, Gratton E, Peterson EM, Zhao W.
Rapid, single bacterial detection from blood using microencapsulated sensors.
15th Annual UC Systemwide Bioengineering Symposium. University of California, Irvine. June 18-20, 2014.
The high mortality of blood stream infections is associated with the ineffectiveness and time-consuming process of bacterial detection and treatment. Unfortunately, blood culture, the gold standard for the detection of bacteremia, takes several days to obtain results. New molecular diagnosis methods, such as polymerase chain reaction (PCR), are often not sensitive enough to detect bacteria that occur at low concentrations in blood (1-100 colony-forming unit (CFU)/mL). Moreover, all these techniques are sophisticated and expensive, and therefore not well-suited for routine testing. Therefore, simple methods are urgently needed for rapid and sensitive identification of bacteria in blood, which has the potential to significantly reduce the mortality rate and the cost of medical care associated with blood stream infections.In this study, we have developed a system that detects bacteria in patient blood at single-cell sensitivity within a few hours. Our system integrates bacterium-detecting DNAzyme sensors, which are obtained by in vitro selection, ... [truncated at 150 words]
Ma N, Chen H, Gratton E, Digman MA.
Spectroscopic profiling and metabolic mapping of cancer cells using endogenous fluorescence.
15th Annual UC Systemwide Bioengineering Symposium. University of California, Irvine. June 18-20, 2014.
Advances in hyper- or multi-spectral based imaging cameras have expanded new areas of cellular research for the last 20 years. Most recently, intensity signals can now be detected with a line spectrograph in which light is transmits over an imaging signal to an ultra sensitive CCD camera with fast readout speed. However, spectrally resolving these images is highly problematic given that most fluorescent emission spectra are broad. In our work, we used Andor’s iXon Ultra EMCCD camera to obtain spectral emission of cancer cell autofluorescence excited with a multiphoton laser source. In parallel we also performed fluorescence lifetime imaging microscopy (FLIM) to map free to bound NADH ratio distribution within single cells. In our approach the raw spectral and FLIM data sets are transformed into sine and cosine components and plotted in a polar coordinate plot called the phasor plot. We can spectrally resolve intrinsic fluorescent species ... [truncated at 150 words]
-
3D orbital tracking of a DNA locus during transcription.
9th International Weber Symposium. Kauai, Hawaii. June 15-20, 2014.
We report on the application of a 3D particle tracking method, namely orbital tracking, to a model cellular system allowing to simultaneously label an actively transcribing genetic locus and the synthesized mRNA using the EGFP-labeled MS2 coat protein. Orbital tracking in a 2-photon microscope allows collecting unprecedented sub-second temporal resolution, hour-long fluorescence trajectories of the newly transcribed mRNA.
We observe distinct regions of active transcription, termed petals, displaying a well defined spatial organization, corralling the denser part of the genetic locus and rotating along its surface.
We apply fluorescence fluctuation analysis to investigate the transcription kinetics of each of these petals; the comparison of experiments and simulations allows highlighting a wide range of polymerase elongation rates, ranging from as low as a few tens basepairs/s to hundreds of basepairs/s. Response of the elongation rate to a common transcription inhibitor is also demonstrated.
Exploiting the trajectory information that we collect for each of the ... [truncated at 150 words]
Anzalone A, Stringari C, Gratton E.
Gold nano-imaging of the cell nucleus.
9th International Weber Symposium. Kauai, Hawaii. June 15-20, 2014.
Metallic nanoparticles such as Gold Nanoparticles (GNPs) have recently become a common fluorescent microscopy tool because they do not bleach or blink upon continuous illumination. Gold nanoparticles are also characterized by an intrinsic feature, the plasmonic enhancement surface, which enhances fluorescence of molecules when in close proximity (few nanometers) to the particle. At the LFD it has been developed the 3D orbital tracking method that utilizes the characteristics of a 2-Photon microscope to study cellular processes. The 3D orbital tracking combined with the plasmonic enhancement of the GNPs provide a reliable method to observe sub-cellular structures, such as chromatin, with a nano-scale resolution. During the movement inside the cell nucleus the gold nanoparticles interact with the surroundings and give fluorescent enhancement of specific fluorophores with different emission spectra. The enhancement due to the proximity of gold NP and fluorophores inside the nucleus appears as emission spikes of different colors presumably ... [truncated at 150 words]
-
Chromatin structure and dynamics.
9th International Weber Symposium. Kauai, Hawaii. June 15-20, 2014.
Chromatin structure, compaction and remodeling at the micro and nanometer scale have fundamental roles in many biological events. Chromatin compaction produces heterogeneity of the cell nucleus which results in structural and transport properties which have been only partially studied. Although the nucleosome structure has been in part deciphered, the topology of chromatin structure at the micron scale remains unresolved. In this work, based on the Gold Enhanced Nano Imaging (GENI) method we studied chromatin organization using the orbital 3D tracking technique applied to 20 nm gold nanoparticles (NPs). This method provides insight of local structure and transport properties at the nano scale by following the trajectories of gold nanoparticle that are trapped in the chromatin. Using two-photon excitation, the fluorescence of fluorophores that are in the nanometer proximity to the gold particle is strongly enhanced. We have recently shown that metallic NPs do not bleach or blink upon continuous illumination, ... [truncated at 150 words]
-
The SimFCS environment for analysis of fluctuations at the micro and nanoscale (Sponsor Talk).
9th International Weber Symposium. Kauai, Hawaii. June 15-20, 2014.
The software package SimFCS is a complete virtual machine for the acquisition, analysis and display of images acquired in different microscopy modalities. The major modules which are part of the SimFCS software comprise 1) the FCS module for analysis of single point FCS capable of analyzing almost all known data formats for diffusion, binding and PCH analysis. 2) A module for analysis of raster scan images for RICS and N&B determination of diffusion, binding, number and brightness from confocal microscope images. 3) A module for analysis of diffusion and MSD (mean square displacement) for images from fast cameras obtained in the TIRF and SPIM mode of operation. 4) A module for 3D particle tracking based on the orbital tracking approach. 5) A comprehensive module for the analysis of FLIM images acquired by most microscopes operating in the time and frequency domain. This part of the software is also used for ... [truncated at 150 words]
Hedde PN, Doerlich RM, Blomley R, Gradl D, Oppong E, Cato ACB, Nienhaus GU.
Stimulated emission depletion-based raster image correlation spectroscopy reveals biomolecular dynamics in live cells.
9th International Weber Symposium. Kauai, Hawaii. June 15-20, 2014.
Raster image correlation spectroscopy (RICS) is an excellent way to study molecular movement such as protein diffusion and transport, or, receptor–ligand interactions within living cells and tissues [1]. From rasterscanned microscopy images, fluctuations in fluorescence intensity can be analyzed with spatio-temporal correlations revealing molecular motions in a spatially resolved manner. However, due to the diffraction-limit, conventional raster image correlation spectroscopy can only separate larger regions of interest; also RICS requires low fluorophore concentrations in the nanomolar range. Here, we applied stimulated emission depletion microscopy (STED) [2] to RICS in order to overcome these limitations. With imaging experiments on model membranes and live cells, we demonstrate that due to the enhanced spatial resolution, STED-RICS offers insight into micron-sized regions of interest as well as access to 10–100 times higher fluorophore concentrations [3].
[1] Digman, M. A. et al., Measuring fast dynamics in solutions and cells with a laser scanning microscope. Biophys. J. ... [truncated at 150 words]
Hinde E, Gratton E, Gaus K.
Imaging the diffusive route of molecules in live cells by fluctuation analysis.
9th International Weber Symposium. Kauai, Hawaii. June 15-20, 2014.
Dynamic changes in higher order chromatin structure modulate the accessibility of DNA toward transcription, DNA repair and replication. The spatiotemporal scale upon which these dynamics occur however, render them ‘invisible’ to live cell imaging. Here we present a novel method to measure the real time accessibility of chromatin that is based on pair correlation analysis of EGFP (an inert tracer) molecular flow throughout the chromatin network. From detected changes in the arrival time of EGFP molecules into and out of the chromatin, this method detects millisecond changes in protein access to condensed chromatin with sub-micron resolution. Using this method we have probed the naturally occurring changes in chromatin compaction during the cell cycle and in combination with laser micro-irradiation, monitored for the first time the in vivo structural rearrangements of chromatin during repair. The pair correlation method thus offers a unique opportunity to probe the directionality of intra-nuclear traffic, by ... [truncated at 150 words]
Malacrida LS, Toledo-Gallo R, Briva A, Denicola A, Gratton E, Jameson DM.
Phasor plots for lifetime and spectrum analysis of LAURDAN and PRODAN emissions in membrane: A new perspective for membrane biophysics studies.
9th International Weber Symposium. Kauai, Hawaii. June 15-20, 2014.
Since its introduction by Weber [1], fluorophores in the PRODAN series have contributed to our understanding of hydration and packing in biological membranes. Based in their solvatochromism properties PRODAN and LAURDAN have long red shifts associated with the dipolar relaxation of the environmental water molecules around the probe. Exploiting this property, here we apply methods based on lifetime determinations and phasor plots as well as steady-state measurements using the spectral phasor approach, for analysis of the behavior of LAURDAN and PRODAN in multilamellar vesicles. The lifetime Phasor approach (Jameson et al., [2]) uses a plot of M.sin(Φ) versus M.cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. With Spectral phasors, introduced by Fereidouni et al [3], the steady-state fluorescence spectrum is Fourier transformed, resulting in two coordinates in x and y (cosine and sine, respectively) used for a scatter plot (Spectral ... [truncated at 150 words]
Moens PDJ, Digman MA, Gratton E.
Modes of diffusion of Cholera toxin bound to GM1 on live cell membrane by image mean square displacement (iMSD) analysis.
9th International Weber Symposium. Kauai, Hawaii. June 15-20, 2014.
The image-Mean Square Displacement technique (iMSD) applies the calculation of the mean square displacement (MSD) used in single molecule tracking to images without resolving single particles. The iMSD plot obtained is similar to the MSD plot obtained using the single-particle tracking technique and is used to reconstruct the protein diffusion law. From these plots, we can determine whether the labeled molecules are undergoing pure isotropic, restricted, corralled, transiently confined or directed diffusion. In our study total internal reflection fluorescence (TIRF) microscopy images were taken of Cholera toxin subunit B (CtxB) membrane labeled cells. We mapped the modes of CTxB diffusion when bound to GM1 in live cell membranes and found different types of diffusion across the cell including isotropic, confined and transiently confined. However, no directed diffusion could be observed. We show that repeated illumination of the same area of the cell leads to a transition of diffusion type from ... [truncated at 150 words]
Ranjit S, Dvornikov A, Jameson DM, Reinhart GD, Gratton E.
Three photon excitation FCS of Phosphofructokinase (PFK).
9th International Weber Symposium. Kauai, Hawaii. June 15-20, 2014.
In this work, three photon excitation fluorescence correlation spectroscopy (FCS) was used to measure oligomerization equilibrium of human phosphofructokinase (PFK). Three photon FCS can provide a new method to study oligomerization of unlabeled proteins by using the endogenously fluorescent tryptophan residues, excitable using a commercially available Ti-Sapphire laser. The fluorescence intensity produced by the three photon excitation was measured using the DIVER microscope set up, originally designed for deep tissue imaging. In this modified home built upright microscope, a large area photomultiplier tube is used as a detector and is placed directly below the sample. The lack of optical elements in the detection path of the microscope results in much improved detection efficiency in the UV spectral region up to about 300 nm, which nicely matches the emission from tryptophan and gives DIVER its extraordinary sensitivity. The three photon excitation autocorrelation decays obtained for Phosphofructokinase (PFK) in presence of fructose-6-phosphate ... [truncated at 150 words]
Ranjit S, Lanzanò L, Gratton E.
Mapping diffusion in a living cell using the phasor approach.
9th International Weber Symposium. Kauai, Hawaii. June 15-20, 2014.
The diffusion of a fluorescent protein within the cell has been measured by using either the fluctuation based techniques (FCS, RICS) or through particle tracking or through FRAP. However none of these methods enable us to measure the diffusion of the fluorescent particle at each pixel of the entire image. Measurement using the conventional single point FCS at every individual pixel results in the long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation we have developed a new method of scanning while constructing fluorescent image of the cell. In this new method of modified raster scanning, the laser scans each individual line multiple times before moving to the next line. This continues until the complete area is scanned. The difference from the RICS approach is in RICS the data is acquired by scanning each frame once and then scanning the image ... [truncated at 150 words]
Wright BK, Jones MR, Digman MA, Gratton E.
Analysis of endogenous nuclear NADH in differentiating myoblasts using the spectral phasor approach.
9th International Weber Symposium. Kauai, Hawaii. June 15-20, 2014.
Analysis of endogenous autofluorescent energy metabolites, for example NADH in the nuclear regions of live cells, aids in determining a cells metabolic status. Detecting the spectral differences of free and bound NADH is currently limited since the NADH spectrum could change depending of the protein that is binding NADH; however applying Spectral Phasor to identify discrete wavelength shifts and discriminated at a pixel level overcomes the need for the a prior knowledge of the spectra. For this investigation, live progenitor rat myoblast cells (L6 cells), were stimulated to differentiate through the reduction of foetal bovine serum (2%) in the cell culture media. Spectral data was acquired using the lambda mode of a Zeiss LSM 710 confocal microscope, exciting the cells at 740nm and analysed with SimFCS. Two emission wavelength maxima; 473nm and 480nm, were selected in both undifferentiated myoblasts and those induced to differentiate. The 473nm wavelengths observed in undifferentiated ... [truncated at 150 words]
Kukreti S, Cerussi AE, Leproux A, Santoro Y, Hsiang D, Mehta R, Tromberg BJ, Gratton E.
Investigation of pathological response to neoadjuvant chemotherapy in breast cancer patients using near-infrared diffuse optical spectroscopic imaging.
ASCI / AAP Joint Meeting 2014. Chicago. April 25-27, 2014.
In patients with locally-advanced breast cancer, neoadjuvant chemotherapy can produce a complete pathological response, which is an important endpoint that correlates with survival. Because many patients do not respond to neoadjuvant chemotherapy, and conventional clinical assessments do not adequately predict pathological response, there is interest in developing imaging methods to monitor and predict response both prior to and during treatment. Diffuse Optical Spectroscopic Imaging (DOSI) provides a non-radiation view of tissue metabolic state based on the intrinsic near-infrared absorption contrast of hemoglobin, bulk lipid and water. Tissues undergoing neoadjuvant chemotherapy display changes in these molecules, but these molecules are not cancer specific. A self-referencing differential spectroscopy analysis of DOSI revealed spectral signatures thought to represent dispositional shifts in NIR absorbers that may be more specific for malignancy. Can these spectral signatures evaluate pathological response in breast cancer tissue treated with neoadjuvant chemotherapy? DOSI methods were utilized to quantitatively measure absorption ... [truncated at 150 words]
Hinde E, Gratton E, Gaus K.
Imaging the diffusive route of molecules in live cells by fluctuation analysis.
Focus on Microscopy 2014. Sydney, Australia. April 13-16, 2014.
Dynamic changes in higher order chromatin structure modulate the accessibility of DNA toward transcription, DNA repair and replication. The spatiotemporal scale upon which these dynamics occur however, render them ‘invisible’ to live cell imaging. Here we present a novel method to measure the real time accessibility of chromatin that is based on pair correlation analysis of EGFP (an inert tracer) molecular flow throughout the chromatin network1. From detected changes in the arrival time of EGFP molecules into and out of the chromatin, this method detects millisecond changes in protein access to condensed chromatin with sub-micron resolution. Using this method we have probed the naturally occurring changes in chromatin compaction during the cell cycle and in combination with laser micro-irradiation, monitored for the first time the in vivo structural rearrangements of chromatin during repair2,3. The pair correlation method thus offers a unique opportunity to probe the directionality of intra-nuclear traffic, by ... [truncated at 150 words]
Yano S, Tome Y, Digman M, Momiyama M, Suetsugu A, Gratton E, Hoffman R.
Color-coded chemotherapy: S/G2-phase-trapping by methioninase pre-treatment, indicated by FUCCI imaging, enables highly effective cancer chemotherapy.
Experimental Biology 2014. San Diego, California. April 26-30, 2014.
FASEB J. 2014; 28(1 Supplement), 923.11.Methionine deprivation by methionine α,γ lyase (methioninase or METase) selectively arrests cancer cells during late S/G2-phase of the cell cycle, where the cancer cells are highly sensitive to DNA-damaging chemotherapy. Fluorescent ubiquitination-based cell cycle indicator (FUCCI) (Cell 132, 487-498, 2008), was used to monitor the onset of the S/G2-phase block due to methionine deprivation effected by METase. The S/G2-phase-blocked cancer cells fluoresced yellow or green, in contrast to cancer cells in G1/G0 which fluoresced red due to FUCCI. Cancer cells, synchronously blocked in S/G2-phase by METase fluorescing yellow-green, were treated with doxorubicin, cisplatin, or 5-fluorouracil. As a control, cancer cells treated with drugs only without rMETAse, were resistant to the drugs. rMETase treatment, followed by chemotherapy, when FUCCI indicated the onset of S/G2 block, was highly effective. Color-coded chemotherapy, whereby the cell cycle of cancer cells is blocked in S/G2-phase, as identified by fluorescent reporters, may be a general approach ... [truncated at 150 words]
Datta R, Kurokawa Y, Digman MA, George SC, Gratton E.
Subcellular level optical metabolic imaging of induced pluoripotent stem cells using endogenous fluorophore.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 200a-201a, 1013-Pos.In this work we show label-free metabolic imaging of induced pluoripotent stem (iPS) cell delineating regions of different metabolic activity at subcellular level. iPS cells, which are derived from adult somatic cells (skin fibroblast in this case) by nuclear reprogramming, possess pluoripotency. Hence, these cells offer a unique platform for stem cell research, drug discovery and testing, disease treatment development and toxicological screening. The phasor approach was applied to endogenous NADH fluorescence lifetime imaging microscopy (FLIM) to map free to bound NADH ratio distribution within single cells. The FLIM phasor distribution shows a clear difference between metabolic states of the nucleus and cytoplasmic regions. The nuclei had a larger fraction of free NADH indicated by a distribution shifted more toward the shorter lifetime as compared to the phasor distribution of the cytoplasm. Also within the nucleus, two distinct lifetime distributions were observed with the perinuclear region having more bound fraction ... [truncated at 150 words]
Gabriel M, Gratton E, Estrada LC.
Spectroscopic properties of intrinsic proteins in collagen samples by using gold-nanoparticles and two-photon excited fluorescence microscopy.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 398a, 2011-Pos.Multiphoton microscopy provides optical sectioning for high-resolution imaging. In biological systems, most multiphoton microscopy studies have relied on two-photon excited fluorescence (TPEF) to produce images. In particular, TPEF from structural proteins has emerged as an invaluable tool for 3D imaging. However, depending on the fluorophores involved, the TPEF could be extremely weak, limiting TPEF use for thick-tissue applications.
The physics and optical properties of noble-metal nanoparticles (NPs) is currently attracting much attention. The signal enhancement of molecules near a metal surface arises from the interaction with surface plasmon (SP) modes. These interactions also result in the shortening of the excited-state lifetime improving the molecules' photo-stability. The optical properties of the environment near a metallic NP are affected by the near-field electromagnetic-field. In the case of fluorescent molecules located at very short distances from a metallic surface, non-radiative energy transfer to SPs in the metal takes place; however, emission enhancement due to ... [truncated at 150 words]
Digman MA, Gratton E, Chen H.
Profiling cancer cell intrinsic fluorescence with the spectral camera- phasor analysis method.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 402a, 2030-Pos.Advances in hyper- or multi-spectral based imaging cameras have expanded new areas of cellular research for the last 20 years. Most recently, intensity signals can now be detected with a line spectrograph in which light is transmited over an imaging signal to an ultra sensitive CCD camera with fast readout speed. However, spectrally resolving these images is highly problematic given that most fluorescent emission spectra are broad and superimposed one with another. In order to spectrally resolve these images with high spectral resolution, we have developed the phasor analysis, used in the first and second harmonic, where each pixel in the image is used to construct a spectral profile that is Fourier transformed to produce the co-ordinates of the pixel in a polar plot. This graphical representation is free of fitting routines and can easily separate linear combinations of multiple spectral components. For this application we used the Andor's iXon ... [truncated at 150 words]
Malacrida LS, Briva A, Vetromile CM, Gratton E, Denicola A, Jameson DM.
Phasor plots and spectral phasor analysis of LAURDAN and PRODAN for membrane heterogeneity studies: new frontiers in membrane biophysics.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 84a, 449-Pos.Since its introduction by Weber [1], fluorophores in the PRODAN series have contributed to our understanding of hydration and packing in biological membranes. Here we apply methods based on lifetime determinations and phasor plots as well as steady-state measurements using the spectral phasor approach, for analysis of the behavior of LAURDAN and PRODAN in vesicles. The lifetime Phasor approach (Jameson et al., [2]) uses a plot of M.sin(Φ) versus M.cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. With Spectral phasors, introduced by Fereidouni et al [3], the steady-state fluorescence spectrum is Fourier transformed, resulting in two coordinates in x and y used for a scatter plot (Spectral phasor).
The temporal Phasor approach shows significant improvement compared with older methods as regards discrimination of the effects of temperature, cholesterol content and drug addition, in our membrane model systems. This approach is very ... [truncated at 150 words]
-
Orbital tracking of single fluorescent particles on a commercial confocal microscope.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 397a, 2005-Pos.Single Particle Tracking (SPT) is a super-resolution technique used to determine the position of fluorescent particles with nanometer precision. The localization is generally obtained by analyzing the spatial distribution of fluorescence intensity emitted by the particle. In fact, the center of the distribution can be determined with an uncertainty which is much lower than the size of the distribution itself. In the orbital tracking method the position of a particle is obtained analyzing the distribution of intensity along a circular orbit scanned around the particle. In combination with an active feedback this method allows tracking of particles in 2D and 3D with millisecond temporal resolution[1]. More recently, the use of orbital tracking to perform imaging has also been proposed[2].
The orbital tracking and the other 3D SPT feedback methods are generally implemented on homebuilt microscopes which are not yet commercially available. On the other hand, commercial setups offer the advantage of ... [truncated at 150 words]
Anzalone A, Stringari C, Gratton E.
Nanoimaging of the cell nucleus using gold nanoparticles.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 68a, 366-Pos.Fluorophores in close proximity to metallic nanoparticles (NPs) are promising systems to develop sensors, especially in the fields of cellular tracking and imaging. Metallic NPs can modify the emission of fluorescent molecules by increasing the excitation and emission rates (plasmonic enhancement). Using the orbital tracking method, here we track 20nm gold NPs inside the nuclei of Chok1 cells that have been transfected with a plasmid expressing H2B-mCherry or H2B-GFP histone proteins. Then the gold nanoparticles are inserted inside the nuclei. We show that, during tracking, gold NPs have different movement inside the nucleus. We observed a confined movement in which the nanoparticles are restricted in specific areas and long directed movement connecting these confined movement areas. During the movement the gold nanoparticles interact with the surroundings and give fluorescent enhancement of specific fluorophores with different emission spectra. The enhancement due to the proximity of gold NP and fluorophores inside the ... [truncated at 150 words]
Plotegher N, Stringari C, Jahid S, Gratton E, Bubacco L.
NADH is an endogenous reporter for alpha-synuclein aggregation in live cells.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 56a, 308-Pos.Alpha-synuclein aggregation is amply investigated for its involvement in Parkinson's disease etiopathogenesis. It has been shown that alpha-synuclein monomers, under pathological conditions, self-assembly to form oligomeric species that further aggregate into amyloid fibrils. Alpha-synuclein fibrils are the main constituent of Lewy Bodies, which are one of the characteristic hallmarks of Parkinson's disease.
Alpha-synuclein aggregation is studied in vitro and in cellular models with the aim to correlate toxicity mechanisms to defined aggregation products. However, the characterization of the aggregation process in cells is a difficult task that typically needs cell lysis or fixation, or the use of exogenous dyes.
Moreover, several different toxic mechanisms were ascribed to alpha-synuclein aggregates, i.e. clearance mechanisms impairment, mitochondrial dysfunctions, oxidative stress, neuroinflammation. In particular, mitochondria seem to be a target for alpha-synuclein to exert its toxicity. Several independent results suggested that alpha-synuclein overexpression and/or aggregation may cause impairment of cellular metabolism due to mitochondrial fragmentation and ... [truncated at 150 words]
Ranjit S, Lanzanò L, Gratton E.
Mapping diffusion in a living cell using the phasor approach.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 202a, 1021-Pos.The diffusion of a fluorescent protein within the cell has been measured by either using the fluctuation based techniques (FCS, RICS) or through particle tracking. However none of these methods enable us to measure the diffusion of the fluorescent particle at each pixel of the image of the cells. Measurement using the conventional single point FCS at each pixel of the image results in the continuous exposure of the cell to the laser and eventual bleaching of the sample.
To overcome this limitation we developed a new method of scanning while constructing fluorescent image of the cell. In this new method of scanning, the intensity trace at each pixel is collected multiple times before it moves to the next pixel. Alternatively, while acquiring the image, the laser scans each individual line multiple times before moving to the next line. This continues until the complete area is scanned. This is different from ... [truncated at 150 words]
-
Focal adhesion axial topography by the z-phasor approach in confocal microscopy.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 202a, 1022-Pos.The protein lateral and axial organization within focal adhesions has been studied by state of the art super resolution methods due to its thin structure, well below diffraction limit. However, to achieve high axial resolution, most of the current approaches rely on either sophisticated optics or diligent sample preparation requiring sample fixation that limits their application. In this report we present a phasor-based method that can be applied to fluorescent samples to determine the precise axial position of proteins using a conventional confocal microscope. We demonstrate that when about a total of 4000 photon counts are collected along a z-scan, axial localization precision close to 10 nm is achievable. We show at 10nm resolution that axial localization of paxillin, FAK and talin is similar at different focal adhesion sites, while F-actin shows a sharp increase in height towards the cell center. We further demonstrated that using line scans we can ... [truncated at 150 words]
di Rienzo C, Gratton E, Beltram F, Cardarelli F.
Fast spatiotemporal correlation spectroscopy to determine protein lateral diffusion laws in live cell membranes.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 224a, 1131-Plat.Here we present a straightforward image correlation analysis method to study the dynamics of fluorescently-labeled plasma-membrane proteins in live cells with high spatiotemporal resolution1. Notably, we don't extract and track each molecule, but we calculate population behavior using all molecules in a given region of the membrane. First, fast imaging of a given region on the membrane is achieved. Then, acquisitions at increasing time delays are correlated, for example each 2, 3, n repetitions. If particles diffuse, the width of the peak of the spatial autocorrelation function increases as the time delay between frames increases. Fitting of the series of autocorrelation functions enables to extract the actual protein ‘diffusion law’ from imaging, in the form of a mean square displacement vs time-delay plot (iMSD). The iMSD yields a quantitative view of the temporal evolution of the average molecular positions with nanometer accuracy, and no need for interpretative models. We demonstrate ... [truncated at 150 words]
Olivera-Couto A, Digman MA, Salzman V, Mailhos M, Gratton E, Aguilar PS.
Eisosomes and plasma membrane domain formation.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 465a-466a, 2353-Pos.A vast body of evidence coming from different microscopy techniques has been instrumental in concluding the long debate on whether biological membranes presented lateral segregation of proteins and lipids. Currently, the existence of membrane domains in both eukaryotes and prokaryotes is common ground. However, the mechanisms that sustain membrane domain formation and maintenance remain largely unknown. Our work is focus on the study of eisosomes, recently discovered plasma membrane domains in S. cerevisiae. We showed that Pil1 and Lsp1, the major proteinaceous components of eisosomes, are able to form self-assemblies that bind and curve membranes both in vivo and in vitro. We also showed that Lsp1 and Pil1 membrane-sculpting abilities are associated with the generation and organization of membrane domains (1,2). Thus, our current work supports the hypothesis that a mechanism for membrane eisosome domain formation is membrane curvature generation directed by Pil1-Lsp1 assemblies. To address this hypothesis we are ... [truncated at 150 words]
-
Diffusion mapping in living cells using camera-based correlation spectroscopy and phasor analysis.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 202a, 1020-Pos.Image correlation spectroscopy (ICS) is a powerful tool to study dynamics in living cells and tissues. However, when using a single point detector, the mapping of molecular movement is either slow or limited to a small field of view due to the sequential, point-by-point data acquisition. With an area detector such as a camera on the other hand, molecular motion can be captured simultaneously for each image pixel, allowing a fast mapping of the entire structure under study.
Usually, image data acquired with a camera is analyzed by correlating the fluorescence time trace captured in each pixel followed by fitting a function modeling the dynamic process. Yet, with ∼216 data points at hand, manual data evaluation is impossible while automatic fitting procedures are prone to errors because they rely on careful adjustment of start parameters and constrains. In particular, a fit to correlation spectroscopy data from regions containing static molecules or ... [truncated at 150 words]
Mieruszynski SP, Digman MA, Gratton E, Jones M.
Determining the quantitative dynamics of nucleic acids in live cells through RICS and iMSD approaches.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 67a, 361-Pos.Addressing the mobility and dynamics of DNA within live cells requires the characterization of individual particles in real time. Two such techniques enabling the quantification of the dynamics and means of motion of fluorescently labeled particles include the Raster Image Correlation Spectroscopy (RICS) and the image-based Mean Square Displacement (iMSD) approaches. In this study, the RICS and iMSD approaches were applied in order to elucidate the mobility and means of motion of DNA lipoplexes formed from varying sized DNA (20bp-5.5kbp). The RICS approach demonstrated that two species of mobility were present within the cytoplasm. Across the DNA fragment sizes, the slower species was not size dependent and were statistically the same. Whereas, faster moving particles which demonstrated a size dependent mobility. Nuclear localized DNA demonstrated to be significantly dynamic compared to other previously published work, ranging from 1.22 μm2/s (5.5kbp) to 3.67 μm2/s (21bp), consistent to nuclear proteins and RNA. ... [truncated at 150 words]
Stringari C, Geyfman M, Wang H, Crosignani V, Kumar V, Takahashi JS, Andersen B, Gratton E.
Circadian metabolic oscillations in the epidermis stem cells by fluorescence lifetime microscopy of NADH in vivo.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 24a, 138-Plat.There is a lack of non-invasive methods to monitor circadian metabolic oscillations of single cells in their native environment. Here we implement a label-free method using NADH as an intrinsic biomarker and the Phasor approach to Fluorescence Lifetime microscopy to measure the metabolic optical fingerprint of single cells during the day-night cycle. For the first time we detect in vivo metabolic circadian oscillations within the stem cells of the epidermis layer. We observe higher ratios of free/bound NADH, i.e. NADH/NAD+, in the night with respect to the day. This difference indicates a glycolytic phenotype associated with high proliferation during the night and an oxidative phosphorylation phenotype associated with low proliferation during the day. We demonstrate that cell-to cell metabolic heterogeneity correlates with circadian phase as measured within the basal epidermal layer by Per1-Venus reporter assay. Finally, we show that NADH metabolic oscillations are Bmal1 dependent.
Work supported with NIH grants P50 ... [truncated at 150 words]
Jahid S, Dvornikov AS, Digman MA, Gratton E.
3D tissue reconstruction by using deep tissue fluorescence imaging system and FLIM.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 400a, 2018-Pos.We have demonstrated a new detection method used in two-photon fluorescence microscope called the DIVER (Deep Imaging via Enhanced-Photon Recovery) with enhanced capabilities for deep tissue imaging. The microscope uses a large area photo-detector to collect scattered emission photons directly from the wide area of a specimen. This detection scheme allows for increasing the imaging depth in tissues by about 6 folds due to a unique detector design and its increased photons collection efficiency compared to conventional methods. The DIVER system is also capable of performing Fluorescence Lifetime Imaging (FLIM), a very powerful tool to segregate various features in cells and tissues. The combination of deep tissue imaging and fluorescence lifetime provides contrasted images based on physiological parameters at depths that are not achievable by conventional microscopes. The DIVER potentially can be useful for imaging of various tissue samples, in particular for human skin cancer diagnostics, providing 3D cellular resolved ... [truncated at 150 words]
-
3D orbital tracking of a DNA locus during the process of transcription.
58th Annual Meeting of the Biophysical Society. San Francisco, California. February 15–19, 2014.
Biophys J. 2014; 106(2, Suppl 1): 394a, 1992-Pos.The temporal resolution currently achieved to track actively transcribing genes by means of fluorescence is of the order of 1-10s. We report on the application of 3D orbital tracking to a previously reported model system allowing to simultaneously label by means of fluorescence the genetic locus and the synthesized mRNA using the EGFP-labeled MS2 coat protein [1]. Our method, previously applied to the tracking of gene arrays in cultured cells [2], has a temporal resolution of 10-100ms, and additionally records the 3D position of the genetic locus by moving along a circular orbit the focused laser beam. Distinct regions of active transcription display a well defined spatial organization, corralling the denser part of the genetic locus. In most cases each region maintains a defined angle in the reference system of the orbit, and the transcriptional activities of different regions are not cross-correlated.
The fluorescence time traces of each of these regions ... [truncated at 150 words]
-
Nanoscale 3D topological structure of chromatin in live cells.
3rd International Symposium Frontiers in Neurophotonics. October 1–4, 2013. Bordeaux, France.
Abstract Book. 2013; : 34.Chromatin structure, compaction and remodeling at the micro and nanometer scale have fundamental roles in many biological events. Chromatin compaction produces heterogeneity of the cell nucleus which results in specific structural and transport properties which have been only partially studied. Although the nucleosome structure has been in part deciphered, the topology of chromatin structure at the micron scale remains unresolved. In this work, based on the Gold Enhanced Nano Imaging (GENI) method we studied chromatin organization using the orbital 3D tracking technique applied to 20 nm gold nanoparticles (NPs) interacting with the chromatin. As the particles move in the proximity of the chromatin, this method provides insight of local structure and transport properties at the nano scale by following the trajectories of gold nanoparticle with nanometer precision. Using two‐photon excitation, the fluorescence of fluorophores that are in the nanometer proximity to the gold particle is strongly enhanced. Metallic NPs do ... [truncated at 150 words]
Digman MA, Dvornikov A, Jahid S.
Deep tissue imaging for cancer detection using FLIM.
2013 Pathways to Cures: Clinical Translational Research Day at UCI. Irvine, California. June 3, 2013.
Abstract Book. 2013; : 24, 40.We have developed a new fluorescence confocal microscope called the DIVER (Deep Imaging via Enhanced-Photon Recovery) with unprecedented capabilities for deep tissue imaging. Our microscope uses 2-photon excitation to image at 3-4 mm in tissues by virtue of a unique detector design that has very large collection efficiency compared to conventional confocal microscopes...
Yano S, Tome Y, Digman M, Momiyama M, Suetsugu A, Gratton E, Hoffman RM.
Cancer cell location in tumor determines cell cycle position and dormancy.
104th Annual Meeting of the American Association for Cancer Research. Apr 6-10, 2013. Washington, DC.
Cancer Research. 2013; 73(8, Supplement 1), 1758.The phase of the cell cycle can determine whether a cancer cell can respond to most drugs. The fluorescence ubiquitination cell cycle indicator (FUCCI) was used to image the cell cycle phase within tumors. In nascent tumors in nude mice, approximately 30% of the cells in the center of the tumor were in G0/G1 and 70% in S/G2/M. In contrast, approximately 90% of cancer cells in the center of an established tumor were in G0/G1 phase. Similarly, approximately 75% of cancer cells far from (>100 μm) tumor vessels were in G0/G1. Although 60-70% of cancer cells near the surface of an established tumor (< 100 μm) or near blood vessels were in S/G2/M, these cells are a small minority of an established tumor. Moreover, tumors that are stable in size for more than six months in nude mice consisted of mostly G0/G1 cells, suggesting they are dormant. The results presented ... [truncated at 150 words]
Yano S, Miwa S, Tome Y, Uehara F, Digman M, Gratton E, Hoffman RM.
Intravital imaging of cancer cell cycle in the bone marrow.
104th Annual Meeting of the American Association for Cancer Research. Apr 6-10, 2013. Washington, DC.
Cancer Research. 2013; 73(8, Supplement 1), 1756.The cell cycle of cancer cells in the bone marrow was imaged using a fluorescence ubiquitination cell cycle indicator (FUCCI). With FUCCI, quiescent cancer cells express mKusabira-Orange fluorescent protein (red) and proliferating cells express mAzami-Green fluorescent protein (green). FUCCI-expressing cancer cells were injected into the bone marrow of tibia of mice. At the early stage after injection (7 days), approximately 90% of the cells were in G0/G1. In contrast, at late stage after injection (28 days more), in the endosteal lesion, approximately 80% of the cancer cells were in S/G2/M phases and in the central zone, approximately 75% of cancer cells were in G0/G1. Thus, cancer-cell location and time in the bone marrow determines cell cycle position and possibly dormancy. Thus, quiescent (dormant) cancer cells in the bone marrow need to be targeted as well as proliferating cancer cells, which is a significant current challenge.
Yano S, Tome Y, Digman M, Momiyama M, Suetsugu A, Gratton E, Hoffman RM.
Imaging of methioninase-induced S/G2-phase-trapping for subsequent effective chemotherapy.
104th Annual Meeting of the American Association for Cancer Research. Apr 6-10, 2013. Washington, DC.
Cancer Research. 2013; 73(8, Supplement 1), 3412.Methionine-dependence of cancer cells may be due to excessive methylation reactions in cancer cells. Deprivation of methionine α,{gamma} lyase (methioninase or METase) selectively arrests cancer cells during late S-phase, where they are highly sensitive to DNA-damaging chemotherapy. Fluorescent ubiquitination-based cell cycle indicator (FUCCI)), was used to monitor the onset of the S/G2-phase block due to methionine deprivation effected by METase. The S-phase-blocked cancer cells fluoresced yellow or green, in contrast to cancer cells in G1 which fluoresced red. Cancer cells, synchronously blocked in S-phase by METase and identified by their yellow-green fluorescence, were administered DNA-damaging chemotherapy drugs such as doxorubicin, cisplatin, or 5-fluorouracil. Treatment of cancer cells with drugs only without methioninase-effected S-phase synchrony, led to the majority of the cancer cell population being blocked in G0/G1 phase (red fluorescent) where they were resistant to the drugs. In contrast, METase treatment, followed by chemotherapy when FUCCI indicated the S/G2 block ... [truncated at 150 words]
Miwa S, Yano S, Hiroshima Y, Tome Y, Uehara F, Mii S, Kimura H, Hayashi K, Tsuchiya H, Digman M, Gratton E, Hoffman RM.
Caffeine modulates the cell cycle in cancer cells to enhance cisplatin efficacy.
104th Annual Meeting of the American Association for Cancer Research. Apr 6-10, 2013. Washington, DC.
Cancer Research. 2013; 73(8, Supplement 1), 703.Caffeine has been shown to enhance the efficacy of chemotherapy, but the mechanism is not well understood. In this study, we investigated cell-cycle modulation induced by caffeine in combination with cisplatinum treatment of human cancer cells. Proliferation inhibition was determined with a clonogenic assay. Mitotic and apoptotic changes were observed by imaging of 143B dual-color cells, in which GFP is expressed in the nucleus and RFP in the cytoplasm. Modulation of the cell-cycle in combination with cisplatinum was observed using time-lapse imaging analyses of HeLa cells expressing a fluorescent cell-cycle indicator, FUCCI (Fluorescent Ubiquitination-based Cell Cycle Indicator). The clonogenic assay showed that caffeine increased the efficacy of cisplatin on inhibition of cell proliferation. Dual-color imaging demonstrated that cisplatin decreased mitosis and induced apoptosis in 143B cells. The combination of cisplatin and caffeine reduced the decrease of mitosis and increased apoptosis. Time-lapse FUCCI imaging showed that cisplatin strongly induced cell-cycle arrest ... [truncated at 150 words]
Miwa S, Yano S, Hiroshima Y, Tome Y, Uehara F, Mii S, Kimura H, Hayashi K, Tsuchiya H, Efimova E, Digman M, Gratton E, Hoffman RM.
Real-time imaging demonstrates cancer cells exposed to UV irradiation either repair their DNA or die.
104th Annual Meeting of the American Association for Cancer Research. Apr 6-10, 2013. Washington, DC.
Cancer Research. 2013; 73(8, Supplement 1), 1769.The DNA repair protein 53BP1 is re-localized in foci after DNA damage. In this study, we imaged the real-time response of 53BP1 linked to GFP to DNA damage induced by UV irradiation. Inhibition of proliferation by UV irradiation was analyzed by a clonogenic assay. The relocalization of GFP fused to the chromatin-binding domain of 53BP1 was imaged after UV irradiation of MiaPaca-2 53BP1-GFP human pancreatic cancer cells. The clonogenic assay demonstrated that UVC dose-dependently inhibited cell proliferation, while UVA and UVB showed little effect. During live-cell imaging, 53BP1-GFP foci formation was observed within 30 min after UVC irradiation. High-dose UVC also induced apoptosis within 24 h after irradiation. The cells treated with UVC irradiation appear to have two different fates: DNA repair or apoptosis. A fraction of the cells underwent apoptosis, and the other fraction repaired their DNA as evidenced by 53BP1 focus formation. The present study demonstrates that some ... [truncated at 150 words]
Arnesano C, D'Amico E, Digman M, Gratton E.
Measurements of fluorescence decay times by the digital parallel frequency-domain method (FLIMbox).
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 347a, 1780-Pos.The digital parallel frequency domain design is a powerful approach that offers the possibility to implement a variety of different applications in fluorescence spectroscopy and microscopy. It allows lifetime measurements in a cuvette, fluorescence lifetime imaging and FCS as well as multi frequency and multi wavelength tissue imaging in small portable medical devices. It dramatically reduces the acquisition time from the several minutes scale to the seconds scale, and performs every signal process in a digital fashion, avoiding any RF emission and it is extremely inexpensive. This development is the result of a systematic study carried on the previous design known as the FLIMbox. The extensive work done in maximizing the performance of the original FLIMbox led us to develop a new hardware solution with exciting and promising results and potentials that were not possible in the previous hardware realizations. The new design permits acquisition of the full harmonic content ... [truncated at 150 words]
Stringari C, Pate KT, Edwards RA, Waterman ML, Gratton E.
Metabolic imaging of colon cancer tumors in vivo by phasor fluorescence lifetime microscopy of NADH.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 342a-343a, 1756-Pos.Here we use a non-invasive method to measure the metabolic phenotype of single colon cancer cells in vivo. By using NADH as optical biomarker and the phasor approach to Fluorescence Lifetime microscopy (FLIM) we identify cancer metabolism related to different rates of glycolysis, cell growth and proliferation of cells. We perform label-free Phasor FLIM on living and actively-perfused xenograft tumors. Colon cancer cells are injected subcutaneously into immune deficient (NSG) mice and tumors are allowed to grow for three weeks. The tumor vasculature is labeled by injecting TRITC Dextran into the tail vein and xenografts are exposed via a skin flap, still perfused by their feeder vessels.
FLIM distinguishes collagen fibers, the tumor stroma, adipocytes, blood vessels and single cancer cells within the living tumor. By measuring NADH lifetime, we quantify the relative concentration of free and bound NADH in single cancer cells, which reflects the cellular redox NADH/NAD+ ratio and ... [truncated at 150 words]
James NG, Digman MA, Ross JA, Barylko B, Wang L, Chen Y, Müller JD, Gratton E, Albanesi JP, Jameson DM.
Mutations associated with centronuclear myopathy (CNM) enhance the size and stability of dynamin 2 clusters in cells.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 618a, 3178-Pos.Dynamin 2 (Dyn2), a ~100 kDa, GTPase that self-assembles into rings around the necks of budding vesicles, has been implicated in generating force for endocytic vesicle scission from the plasma membrane. Self-association of Dyn2 at the site of membrane invagination, and subsequent catalysis of membrane scission, is tightly coupled to GTP binding and hydrolysis. Mutations that affect the stability of Dyn2 polymers have been linked to autosomal dominant forms of CNM, a congenital disorder characterized by muscle weakness and wasting. In vitro biochemical analysis revealed that CNM-causing Dyn2 mutants express enhanced GTPase activity relative to wild-type Dyn2, and form polymers that are more resistant to GTPase-dependent disassembly. We compared the physical properties of assembled wild-type and CNM-linked mutant forms of Dyn2 in the cytosol and plasma membrane of living cells using fluorescence fluctuation spectroscopy (FFS). Our data indicate that unassembled wild-type Dyn2 is a tetramer in the cytosol, whereas CNM-associated ... [truncated at 150 words]
Lanzanò L, Giral H, Levi M, Gratton E.
Subcellular imaging of protein-protein interactions in live epithelial cells using single particle tracking-FLIM-FRET.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 680a, 3496-Pos.Phosphate (Pi) homeostasis is crucial for many cellular processes. Pi absorption in the kidney proximal tubule and in the intestine is mainly mediated by the type II family of the sodium-dependent phosphate co-transporters (NaPi) which are located in the apical membrane microvilli. Interaction with scaffolding PDZ domain containing proteins is thought to play a central role in the regulation of the abundance and activity of the NaPi transporters, in response to dietary and hormonal stimuli.
We investigated protein-protein interactions between NaPi and PDZ proteins in cellular models of the proximal tubule and the intestine using the FLIM detection of FRET [1]. Also, we have recently introduced the nSPIRO method which generalizes the principles of Single Particle Tracking to the imaging of extended subcellular structures like the apical membrane microvilli [2]. Using this method we can image the microvilli with high resolution even if they are moving.
Here we combine the tracking of ... [truncated at 150 words]
Cardarelli F, Lanzanò L, Gratton E.
Super-resolution by feedback imaging: mechanisms of translocation through the nuclear pore complex.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 120a, 616-Pos.Nuclear pore complexes (NPCs) are the gateways for nucleocytoplasmic exchange. Because single molecules undergo rapid transport, it is challenging to follow their motion in live cells. Hence fundamental questions remain in regard to the nanomechanical basis of selective gating of molecules through the NPC in live cells.
We set out to address these issues by a combination of fluorescence correlation spectroscopy (FCS) and real-time tracking of the center of mass of single NPCs in live cells. The center of mass tracking allows us to create an “Einstein trap” in which the thermal motion of the entire pore is compensated so that we observe the shuttling of single molecules in the reference frame of the pore. Using this setup we demonstrate that the transport of Karyopherin-ß1 (Kapß1) receptor is regulated to produce a characteristic narrow correlation time within the NPC, which is the signature of directed-motion events. We also show that the ... [truncated at 150 words]
Hinde E, Cardarelli F, Chen A, Khine M, Gratton E.
Tracking the mechanical dynamics of stem cell chromatin.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 201a, 1030-Plat.A plastic chromatin structure has emerged as fundamental to the self-renewal and pluripotent capacity of embryonic stem (ES) cells. Direct measurement of chromatin dynamics in vivo is however challenging as high spatiotemporal resolution is required. Here we present a new tracking based method which can detect chromatin fiber movement and quantify the mechanical dynamics of chromatin in live cells. We use this method to study how the mechanical properties of chromatin movement in hESCs are modulated spatiotemporally during differentiation into cardiomyocytes (CM). Notably, we find that pluripotency is associated with a highly discrete, energy-dependent frequency of chromatin movement, that we refer to as a “breathing” state. We find that this “breathing” state is strictly dependent on the metabolic state of the cell and is progressively silenced during differentiation, thus representing a hallmark of pluripotency maintenance. This is a result that could not have been observed without the nanometer resolution provided ... [truncated at 150 words]
-
Axial super resolution topography of focal adhesion by confocal microscopy.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 535a, 2747-Plat.Focal adhesions mediate cell-ECM interaction through a complex protein network and have been well studied in terms of protein functions and interactions. The protein organization within focal adhesions was a subject of state of the art super resolution methods due to its thin structure well below diffraction limit. However, most of the current super resolution approaches rely on either sophisticated optics or photoactivable compounds, limiting their application.
In this work we present a phasor-based method to determine the precise axial position of focal adhesion proteins using conventional confocal microscope. 3D image stacks with small axial step size (50 nm or lower) were acquired. For each pixel, the intensity along the axial axis was Fourier transformed. The first harmonic of the transform was represented as phasor, and the angle difference was used for calculating the relative axial center-of-mass position of the fluorescence compound. The resolution of the axial position depends on the ... [truncated at 150 words]
Crosignani V, Dvornikov A, Gratton E.
Deep imaging of biological tissue by ultra-efficient photon collection.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 336a, 1722-Pos.We present an upright two-photon fluorescence microscope that is capable of imaging in turbid media up to 3mm depth with micron resolution. The system utilizes a high power Ti:Sa Mai Tai laser with a group velocity dispersion compensator (DeepSee) for two-photon fluorescence excitation. The especially designed fluorescence detector, which is a key feature of the system, is capable of collecting fluorescence photons from the wide surface area (25mm diameter) of the specimen. This novel detection scheme has proven to be extremely efficient in the collection of fluorescence photons scattered by turbid media and allows about 6 fold increase in imaging depth when compared with conventional two-photon microscopes. The system is also equipped with a second fluorescence detector that allows its use as a conventional two-photon microscope and the comparison of the data acquired by both detection methods. In addition, the presented microscope is coupled to the FLIMbox (ISS, Inc.) and ... [truncated at 150 words]
Digman MA, Jahid S, Chiang L, Gratton E.
Determination of protein complexes with NADH in live cells.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 682a, 3506-Pos.The ratio of free/bound NADH is used to measure the redox state of the cells. This ratio does not provide specific information about which proteins are involved in binding of NADH. In vitro, FRET between tryptophan containing proteins (excited between 280-300nm) and the NADH co-factor (emitting between 400-450 nm) has been exploited to detect the ratio of free/bound NADH to specific proteins. In live cell microscopy this approach is difficult to implement because it requires UV excitation. If a specific protein interacting with NADH has an absorption or fluorescent moiety, this will result in the quenching of the NADH fluorescence. However, since there is a large amount of NADH, this decrease of fluorescence is difficult to associate to the specific protein. Instead, when FRET occurs between NADH and a fluorescently tagged protein there will be a sensitized emission of the acceptor molecule which is easy to detect. Here we develop ... [truncated at 150 words]
Altamore I, Lanzanò L, Gratton E.
Dual channel detection of ultra low concentration of bacteria in real time and via scanning FCS.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 347a, 1781-Pos.The rapid quantification and identification of infectious disease agents is of primary importance for medical diagnosis, public health, food safety and environment monitoring. Here we describe an alternative, simple and rapid method to detect very low concentrations of bacteria in water. Our device consists of a small confocal microscope with a horizontal geometry with large pinhole and a holder for cylindrical cuvettes. Two motors provide a rotational and slower vertical inversion motion of the cuvette, so as to scan a total volume of 1ml/min. The device looks like a simplified flow cytometer without flow. Bacteria are stained by two nucleic acid dyes that fluoresce green and red and excited with two lasers. When a bacterium passes through the observation volume emits both red and green fluorescence. The light emitted from the sample is directed by a system of lens toward a dichroic beam splitter and then separated into two light ... [truncated at 150 words]
-
Exploring the chromatin architecture in living cells by minutes-long tracking of gold nanoparticles.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 582a, 2999-Pos.Investigating the chromatin compaction on the micro(nano)-meter scale, has become a question of interest to understand many cellular processes. Previous evidence suggests that the cell nucleus is spatially heterogeneous and with inaccessible regions mainly due to a high concentration of chromatin. However, the in vivo 3D picture of the nuclear structure remains unclear. In this work, we studied chromatin organization applying the orbital 3D tracking technique to 20 nm gold nanoparticles (NPs) previously incorporated inside the nucleus of NIH3T3 live cells. We have recently shown that metallic NPs do not bleach or blink upon continuous illumination, are extremely stable, very bright and their luminescence spans over the visible spectrum. These characteristics allow us to track them for minutes thus providing 3D trajectories appreciably longer than those based on fluorescent proteins or quantum dots. For this study we have analyzed the motion of 60 NPs. Each one provided us with a ... [truncated at 150 words]
Gratton E, Digman MA, Stringari C, Arnesano C.
FLIM phasor analysis for time-domain and frequency-domain data.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 347a, 1779-Pos.The phasor analysis of FLIM images provides a fit free global view of molecular species and their interaction in cells and tissues. Different techniques are used to collect the original data either in the time domain or in the frequency domain. The “phasor transformation” which is based on the calculation of Fourier components should in principle make the phasor plot independent of the domain of data collection. However, technical differences between the modalities of data acquisition in various instruments result in slightly different phasor calculations. In this poster we discuss the origin of the variations between the different methods of data acquisition. In particular we compare data obtained with the classical analog frequency domain instrument, data obtained with the FLIMbox principle that is based on a digital equivalent of the frequency domain instrument and data obtained with the popular time-correlated single photon counting instrument. We discuss how to minimize these ... [truncated at 150 words]
Golfetto O, Hinde E, Gratton E.
Laurdan spectral phasor detects membrane micro-heterogeneity and lipid domains in live cells.
57th Annual Meeting of the Biophysical Society. Philadelphia, Pennsylvania. February 2–6, 2013.
Biophys J. 2013; 104(2, Suppl 1): 345a, 1768-Pos.The fluorescent probe Laurdan is sensitive to membrane lateral packing and lipid order. Laurdan is commonly used in model and biological membranes to report on different lipid phases. Here we describe a novel approach to detect Laurdan spectral changes due to membrane packing in vivo: the Laurdan spectral phasor. By Fourier transformation of Laurdan’s spectrum into a spectral phasor we obtain two coordinates for each pixel of an image. The linear combination of two spectral components always falls in the line joining the phasors representing the two components, which allows quantification of the relative contribution of the different lipid phases. If, however, there is a different environment for Laurdan in a pixel, the phasor of that pixel cannot be represented by the linear combination of the phasor of two components. Therefore, non-linear environment interactions can be immediately detected. We use this approach to perform a comprehensive analysis of membrane heterogeneity ... [truncated at 150 words]
Salih A, Geny D, Cutrale F, Digman MA, Diamond S, Carrol A, Dalton S, Larkum A, Gratton E, Gudge S, Kerr I.
Corals as solar modulators in ambient sunlight and following coral bleaching.
12th International Coral Reef Symposium. Cairns, Queensland, Australia. 9-13 July 2012.
Program Book. 2012; .Arnesano C, Santoroa Y, Gratton E.
Digital parallel frequency-domain spectroscopy for tissue imaging.
Optics Within Life Sciences (OWLS). Genova, Italy. July 4-6, 2012.
OWLS 2012 Abstract Book. 2012; : 30.Near-infrared (NIR) (650–1000 nm) optical properties of turbid media can be accurately quantified noninvasively using methods based on diffuse reflectance or transmittance1, such as frequency domain photon migration2 (FDPM). For instance, Diffuse Optical Spectroscopy (DOS) is a noninvasive technique that is commonly used to provide biochemical information on hemoglobin, bulk lipids and water concentration by NIR tissue absorption and scattering. DOS does not require exogenous contrast, and rapidly provides quantitative, functional information about tumor biochemical composition. Conventional FDPM techniques are based on white-light steady-state (SS) measurements and the acquisition of frequency-domain (FD) data at several wavelengths using laser diodes, to measure broadband NIR scatter-corrected absorption spectra of turbid media3. These techniques are limited by the number of wavelength points used to obtain the FD data. We developed a new method to improve the acquisition of optical parameters of the examined tissues, based on digital parallel acquisition in FD.
With our system, ... [truncated at 150 words]
Battisti A, Digman MA, Gratton E, Storti B, Beltram F, Bizzarri R.
Intracellular pH measurements made simple by genetically encoded fluorescent probes and phasor approach to FLIM.
Optics Within Life Sciences (OWLS). Genova, Italy. July 4-6, 2012.
OWLS 2012 Abstract Book. 2012; : 35.Intracellular pH (pHi) is a crucial parameter in the regulation of many cell functions, being related to metabolism and growth, ion transport and homeostasis, endocytosis, muscle cellular contractility, apoptosis and cancer, impairment of posttranslational modifications of secreted proteins, neuronal excitability, cell-cell coupling, and signal cascades. Tools for a precise measurement of pHi are then required to monitor changes in the intracellular environment. In this field, fluorescence microscopy has been extensively applied to obtain high-resolution spatio-temporal pHi maps in living cells thanks to genetically encoded pH-sensitive fluorescent proteins [1,2]. Indeed, these indicators can be expressed in cells, tissues, and whole organisms as fusion chimeras with specific protein moieties targeting subcellular compartments characterized by interesting pHi homeostasis. Here we show a versatile pHdependent GFP mutant (E2GFP), whose pKa falls within the physiological range, which has been used to monitor pH changes via fluorescence lifetime imaging (FLIM) [3]. FLIM is a concentration-independent technique ... [truncated at 150 words]
Coppola S, Cardarelli F, Pozzi D, Estrada LC, Digman MA, Gratton E, Bifone A, Caraccioloa G.
Intracellular trafficking of multicomponent lipoplexes: a fluorescence microscopy study.
Optics Within Life Sciences (OWLS). Genova, Italy. July 4-6, 2012.
OWLS 2012 Abstract Book. 2012; : 49.We investigated the role of the actin and microtubule networks on the uptake and cytoplasmic transport of multicomponent (MC) cationic liposome-DNA complexes in CHO-K1 live cells. Three-dimensional single particle tracking (SPT) allowed mean square displacement-based classification of intracellular modes of motion of complexes. In most cases (˜ 80%) the motion was classified as directed, while Brownian motion was observed much less frequently. Treatment with actin- (Latrunculin B) and microtubule-disrupting (Nocodazole) reagents indicated that intracellular trafficking of complexes predominantly involves microtubule-dependent transport. Confocal laser scanning microscopy (CLSM) on fluorescently-labeled complexes in the presence of LysoSensor (a pH-dependent lysosome marker) was applied to investigate the final fate of complexes. CLSM showed that, in untreated cells, most of the complexes were targeted to the lysosomes for degradation. Co-localization analysis of CLSM images after treating the cells with both types of inhibitors revealed that the actin network has a major effect on the initial ... [truncated at 150 words]
Golfetto O, Hinde E, Gratton E.
Laurdan fluorescence lifetime as a biosensor for membrane fluidity.
Optics Within Life Sciences (OWLS). Genova, Italy. July 4-6, 2012.
OWLS 2012 Abstract Book. 2012; : 59.Investigating changes in cellular membranes became a question of interest since recognition of their key role(s) influence in cell life. The fluorescent probe laurdan has been extensively used in membrane studies and its spectral properties have been investigated thoroughly. Laurdan generalized polarization (GP) became a common tool for evaluating quantitatively the order and the fluidity of membranes, and the coexistence of domains with different rigidity which could be associated with the presence and influence of cholesterol in the membrane. However when applied to cellular membrane studies GP analysis is limited given the compositional complexity of biological membranes. The phasor phasor approach to laurdan lifetime can help us detect and identify the fractional contribution of different components, such as cholesterol.
In this work the membrane marker laurdan proved to be a useful tool to investigate membrane order, monitor the fluidity in biological cells, even upon external stimulation, and (used in conjunction with ... [truncated at 150 words]
Suetsugu A, Digman M, Sabatini F, Moriwaki H, Saji S, Gratton E, Hoffman RM.
Raster-image-correlation spectroscopy of Paxillin-GFP-expressing breast cancer cell in vitro and in vivo.
103rd Annual Meeting of the American Association for Cancer Research. Mar 31-Apr 4, 2012. Chicago, IL.
Cancer Research. 2012; 72(8, Supplement 1), 5183.Raster-image-correlation spectroscopy (RICS) is a noninvasive technique to detect and quantify events in the living cell, including concentrations of molecules and their diffusion coefficients. Any cell containing a fluorophore that can be imaged with a laser scanning microscope can be analyzed with RICS. We obtained RICS images with an Olympus FluoView FV1000 confocal microscope using Olympus FluoView software to acquire data and SimFCS software to perform RICS analysis. Paxillin is involved in the assembly of focal adhesions, which was linked to green fluorescent protein (GFP) for the current study. In this study, we describe RICS of paxillin-GFP expression in breast cancer cells (MDA-MB-231) in vitro and in vivo. Slow-moving membrane-bound paxillin proteins were measured in live breast cancer cells in vitro. Paxillin-GFP-expressing breast cancer cells (1x106) were injected in the epigastric cranials vein of the nude mouse. Paxillin-GFP-expressing breast cancer cells became attached to the inner vessel wall within 3 ... [truncated at 150 words]
Lanzanò L, Caldas Y, Giral H, Digman MA, Levi M, Gratton E.
Tracking of single microvilli to study regulation of the intestinal phosphate transporters.
FASEB J. 2012; 26(Meeting Abstracts), 1068.15.Intestinal phosphate (Pi) uptake is a key mechanism in systemic Pi homeostasis. Modulation of intestinal Pi transport has been recently recognized as an important target in prevention of hyperphosphatemia and the associated cardiovascular complications in Chronic Kidney Disease. Intestinal Pi absorption is mainly mediated by the sodium-phosphate co-transporter NaPi2b expressed in the intestinal apical microvilli. Recent evidences show that agonists of the LXR nuclear hormone receptor inhibit intestinal NaPi transport activity, however, the detailed mechanisms of how LXR decreases intestinal NaPi transport activity, including the role of changes in membrane cholesterol or PDZ interacting proteins, remain to be determined.
We study these processes at the molecular level applying the Modulation Tracking nanoimaging method to microvilli of live cells expressing fluorescently tagged NaPi2b. Tracking single microvilli we measure their motion, image NaPi2b distribution on the membrane and perform Fluorescence Correlation Spectroscopy techniques (RICS, N&B) in steady state, even if the microvilli are ... [truncated at 150 words]
Crosignani V, Dvornikov AS, Gratton E.
Ultra-deep imaging with cellular resolution: enhanced two-photon fluorescence microscopy with the use of a wide area photodetector.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 199a, 1001-PosB787.We have previously shown that the use of a wide photocathode area PMT as a detector in a two-photon fluorescence microscope allowed us to image in turbid samples up to the depth of about 2.5 mm with cellular resolution. This detection scheme enables a very efficient collection of fluorescence photons directly from the wide (1” diameter) area of the sample, which considerably increases the detection system sensitivity in comparison to a traditional two-photon microscope, where fluorescence is collected by the same objective lens used for excitation. Because the imaging depth depends on the ability of the system to sense weak fluorescent signals, this new detection method significantly enhances the imaging depth. We have recently built a new experimental system that works in the upright configuration, which is best suited for experiments on live animals. The system employs a high power Ti:Sa Mai Tai laser with a group velocity dispersion compensator ... [truncated at 150 words]
-
Using metallic nanoparticles for nanometer-resolution optical imaging in living cells: photophysical properties and applications.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 209a, 1056-PosB842.Labeling cells and tissues with fluorescent probes such as organic dyes and quantum dots (QDots) is a widespread and successful technique for studying molecular dynamics both in vitro and in vivo. However, those probes usually suffer from photophysical/photochemical processes, such us blinking and photobleaching, limiting their utilization. The main challenges in probe design are to improve single molecule fluorescence detection, and to provide higher stability against photobleaching. In the last few years, metallic nanoparticles (NPs) of various sizes, shapes, and materials have been used as new labels for cellular microscopy. This is in part because, unlike common organic dyes and QDots, metallic NPs do not bleach or blink upon continuous illumination, are extremely stable, very bright and their luminescence spans over the visible spectrum. Their excellent optical and chemical properties make them an attractive contrast agent for cell imaging both in vitro and in vivo. In this work we show ... [truncated at 150 words]
Lanzanò L, Caldas Y, Giral H, Digman MA, Levi M, Gratton E.
Tracking of single microvilli to study regulation of the intestinal phosphate transporters.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 216a, 1093-Plat.Intestinal phosphate (Pi) uptake is one of the key mechanisms of systemic Pi homeostasis. Modulation of intestinal Pi transport has been recently recognized as an important target in prevention of hyperphosphatemia and the associated cardiovascular complications in Chronic Kidney Disease. Intestinal Pi absorption is mainly mediated by the type IIb sodium-phosphate co-transporter NaPi2b which is expressed in the intestinal apical microvilli. Recent evidences show that agonists of the LXR nuclear hormone receptor inhibit intestinal NaPi transport activity, NaPi2b protein and mRNA abundance, and decrease serum Pi level [1]. However, the detailed mechanisms of how LXR decreases intestinal NaPi transport activity, including the role of changes in membrane cholesterol or PDZ interacting proteins, remain to be determined.
In order to study these processes at the molecular level we apply the Modulation Tracking nanoimaging method [2] to microvilli of live cells expressing fluorescently tagged NaPi2b. This technique generalizes the principles of Single Particle ... [truncated at 150 words]
Gratton E, Hinde E, Digman MA.
Analysis of FRET biosensor distribution in 3D by the phasor approach to FLIM.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 234a-235a, 1183-Plat.The phasor approach to analyze FLIM images has several advantages with respect to the classical multi exponential of the decay at each pixel of an image. More importantly, the phasor approach lends itself to simultaneous correlation of lifetime changes with spatial localization. Here we describe an application of FRET image analysis using the Rac and RhoA biosensors in which the specific distribution of the sensor in a cell is important to establish its activation. Specifically we study cells in a 3D matrix in which the activation of the Rac and RhoA biosensor could have a different distribution than in 2D. In order to simultaneously measure several 3D locations we use a method in which we measure FRET along a 3-dimensional line which encompasses different parts of the cell. The measurement of the decay at each point of a 3D line can be done very fast (in millisecond) potentially revealing the ... [truncated at 150 words]
Coppola S, Estrada LC, Digman MA, Pozzi D, Gratton E, Caracciolo G.
Intracellular trafficking of lipid gene vectors investigated by three-dimensional single particle tracking.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 378a-379a, 1925-PosB695.Three-dimensional single particle tracking (SPT) was applied to investigate the intracellular trafficking of multicomponent (MC) lipoplexes in CHO-K1 cells. In untreated (NT) cells, we have found that: (i) intracellular lipoplex motion was either directed or Brownian;(ii) the occurrence of directed motion was more frequent (more than 70%) than the Brownian one;(iii) within experimental error, the Brownian motion (D∼0.7•10-3 um⊥2/s) was faster than the directional movement (D∼0.35•10-3 um⊥2/s);(iv) the directed motion mean velocity was about v=0.032 um/s;(v) the calculated three-dimensional asphericity, A3, was close to unity denoting the privileged occurrence of movement along a direction. To elucidate the role of the cytoskeleton structure in the lipoplex trafficking, cells were treated with cytoskeleton (actin microfilaments and microtubules) polymerization inhibitors (Latrunculin B and Nocodazole, respectively). In inhibitor-treated cells, we have found that: (i) the percentage of directional movement decreased balanced by the simultaneous increase in the occurrence of Brownian motion;(ii) reduction of directional ... [truncated at 150 words]
Chiu CL, Aguilar JS, Digman MA, Gratton E.
Measuring actin flow in cell protrusion in 3D.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 47a, 237-PosB23.Cells generate forces through the cytoskeleton that are transmitted to the extracellular matrix via cell adhesion in order to adhere and migrate. Though cellular force generation has been studied in detail in 2D environment, less is known about cytoskeleton dynamics of cells embedded in natural 3D matrices.
Fluorescent Speckle Microscopy (FSM) has been used to capture high-resolution images of actin turnover dynamics within living cells in 2D. However, this method is not applicable when cells are in 3D environment due to lower resolution and signal level.
In this work we developed a new method to capture actin flow in 3D with high spatial-temporal resolution. Modulation tracking and correlation spectroscopy techniques were combined to show the directional flow of actin in 3D live cell. MDA-MB 231 cells with actin-GFP expression were cultured in type I collagen. The laser bin was oscillating back and forth at nearby two cross sections on cell protrusion, while ... [truncated at 150 words]
Cardarelli F, Lanzanò L, Gratton E.
Molecular springs in the nuclear pore complex of live cells.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 526a, 2678-PosB448.Nuclear pore complexes (NPCs) are the gateways for nucleocytoplasmic exchange. Measurements of molecular transport through NPCs may provide valuable information to unravel the mechanism of communication between the nucleus and the cytoplasm. Unfortunately, because single molecules undergo very rapid transport, it is challenging to follow their motion in live cells. We set out to address the nanomechanical basis of pore function in intact cells by a combination of fluorescence correlation spectroscopy (FCS) and real-time tracking of the center of mass of single NPCs. We find the dynamics of the nucleoporin Nup153 to be regulated at the nanoscopic level so as to produce rapid, discrete exchange between two separate positions within the NPC. By means of the pair correlation function (pCF) analysis we are able to separate the two compnonents of Nup153 exchange: a fast collapse into compact molecular conformations (cytoplasm-to-nuclues) and a slightly slower release into extended conformations (nucleus-to-cytoplasm). We ... [truncated at 150 words]
Wright B, Andrews LM, Markham J, Jones MR, Stringari C, Digman MA, Gratton E.
The spatial mapping of the metabolic cofactor NADH within live progenitor stem cells.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 576a, 2927-PosB697.NADH is a naturally occurring bi-product and regulatory metabolite associated with cellular respiration. The quantification using the difference lifetime of autofluorescence of free and bound NADH has the potential to enhance the understanding of a range of cellular processes including apoptosis, cancer pathology and enzyme kinetics. Fluorescence lifetime imaging microscopy (FLIM) enables not only examination of the spatial location of the cofactor within live cells but also of its state.
Here we describe the use of phasor FLIM to spatially map the fluorescence lifetimes of NADH in both free and bound form within live undifferentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. The phasor representation enables the possibility for a direct comparison of either optical sections (i.e. different focal planes) of one cell or multiple cells to enable a global analysis.
A comparison of myoblast cells induced ... [truncated at 150 words]
Arnesano C, Santoro Y, Gratton E.
Digital parallel acquisition in frequency domain for the characterization of tissue spatial heterogeneities.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 591a, 3009-PosB779.Near-infrared (NIR) (650-1000 nm) optical properties of turbid media can be accurately quantified noninvasively using methods based on diffuse reflectance or transmittance, such as frequency domain photon migration (FDPM). For instance, Diffuse Optical Spectroscopy (DOS) is a noninvasive technique that is commonly used to provide biochemical information on hemoglobin, bulk lipids and water concentration by NIR tissue absorption and scattering. DOS does not require exogenous contrast, and rapidly provides quantitative, functional information about tumor biochemical composition. Conventional FDPM techniques are based on white-light steady-state (SS) measurements and the acquisition of frequency-domain (FD) data at several wavelengths using laser diodes, to measure broadband NIR scatter-corrected absorption spectra of turbid media. These techniques are limited by the number of wavelength points used to obtain the FD data. We developed a new method to improve the acquisition of optical parameters of the examined tissues, based on digital parallel acquisition in FD. With our ... [truncated at 150 words]
Stringari C, Edwards R, Watermen M, Pate K, Donovan PJ, Gratton E.
In vivo metabolic mapping of stem cells and differentiated progeny in small intestine and colon crypts by phasor fluorescence lifetime microscopy.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 618a, 3138-Plat.We have performed label-free Phasor Fluorescence lifetime microscopy (FLIM) to reconstruct the three dimensional metabolic signature of small intestine and colon tissue in vivo. Our method provides a label-free identification and metabolic mapping of stem cells during differentiation. Freshly excised tissues are imaged with two photon microscopy and FLIM within two hours. Lgr5-GFP mice are used to mark the Lgr5+ stem cell population at the base of small intestine (SI) and colon crypts. Using Phasor FLIM analysis of live tissue excited at 880nm and 740nm, we identify and map the concentration of different intrinsic metabolic fluorophores and extracellular matrix elements such as NADH, FAD, and collagen. We observe that different compartments of the tissue are defined by unique Phasor FLIM signatures. We can distinguish collagen fibers at the base of the crypts, the lamina propria, the vascular network and the epithelium. The FLIM signature at the base of the crypt ... [truncated at 150 words]
Golfetto O, Hinde E, Gratton E.
Laurdan fluorescence lifetime as a biosensor to detect differences and changes in membrane of living cells.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 85a, 431-PosB217.The cellular membrane directs and influences cell function through its structural and dynamical properties. It is generally recognized that the presence of cholesterol and lipid rafts in the phospholipid bilayers not only determines the physical rigidity of the membrane but also modulates the membrane fluidity and the capability for the cell to exchange lipid molecules. These are two fundamental factors which can explain the different ability of cells to move as well as reproduce and therefore play an important role in pathogenesis.
The lipophilic probe Laurdan has been used extensively to study synthetic and natural membranes since Laurdan is sensitive to membrane packing. The emission spectrum of Laurdan is shifted toward blue in the ordered lipid phase of the membrane (more rigid) and toward the green in the disordered lipid phase, the shift can be quantified by calculating the generalized polarization (GP).
Here we explore the fluorescence lifetime of Laurdan at different ... [truncated at 150 words]
Digman MA, Suetsugu A, Sabatini F, Hoffman RM, Gratton E.
Fluorescence lifetime imaging microcopy of extravasating cancer cells in the mouse microenvironment.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 191a, 961-PosB747.Cell migration is fundamental for cancer metastasis, wound healing, stem cell differentiation and development. Here we explore the tumor microenvironment of MDA-MB-231 breast cancer cells using the phasor approach to FLIM in order to map the fluorescence lifetime of autofluorescencent intrinsic bio-chemical species in living tissues. We compared aggressive cancer cells with host mouse cells as they extravasated from the blood vessel wall into surrounding tissue. To visualize the tumor cells we engineered MDA-MB-231 breast cancer cells to express paxillin-GFP, a focal adhesion protein involved in cell migration. To image the response of cancer cells and the surrounding tissue during extravasation, tumor cells were injected in the blood stream of nude mice and imaged using 2-photon excitation to excite EGFP at 900 nm and at 760nm to detect autofluorescence in the microenvironment. A skin flap was opened to visualize blood vessels and recognize the position of colonies of the ... [truncated at 150 words]
-
Inclined single plane illumination microscopy (iSPIM).
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 192a, 964-PosB750.Current need in biomedical sciences requests live imaging with higher spatial and temporal resolution in the 3D environment. Furthermore, live sample acquisition increases the technical challenges required for 3D imaging. Ideal features of a system for this purpose include high imaging speed, high contrast ratio, low photo-bleaching and photo-toxicity, good resolution in a 3D context and mosaic acquisition for large samples.
Our work presents the inclined illumination implementation of Single Plane Illumination Microscopy, introduced by Stelzer et al (2004). SPIM has been gaining importance in the biomedical field over the years, with its load of technical challenges but has been limited by construction and sample preparation. Based on the same principle introduced by Stelzer, but with a configuration similar to Total Internal Reflection Fluorescence basis, Dunsby (2008) proved the possibility to build an Oblique Single Plane Illumination Microscope (OSPM), also known as Oblique Light Sheet Microscope on a completely lab-made system ... [truncated at 150 words]
Hinde E, Digman MA, Welch C, Hahn KM, Gratton E.
Millisecond spatiotemporal dynamics of FRET biosensors by the pair correlation function and the phasor approach to FLIM.
56th Annual Meeting of the Biophysical Society. San Diego, California. February 25-29, 2012.
Biophys J. 2012; 102(3, Suppl 1): 198a-199a, 997-PosB783.We multiplex the phasor approach to biosensor FRET detection by fluorescence lifetime imaging microscopy (FLIM) with pair correlation function (pCF) analysis along a line scan acquisition, to measure the spatiotemporal dynamics of Rac1 and RhoA activation at the front and back of the cell upon growth factor stimulation. We recently demonstrated the phasor approach to biosensor FRET detection by FLIM as a method that is robust towards biosensor design (single and dual chain) as well as the fluorescence artifacts inherent to the cellular environment. Using a frame mode acquisition we were able to map the spatial localization and quantify the fractional contribution of the free and bound state of a dual chain biosensor or the low and high FRET species of a single chain biosensor in each pixel of an image. To increase temporal resolution we find that line acquisition of FLIM data increases the total pixel integration and allows ... [truncated at 150 words]
Giral H, Caldas Y, Lanzanò L, Gratton E, Levi M.
NHERF-1 modulates intestinal NaPi transporter NaPi-2b expression in apical microvilli.
American Society for Cell Biology (ASCB) Annual Meeting. Denver, Colorado. December 3-7, 2011.
The regulation of phosphate (Pi) homeostasis is maintained by the coordinated function of the renal and intestinal phosphate transporters. Several PDZ (PSD-95/discs large/ZO-1 homologous) domain proteins, including NHERF-1, PDZK1, ShanK2, and PIST play an important role in the regulation of the renal sodium-phosphate (NaPi) co-transporters (NaPi-2a and NaPi-2c). The main mediator of intestinal sodium dependent transcellular Pi transport, NaPi-2b, also contains a PDZ-binding motif consensus in the C-terminal region. However, interactions of the transporter NaPi-2b with PDZ proteins have been not described and their potential role in regulation of the intestinal transporter is not known. For this purpose we performed studies with knock-out (KO) mice models and cell culture to determine a potential role for NHERF-1 and PDZK1 in the regulation of NaPi-2b.
To study the putative interaction between NaPi-2b and PDZ proteins we determined the Forster Resonance Energy Transfer (FRET) by using Fluorescence Lifetime Imaging Microscopy (FLIM). OK cells, an ... [truncated at 150 words]
Andrews LM, Jones MR, Hinde E, Digman MA, Gratton E.
Unlocking the intranuclear dynamics of RNA: an image derivative approach.
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
Despite technological advances, the dynamics of RNA species within the nucleus and cytoplasm are not widely characterised. We have developed a model to analyse the spatial and temporal behaviour of RNA in live cells based on in situ staining of RNA, Raster Image Correlation Spectroscopy (RICS) to map diffusion and trajectory as well as an image derivative analysis to identify regions of transcriptional barriers. For this investigation, Chinese Hamster Ovary K1 cells (CHO-K1) were transfected with eGFP. Selected cell samples were then dual labelled with the fluorescent probe Pyronin Y to aid in the visualisation of RNA. Image data was captured with a Zeiss LSM 710. A RICS analysis assisted in the production of correlations which were algorithmically fit to determine the diffusive properties of each analysed region of interest within the cells. The eGFP alone was identified to diffuse between 3.85 - 8.71 ... [truncated at 150 words]
Cardarelli F, Lanzanò L, Gratton E.
Tracking nuclear pore complexes in live cells: single molecule transport and beyond.
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
Nuclear pore complexes (NPCs) mediate the exchange of macromolecules between the nucleus and the cytoplasm. No methods proposed thus far, however, have the capability to measure the transport of single molecules through single pores in intact cells. Hence fundamental questions remain as to how molecules physically perform facilitated transport and which is the nanomechanical basis of their selective gating through the NPC in live cells.
To address these issues we combine fluorescence correlation spectroscopy (FCS) to real-time tracking of the center of mass of single NPCs to study the nanomechanical basis of nucleocytoplasmic transport in live cells. The center of mass tracking allow us to create an “Einstein trap” in which the thermal motion of the entire pore is compensated so that we can observe the shuttling of single molecules in the reference frame of the pore. Using this experimental protocol we are able to demonstrate that the transport of Karyopherin-ß1 ... [truncated at 150 words]
Celli A, Sánchez SA, Gratton E, Mauro T.
Detection of Calcium gradients in live ex vivo human epidermis using phasor analysis of two-photon excitation Fluorescence Lifetime Imaging (FLIM).
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
Ionic gradients regulate the function and the structure of many tissues and organs. However, traditional techniques used to measure ionic concentrations in tissue (such as Proton induced X-ray Emission or ion capture cytochemistry) are often highly invasive (requiring fixation and dehydration of the specimen) and offer poor spatial resolution. In skin, Ca2+ gradients have been shown to regulate epidermal barrier function, homeostasis and repair after perturbation, but a clear understanding of the mechanisms regulating such gradients is still lacking. We applied the phasor analysis approach to two-photon excitation FLIM measurements of Calcium Green 5N to quantify and localize Ca2+ pools in human epidermis. The high spatial resolution offered by this method and the data analysis performed allowed us to measure intracellular average calcium concentrations directly in tissue and detect a previously unknown heterogeneous distribution of Ca2+ concentrations in the proliferative epidermal basal layer. More importantly, this approach revealed that the ... [truncated at 150 words]
Digman MA, Aguilar JS, Gratton E, Suetsugu A, Hoffman RM.
Fluorescence correlation spectroscopy in 3D and in live tissue.
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
Recent developments in fluctuation correlation spectroscopy using laser based imaging have had a large impact in obtaining quantitative spatio-temporal information of protein dynamics at the level of single cells. The study of cells in tissues requires additional methods to penetrate in tissues and for compensation of rhythmic movements in live animals. The spatiotemporal fluctuation approach is in principle non-invasive. It can capture complex biological functions in real time, give insights into microscopic single cellular states and if done in whole organisms, maintain crucial biological network intact without altering cellular fate. We have developed new nano-imaging method which is based on steering a two-photon laser beam around cellular protrusions to look at protein dynamics in cells during extravasation. We are using a mouse model in which we inject cancer cells in the venous blood stream. The cancer cells (MB MDA231) have a cytoplasmic (Paxillin-EGFP) and/or a nuclear marker (H2B-mCherry). ... [truncated at 150 words]
-
The gold enhancement nano-imaging method: a 3D super-resolution technique for imaging of filamentous actin by directed motion of gold nanoparticles.
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
More than 100 years ago, Ernst Abbe established that diffraction of light prevents optical-based microscopes from having spatial resolution beyond a value comparable to the illumination wavelength. However, since the mid-90s, several super-resolution techniques have emerged. Stimulated Emission Depletion (STED), Saturated Structured-Illumination Microscopy (SSIM), Photo-Activated Localization Microscopy (PALM) and Stochastic Optical Reconstruction Microscopy (STORM) are some of the imaging techniques now available. In this contribution, I will present a new optical super-resolution method based on a nanoparticle (NP) tracking/pushing principle. Although the image of a single NP is diffraction-limited, the precision of determining its position scales with the inverse of the square root of the number of collected photons. Using the gold enhancement nano-imaging (GENI) method, we can push single gold NPs along biological fibers by using a two-photon microscope and the localized surface plasmon at the NP. Gold NPs binds to collagen and actin but it can gently slide ... [truncated at 150 words]
-
Following single molecules in cells and tissues by correlation spectroscopy.
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
Methods in Fluorescence Correlation Spectroscopy (FCS) allow us to detect and characterize the behavior of single molecules directly in cells and in tissue. However the “tracking” of single molecules is a much more difficult operation. Single particle tracking (SPT) in 2D and in 3D is a well-established method in biophysics. The application of SPT requires bright and isolated particles so that the trace of the particle can be recognize in successive image frames. By combining the sensitivity of FCS to single molecules and methods for spatial cross-correlations we show that we can produce a characteristic signature through an analysis method that allow us to determine specific paths that molecules follow in complex environments such as the cell interior and specifically the nucleus. In this talk we present the statistical basis of the pair correlation function approach and the application of this method to the flow ... [truncated at 150 words]
Hinde E, Digman MA, Welch C, Hahn KM, Gratton E.
Biosensor FRET detection by the phasor approach to fluorescence lifetime imaging microscopy (FLIM).
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
FRET based biosensors report on the concentration of a ligand by switching between a high FRET (HF) and a low FRET (LF) state. In principle this change of state can be quantified using either an intensity based ratiometric method of analysis or by monitoring the change in donor lifetime. In practice this measurement is difficult because changes in the concentration of the LF and HF states are often small, and a significant portion of the biosensors present are not optically active due to photobleaching or protein synthesis malfunction. Furthermore in every pixel of a FRET image we have a different fractional contribution of fluorescence not derived from the biosensor (i.e. cell auto-fluorescence) as well instrumental fluorescence background. Clearly determination of the concentrations of the multiple fluorescent components at any one pixel in a FRET image cannot be accomplished using only two emission wavelengths. It is however possible to determine ... [truncated at 150 words]
Lanzanò L, Digman MA, Giral H, Levi M, Gratton E.
Modulation tracking of single microvilli: imaging protein distribution and dynamics.
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
Brush border microvilli (MV) are specialized cell protrusions that provide an increase in the surface area for the absorption of nutrients. In the kidney proximal tubule, reabsorption of inorganic phosphate (Pi) occurs via the sodium gradient-dependent Pi (NaPi) cotransport proteins localized in the MV membrane. The proper localization and regulation of the NaPi transporters on the MV are related to a set of specific protein-protein interactions. These include interaction with the PDZ proteins, a family of proteins generally acting as scaffolders of subcellular structures, and mediating the interaction with the actin-based MV cytoskeleton.
MV are mobile structures which are microns long with a lateral size of the order of 100nm, and can be difficult to study with conventional imaging. We image MV in live cultured proximal tubule cells using the Modulation Tracking (MT) method that, instead of scanning following a predetermined pattern, uses a feedback approach to adapt the scan to ... [truncated at 150 words]
Stringari C, Donovan P, Gratton E.
In vivo metabolic mapping of stem cells and cancer cells by phasor FLIM.
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
Here we present a novel label-free method for deriving metabolic maps of cells and living tissues in vivo. We use the phasor approach to fluorescence lifetime imaging and intrinsic biochemical fluorescence biomarkers such as NADH, flavins, retinoids and porphyrin. Metabolic signatures of living tissues are obtained by calculating the phasor fingerprint of cells and by mapping the relative concentration of FAD, free and bound NADH. Phasor fingerprints of embryonic and adult stem cells cluster according to their differentiation state, both in vitro and in a living tissue such as the C. Elegans germ line. This method can detect small changes in metabolic signatures of colon cancer cells with different phenotypes and identify highly metabolically active cells that correspond to the stem cells at the base of living crypts from colon and small intestine. Phasor FLIM provides a label-free, fit-free and sensitive method to identify different metabolic states of cells and ... [truncated at 150 words]
Liao SC, vandeVen MJ, Periasamy A, Gratton E, Barbieri BB.
Digital frequency domain for fast FLIM measurements and phasor analysis: applications to FRET pairs.
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
Fluorescence Lifetime Imaging (FLIM) in combination with a variety of fluorescence techniques is becoming an important tool in cell biology for the study of dynamic interactions of molecular species, for example protein-protein interactions, and for the intracellular mapping of ion concentration and pH imaging. We present FLIM data obtained with a new method called "Digital Frequency Domain" (DFD). It offers superior sensitivity, which results in a factor of four reduction in the time required for image acquisition as compared to the traditional approach using time-correlated single photon counting (TCSPC). This improves capturing images of motile samples, eg. live cells.
We report the measurement of Förster Resonance Energy Transfer (FRET) efficiency by using FLIM on mouse pituitary GHFT1 cells labeled with Cerulean (donor) and Venus (acceptor) fluorescent proteins. Several genetic constructs were developed tethering these fluorescent proteins via different amino acid linkers. A standard ISS FastFLIM unit was ... [truncated at 150 words]
-
The SimFCS program for data acquisition and analysis.
8th International Weber Symposium. Kauai, Hawaii. June 12-17, 2011.
SimFCS is a comprehensive platform for data fluorescence data analysis, simulation and data acquisition. SimFCS contains drivers for data acquisition, algorithms for data reduction and methods for data display and export. Different technologies work together to provide new kinds of information, which is not available using each one independently. The challenge is both technical: combine technologies to achieve new functions; and scientific: plan experiments that could make use of the combined new capabilities. By designing a software platform that is agile and can access information from different modalities of data acquisition, we can join different conceptual measurements in the same platform. For example, while performing tracking experiments, we can also acquire and analyze the data for the diffusion of molecules in the proximity of the particle being tracked, or detect binding. As another example, since data are collected and analyzed by the same software, we can rapidly manipulate the data ... [truncated at 150 words]
Brown CM, Dalal RB, Digman MA, Horwitz AR, Gratton E.
Measuring fast dynamics from laser scanning microscope images: raster image correlation spectroscopy (RICS).
38th Annual Meeting of the Microscopical Society of Canada. June 7-10, 2011. Ottawa, Canada.
Raster image correlation spectroscopy (RICS) is a novel technique for measuring molecular dynamics and concentrations from fluorescence confocal images [1]. Basically spatial correlation functions can be calculated for images or regions of interest (ROIs) within images from the spatial intensity fluctuations. When the characteristics of a confocal laser scanning microscope such as pixel dwell time, interline time and pixel size are known these movements of the laser beam can be taken into account when fitting the spatial autocorrelation function (ACF) and the decay of the correlation function due to molecular movements of fluorescent dyes molecules, proteins or lipids can be determined by fitting the spatial ACF. In addition, the amplitude of the spatial ACF can be used to determine concentrations of fluorescently labelled molecules. Simulations show that the shape of the spatial ACF truly reflects the dynamics of the molecular motions being measured and that RICS can be used to ... [truncated at 150 words]
Digman M, Aguilar JA, Gratton E, Suetsugu A, Hoffman RM.
Real-time imaging of 3-dimensional cancer cell movement in tissues.
AACR 102nd Annual Meeting 2011. April 2-6, 2011. Orlando, FL.
Our knowledge of how cells move in 3D in tissues is limited due to the lack of imaging methods that can produce 3D images fast enough and with sufficient resolution. Cancer cells migrate in 3D by forming adhesion points at the end of very long cellular protrusions. These protrusions are very thin and it is difficult to visualize adhesions along the protrusion surface. Conventional 3D stack reconstruction has relatively low resolution unless it is done using many frames. This results in a very slow acquisition in 3D confocal microscopy. Faster methods of 3D data acquisition (spinning disk microscopy) cannot be easily implemented since there is significant amount of scatter in tissues. A major obstacle in imaging adhesions is to find and track them so that they will not go out of focus. We are developing a new method which is based on orbiting imaging around cellular protrusions to visualize protein ... [truncated at 150 words]
Suetsugu A, Digman M, Gratton E, Moriwaki H, Saji S, Bouvet M, Hoffman RM.
Real-time imaging of Paxillin-GFP expression in breast cancer cells in vitro and in vivo.
AACR 102nd Annual Meeting 2011. April 2-6, 2011. Orlando, FL.
Paxillin is involved in the assembly of focal adhesions. In this study, we describe real-time imaging of paxillin-GFP expression in breast cancer cells (MDA-MB-231) in vitro and in vivo. In vitro, round cancer cells had greater paxilin movement than stretched cancer cells as seen by fluorescence imaging. When paxillin-GFP breast cancer cell (1×106) were injected in the epigastric cranials vein, round cancer cells had GFP-expressing protrusions. Many paxillin-GFP-expressing breast cancer cells stretched. The cancer cells extravasated and subsequently grew around the outer surface of the blood vessel after one week. Migration of single cells in vivo was observed by fluorescence microscopy. Two weeks after cell injection, paxillin-GFP breast cancer cells migrated along the vessel wall. Most of paxillin-GFP cancer cells were stretched and were not mobile. With anti-VEGF treatment, paxillin was observed in round structures rather than stretched structures and paxillin movement within the cell were arrested. The results show ... [truncated at 150 words]
Digman MA, Aguilar JS, Gratton E, Suetsugu A, Hoffman RM.
Two-photon imaging of cancer cell extravasation in live mice.
AACR 102nd Annual Meeting 2011. April 2-6, 2011. Orlando, FL.
MDA-MB-231 breast cancer cells were engineered to express cytoplasmic paxillin-GFP and nuclear H2B-mCherry. In order to image extravasation, the cancer cells were injected in the blood stream of nude mice. Using 2-photon excitation microscopy we can simultaneously excite the two probes and also visualize the autofluorescence of tissues. A skin flap was opened to visualize blood vessels and recognize the position of the cancer cells. Two-photon imaging showed that after an initial phase in which the cells are non-adherent, some cells spread on the internal surface of the capillaries. Days later some cells started to appear on the external side of the capillary. The extravasated cells extend very long protrusions into the tissue. The goal was to determine if at the end of the long protrusion, if it is possible to observe the formation of focal adhesions by imaging paxillin-GFP. Preliminary results show that when cells start to adhere to ... [truncated at 150 words]
Hinde E, Digman MA, Hahn K, Gratton E.
FRET based biosensor: a comparative study of the phasor approach to FLIM with ratiometric analysis in live cells.
International Bunsen Discussion Meeting - FRET in Life Sciences. Göttingen, Germany. March 27-30 2011.
Förster FRET 2011 Book of Abstracts. 2011; : 32.Several genetically encoded biosensors report on the concentration of a ligand by switching between a high FRET (HF) and a low FRET (LF) state. In principle this change of state can be monitored either using ratiometric methods or by the change of the lifetime of the donor. Often the changes in the concentration between the two forms of the biosensors are small and there are artifacts because not all biosensor molecules are active. Therefore there is interest in developing methods to accurately determine in vivo the relative amount of the HF and LF species as a function of time and space. A technical challenge of using FRET biosensors is the exact determination of the high and low FRET states in cells (calibration). Another problem is the recognition of the several sources of fluorescent artifact that are present in live cells and that will make the determination of the relative concentration ... [truncated at 150 words]
Stringari C, Cinquin A, Cinquin O, Donovan P, Gratton E.
In vivo identification of changes in metabolic state as stem cells differentiate, by phasor analysis of fluorescence lifetime imaging.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 183a, 1008-Plat.In stem cell research there is a high demand of techniques to investigate self-renewal and differentiation mechanisms and to develop stem-cell-based therapies for regenerative medicine. Here we develop a label-free method to identify and classify stem cells and differentiating cells according to their metabolic state. We use the phasor approach to fluorescence lifetime imaging and intrinsic biochemical fluorescence biomarkers such as NADH, flavins, retinoids and porphyrin. The organ studied is the C. elegans germ line, expressing a histone-GFP fusion protein that allows us to identify the differentiation state of the germ cells. We calculate the average phasor value of the intrinsic fluorescence of germ cells and we plot the cell phasor fingerprints in a scatter diagram. Cell phasor fingerprints cluster according to their differentiation state. Different metabolic fingerprint of cells reflect changes in binding sites of NADH with different coenzymes during differentiation. The phasor approach to lifetime imaging provides a ... [truncated at 150 words]
Chiu CL, Aguilar JS, Gratton E, Digman MA.
Focal adhesion formation on cell protrusions in 3D shown by modulation tracking.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 226a, 1236-Pos.Focal adhesion formation of cells in the 2D environment has been studied in great details. However, in the natural 3D environment, focal adhesion proteins may have distinct protein dynamics, and cells may employ different mechanism for cell movement. Instead of forming filopodial or lamellipodial protrusions as seen on conventional culture dish, when embedded in collagen gel, MDA-MB-231 cells produce long protrusions that grab extra cellular matrix and guide the movement. Here we applied an optical imaging system called modulation tracking (MT) that enables the study of protein dynamics in 3D and shows the nanometer-scale resolution feature that cannot be detected using conventional confocal microscope. MT is done by moving a laser spot along the protrusion and detecting the fluorescence as the spot approaches the surface. Both fluorescent and second harmonic generation signals from collagen are simultaneously recorded. To study the cell protrusion in 3D, MDA-MB-231 cells were transfected with fluorescent ... [truncated at 150 words]
Cardarelli F, Lanzanò L, Gratton E.
Tracking single nuclear pore complexes in live cells: importin ß transport and beyond.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 249a, 1367-Pos.The molecular details of transport through nuclear pore complexes (NPCs) have been well characterized, including the key role of Importin Beta (ImpB) receptor. However, the overall system behavior in intact cells is difficult to analyze because of its highly dynamic nature. Contrary to the common single particle tracking (SPT) approach that tracks an isolated particle as it moves through the pore, we set out to track the center of mass of the entire single NPC. While we track the pore, single molecules pass through it, and their location and dynamics are analyzed by fluorescence correlation spectroscopy (FCS). By this unconventional approach we find that ImpB transport is regulated so as to produce a characteristic bump in the autocorrelation function at the NPC. This regulation is spatially restricted to the pore, dependent on ImpB properties, pore structure, and metabolic energy. Combined to simulations our results suggest that ImpB movement within the ... [truncated at 150 words]
Sánchez SA, Vazquez R, Maté S, Bakás L, Gratton E, Herlax V.
Effects of alpha hemolysin from E.Coli on erythrocytes from different species.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 339a, 1845-Pos.Alpha-hemolysin (HlyA)is one of the key virulence factors released by E. coli strains. This toxin causes lysis of various mammalian cells, including erythrocytes of different animal species.
The hemolytic activity of the toxin on rabbit and sheep erythrocytes was determined, showing that rabbit is the most susceptible specie. Calcium concentration inside the erythrocytes while incubated with sublytic concentrations of HlyA was monitored using two-photon fluorescence Lifetime Imaging (FLIM), the calcium indicator Calcium Green 1 and the phasor analysis method. HlyA induces an increase in calcium concentration in both erythrocytes, but the increment in rabbit is 4 times higher and faster than in sheep. Two-photon Laurdan Generalized Polarization (GP) was used to determine the fluidity of the membrane (measured as the membrane water content) in the presence and absence of HlyA in live erythrocytes. The GP value for the sheep erythrocytes membranes was higher that the ones from rabbit and after incubation ... [truncated at 150 words]
-
A 3D nano-imaging method using nanoparticles as optical antennas.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 468a, 2536-Pos.Under resonant illumination, metallic nanoparticles (NPs) exhibit a huge enhancement and confinement of the electromagnetic field that make them unique for several high spatial resolution scientific and technological applications such as molecular biology, nanolithography and DNA sequencing. We describe a method to move in a controlled manner, in 3D, and with nanometer resolution, gold NPs along a collagen fiber. As the NP moves along the fiber, it spectroscopically interacts with the intrinsic fluorophores present in the fiber enhancing the weak fluorescence of the collagen and mapping their 3D structure with high spatial and temporal resolution. We distinguish between conventional 3D Orbital Tracking, and the nano-imaging tracking technique introduced in this work. In 3D Orbital Tracking, the laser performs an orbit which follows the moving NP by continuously changing its position according to the NP movement. On the contrary, in this work, we move the NP by slowly moving the orbit. ... [truncated at 150 words]
Lanzanò L, Fwu P, Digman MA, Giral H, Levi M, Gratton E.
Nanometer scale fluorescence imaging of single microvilli.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 470a, 2545-Pos.Brush border microvilli (MV) are highly specialized finger-like protrusions emanating from the apical surfaces of certain types of epithelial cells. In the kidney proximal tubule they provide an increase in the surface area for the absorption of nutrients. Renal tubular reabsorption of inorganic phosphate (Pi) occurs via the sodium gradient-dependent Pi (NaPi) cotransport proteins localized in the MV membrane. The localization and the regulation of the NaPi transporters on the MV are related to a set of specific protein-protein interactions. These include interaction with the PDZ proteins, a family of proteins generally acting as scaffolders of subcellular structures, and mediating the interaction with the actin-based MV cytoskeleton.
Here we use the Modulation Tracking (MT) optical method to image small protrusions in live cells and apply it to the study of MV in live cultured proximal tubule cells. MV are several microns long with a lateral size of the order of 100nm, ... [truncated at 150 words]
Caracciolo G, Pozzi D, Marchini C, Montani M, Amici A, Digman MA, Sánchez SA, Gratton E, Capriotti AL, Laganà A.
Novel mechanisms of cell uptake in lipid-mediated gene delivery.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 601a, 3247-Pos.The mechanism of cell uptake in lipid mediated gene delivery was investigated in NIH3T3 and CHO cell lines. We show that different endocytic pathways are activated by shape coupling between lipoplex and membrane lipids. Our results suggest that tailoring the lipoplex lipid composition to the patchwork-like plasma membrane profile could be a successful machinery of coordinating the endocytic pathway activities and the subsequent intracellular processing. Transfection experiments performed at 4°C, when endocytosis does not take place, show that a novel class of highly efficient multicomponent lipoplexes enter cells by a temperature-independent fusion-like mechanism. In vivo, plasma proteins bind to lipoplex surface and create a rich ‘protein corona’ that is recognized by cells and other biological structures. The ‘protein corona’ associated to lipoplexes after interaction with human plasma was found to be much richer in basic immunoglobulins gamma proteins (Ig-Gs) than that of pure lipid vesicles in the absence of DNA. ... [truncated at 150 words]
Digman MA, Chiu CL, Aguilar JS, Gratton E.
Following actin fibers in 3D during cell migration in collagen matrices.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 616a-617a, 3337-Pos.Actin polymerization is a major mechanism for the production of the force necessary for cell migration in 2D. The polymerization of actin and its retrograde motion at the leading edge of cell moving in 2D has been studied in great detail as well as the interaction of actin with focal adhesions. When cells grow in 3D collagen matrices, the extending lamellipodial protrusion is more difficult to visualize and it is likely not relavent for the movement of the cell over large distances. We use the modulation tracking 3D method to accurately image the cell protrusion. This method is capable of producing detailed images of 3D structures at the nanoscale and at the same time measure diffusion and aggregation of molecules in these structures. In 3D, cells produce very long protrusions that presumably grab on the surrounding collagen fibers to propel the rest of the cell body in a specific direction. ... [truncated at 150 words]
Hinde E, Cardarelli F, Digman MA, Gratton E.
The impact of nuclear architecture on EGFP diffusion revealed by pair correlation analysis.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 67a, 351-Pos.The diffusion of molecules within the nucleus is obstructed by the steric constraints imposed by the nuclear environment. The extent to which nuclear architecture directs the diffusive route taken by these molecules is of significant interest. No methods proposed thus far have the capability to measure overall molecular flow in the nucleus of living cells. Here we apply the pair correlation function analysis (pCF) to measure molecular anisotropic diffusion in the interphase and mitotic nucleus of live cells. In the pCF method we cross correlate fluctuations at several distances and locations within the nucleus, enabling us to define migration paths and barriers to diffusion. We use monomeric EGFP as a prototypical inert molecule and measure its flow in and between the different nuclear environments.
For the interphase nucleus we observe two disconnect molecular flows associated with high and low DNA density, which cause the chromatin to behave as a channeled network. ... [truncated at 150 words]
Crosignani V, Dvornikov AS, Gratton E.
Two-photon imaging in turbid media to a few mm depths.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 141a, 761-Pos.We present a new detection method for two-photon fluorescence imaging of turbid media that extends imaging depth to few mm. Compared to conventional two-photon microscopy, the imaging depth enhancement is achieved by use of a more efficient detection of fluorescence, propagating in multiple-scattering media. The imaging depth in turbid media depends on two major factors: the ability of the imaging system to deliver necessary excitation light power to a certain depth to induce two-photon fluorescence and the ability of the detection system to collect and detect fluorescence photons scattered by the media. In two-photon imaging, usually, fluorescence is collected by the same microscope objective that is used for excitation. This optical scheme has its principal limitation in efficiency of fluorescence detection because of the narrow angle and area from which fluorescence photons can be collected. As a result, the maximum depth of tissue imaging that was reached so far by ... [truncated at 150 words]
Gratton E, Digman MA, Lanzanò L.
Reconstruction of nanostructures in cells by the modulation tracking optical method.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 142a, 767-Pos.Resolving biological structures at the nanoscale has been a subject of intense recent research. Essentially two approaches have been proposed, one based on shaping the effective illumination profiles using stimulated emission (STED) and the other using the determination of the center of mass emission by single molecules (PALM and STORM). Both approaches have been shown to produce high quality images with resolution on the order of 20 nm. The STED method is ultimately based on using a raster scan approach to produce an image, which is quite inefficient for imaging objects in 3D and the PALM method is very slow for obtaining 3D structures of the size of microns. We have developed the modulation tracking method which is based on a feedback principle that is capable of producing 3D images with resolution in the 20 nm range. With simulations of 3D objects we show how the images are acquired in ... [truncated at 150 words]
Itano MS, Liu P, Neumann AK, Steinhauer C, Zhang F, Gratton E, Tinnefeld P, Parak WJ, Thompson NL, Jacobson KA.
Superresolution imaging and molecular dynamics of dc-sign in domains on the plasma membrane.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
DC-SIGN, a Ca2+-dependent transmembrane lectin, is found assembled in microdomains on the plasma membranes of dendritic cells. These microdomains bind a large variety of pathogens and facilitate their uptake for subsequent antigen presentation. In this study, DC-SIGN dynamics and distribution in microdomains have been explored with several fluorescence microscopy methods and compared with those for influenza hemagglutinin (HA), which is also found in plasma membrane microdomains. Fluorescence imaging indicated that DC-SIGN microdomains may contain other C-type lectins and that the DC-SIGN cytoplasmic region is not required for microdomain formation. Superresolution Blink Microscopy indicated that DC-SIGN domains are very small (<100 nm in diameter), while HA domains assemble on a large range of length scales (tens of nm to greater than 1 µm). Fluorescence recovery after photobleaching (FRAP), line-scan fluorescence correlation spectroscopy and defined valency quantum dot single particle tracking measurements showed that full-length and cytoplasmically truncated DC-SIGN is essentially immobilized ... [truncated at 150 words]
-
Cerebral oxygenation during anesthesia.
55th Annual Meeting of the Biophysical Society. Baltimore, Maryland. March 5-9, 2011.
Biophys J. 2011; 100(3, Suppl 1): 94a, 507-Pos.Cerebral oxygenation during anesthesia: correlation with blood pressure and cardiac output.
During surgery, the anesthesiologist is tasked with monitoring, manipulating and maintaining the patient’s key physiological parameters, especially of critical organs such as the brain. Generally, cerebral tissue oxygenation is not explicitly tracked, but a measure of Mean Arterial Pressure (MAP) is considered an adequate surrogate marker for sufficient cardiac output to satisfy cerebral oxygen demand. Avoidance of cerebral ischemia is an important consideration. Consequently, we have used a tissue oximeter (Oxipex TS; ISS, Inc.) to non-invasively and quantitatively monitor cerebral oxygenation (bilateral; frontal lobes) during surgery in more than two dozen patients (under IRB approved protocols). If blood pressure declines after inducing anesthesia, then the anesthesiologist pharmacologically intervenes with a pressor such as Ephedrine or Phenylephrine. Phenylephrine acts as a vasoconstrictor, whereas ephedrine stimulates cardiac output. We were intrigued by our preliminary observations that cerebral oxygenation (and oxygenated Hemoglobin concentrations) ... [truncated at 150 words]
-
Laurdan generalized polarization microscopy for the study of cell membrane heterogeneity.
X Encuentro Latinoamericano De Fotoquímica Y Fotobiología (XELAFOT). La Serena, Chile. October 10-14, 2010.
XELAFOT Abstract Book. 2010; : 5, C4.Fluorescence techniques such as fluorescence lifetime or simple intensity measurements were originally developed to be used in a cuvette fluorometer. The implementation of these techniques in the microscope added to the measurements the spatial resolution needed for cellular studies but also forced researchers to change the experimental design and the analytical methods used. One of these techniques, Laurdan Generalized Polarization is presented here.
Laurdan (6-lauroyl,1-2-dimethylamino naphthalene) is a fluorescent dye designed and synthesized in 1979 by Gregorio Weber to study the phenomenon of dipolar relaxation. Polarity changes are detected by shifts in the Laurdan emission spectrum, and the Generalized Polarization function (GP) was defined as a way of measuring the wavelength displacements. Changes in GP values when Laurdan is within a lipid bilayers in either fluid or gel phase, extended the use of the technique to the field of membrane dynamics and protein-lipid interaction. In 1997 GP measurements were done for ... [truncated at 150 words]
Aguilar LF, Cuevas F, Sotomayor CP, Soto-Arriaza M, Sánchez SA.
Effect of Na,K-ATPase reincorporated into GUV models upon segregation domains: study by two photon microscopy.
X Encuentro Latinoamericano De Fotoquímica Y Fotobiología (XELAFOT). La Serena, Chile. October 10-14, 2010.
XELAFOT Abstract Book. 2010; : 40, P3.In the present work, we study the effect of Na,K-ATPase in lipid segregated domain when it is incorporated into giant unilamellar vesicles (GUVs) formed by DPPC:DOPC lipid mixture in absence and presence of different content of cholesterol. The result was obtained measuring GP Laurdan image. The protein reconstitution into GUVs was realized by adapting Cornelius methods. This reconstitution was realized through incubation of protein previously solubilized with lipid:detergent solution at 1:1,2 ratio. After that, detergent was removed by hydrophobic polystyrene polymer (Bio-Beds) when the lipid solution was maintained for 12 hr at 4°C to constant stirrer. GUVs of reconstituted Na+,K+-ATPase into different lipid-cholesterol mixtures to grow the Giant Unilamelar Vesicles using the conventional electroformation method with some modifications to avoid the dehydration of the protein. In the presents work, we used Twophoton microscopy to obtain Laurdan GP imaging to study membrane segregated domains. Laurdan [6-lauroyl-2-(dimethylamino) naphthalene] is used as a ... [truncated at 150 words]
-
Espectroscopia de Correlacion de Campo Cercano: Que es? Por que es importante? Para que sirve? (plenary talk).
95th Annual Meeting of the Argentinean Physical Society. Malargüe, Argentina. September 28-October 1, 2010.
AFA2010 Librode de Resumenes. 2010; : 18.
-
Nanometer-scale optical imaging of collagen fibers using gold nanoparticles.
15th International Symposium on Small Particles and Inorganic Clusters (ISSPIC-XV). Oaxaca City, Mexico. September 19-24, 2010.
ISSPIC-XV Book of Abstracts. 2010; : 126, A-05-07.
Azartash K, Kwan JT, Paugh JR, Nguyen AL, Jester JV, Gratton E.
Preliminary tear film thickness data in humans measured with a novel technique.
Association for Research in Vision and Ophthalmology (ARVO 2010). Fort Lauderdale, Florida. May 2-6, 2010.
Purpose: The purpose of this work was to gather normative and repeatability data in normals and dry eye subjects using a new, non-invasive method to measure the thickness of pre-corneal tear film.
Methods: Human subjects were screened for dry eye using a battery of clinical tests and classified as dry or normal. Tear film thickness of the inferior paracentral corneal area was measured with spatial correlation analysis. The technique is termed Fluctuation Analysis by Spatial Image Correlation (FASIC), wherein a series of images are obtained using laser illumination and a cMOS camera. The spatial correlation image of laser speckle is calculated for every frame. A sinusoidal background due to interference caused by the tear film appears in this spatial correlation image together with other features. We developed a mathematical model to obtain the thickness of the tear film from this sinusoidal background.
Results: Mean tear thickness values (± SD) were 3.08 μm ... [truncated at 150 words]
Giral H, Caldas Y, Lanzanó L, Gratton E, Sutherland E, Blaine J, Okamura K, Verlander J, Levi M.
Role of PDZ proteins in the differential regulation of the proximal tubule NaPi transporters.
Experimental Biology 2010. Anaheim, California. April 24-28, 2010.
FASEB J. 2010; 24(Meeting Abstracts), 699.2.The Na-dependent phosphate (NaPi) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of Pi and maintenance of Pi homeostasis. The transporters show differential regulation under dietary and hormonal stimuli suggesting different regulatory pathways controlling the endocytosis, stability, or functionality of the transporters. The scaffolding NHERF family of PDZ proteins has been involved in the regulation of NaPi-2a and NaPi-2c. We propose that differences in the molecular interaction with these PDZ proteins are related with the differential adaptation of the transporters. We studied the specific interaction of NaPi-2a and NaPi-2c with NHERF-1 and –3 in the OKP cells by FLIM-FRET lifetime measurements. Results showed a direct interaction of NaPi-2a with NHERF-1 (FRET occurrence), while there was no interaction between NaPi-2c and NHERF-1. In contrast NaPi-2c showed significant FRET with NHERF-3 protein. According with these results, adaptation of the NHERF-3 KO mouse to chronic low Pi diets was ... [truncated at 150 words]
Lanzanò L, Fwu P, Giral H, Levi M, Gratton E.
Dynamic imaging of single microvilli in opossum kidney cells.
Experimental Biology 2010. Anaheim, California. April 24-28, 2010.
FASEB J. 2010; 24(Meeting Abstracts), 1002.6.Reabsorption of inorganic phosphate (Pi) in the renal proximal tubule is fundamental to overall Pi homeostasis. Its regulation occurs via the apical brush border membrane (BBM) sodium gradient-dependent Pi (NaPi) cotransport proteins. Distinct families of NaPi cotransporters show differential regulation under dietary and hormonal stimuli, but how this is accomplished at a molecular level is not yet fully understood.
The application of modern fluorescence correlation spectroscopies to the BBM of living cultured Opossum Kidney (OK) cells expressing NaPi cotransporters with different GFP constructs could provide useful information on the dynamics of the proteins, including diffusion, aggregation and interaction with other proteins. However, BBM microvilli show a relatively fast motion that makes the use of fluctuation spectroscopy difficult.
We developed an optical imaging technique called Modulation Tracking (MT) in which we track the center of mass of the microvillus at an arbitrary point along its length via orbital scanning while the laser ... [truncated at 150 words]
Lin S, Vyas KJ, Nguyen QM, Uch S, Michalos A, Gratton E, Srinivasan R.
Changes in physiological and bioenergetic markers associated with stationary meditation.
Society for Acupuncture Research - Translational Research in Acupuncture (SAR 2010). Chapel Hill, North Carolina, USA. March 19-21, 2010.
Common mediation practices can be classified as those carried out in a stationary position (e.g., standing or sitting Qigong meditation) and those involving body movements (e.g., Tai Chi. The aim of this study is to investigate changes in various physiological and bioenergetic markers associated with stationary meditation, and compare them with some of those associated with moving meditation. First, we found that the slow, soft, circular hand-arm movements of Chen Style Single Hand Silk Reeling exercise significantly increased cutaneous blood flow by up to several fold measured as “Flux” with laser Doppler flowmetry at the PC8 acupoint at the center of the palm. This type of change was due to elevation of the “Speed” parameter in the measurement (“Flux” = “Speed” x “Concentration”). This change was accompanied by a ~15% increase in bioenergy measured as biophoton emission from the palm with a single photon counting system. In contrast, when we ... [truncated at 150 words]
-
Nanometer-scale imaging of collagen fibers using gold beads.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 179a, 926-Pos.The 3D spatial position of a particle can be determined by scanning the excitation volume of a 2-photon microscope in a three-dimensional orbit around the particle and by subsequently analyzing the fluorescence intensity profile along the orbit. We track the movement of gold beads moving along collagen fibers by 3D particle tracking method. As the particle moves on the fiber, the particle trajectory maps the substrate with high-resolution (2-20 nm). When the particle moves in close proximity to the collagen, it locally couples and excites to the weak fluorophores on the collagen. This method provides the possibility to characterize the interactions between particle and substrate even further. We can obtain the dynamic structure information of collagen fibers with nanometer resolution in real time. More interestingly, the gold beads move not at random but in specific directions under two photon laser excitation. We were able to move the gold particle very ... [truncated at 150 words]
-
In vivo imaging of single-molecule translocation through nuclear pore complexes by pair correlation functions.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 751a, 3903-Pos.Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoproteins across the double-membrane nuclear envelope. We recently introduced a method based on pair correlation functions (pCF) which measure the time the same molecule takes to migrate from one location to another within the cell 1. The spatial and temporal correlation among two arbitrary points in the cell can provide a map of molecular transport, and also highlight the presence of barriers to diffusion with very high time resolution (in the microsecond scale) and spatial resolution (limited by diffraction).
Here we report the use of this method to directly monitor a model protein substrate undergoing transport through NPCs in living cells, a biological problem in which SPT has given results that cannot be confirmed by traditional FCS measurements because of the lack of spatial resolution. Our substrate is composed by a GFP linked to a functional nuclear localization sequence (NLS) ... [truncated at 150 words]
Ossato G, Digman MA, Lukacsovich T, Marsh JL, Gratton E.
N&B and cross-N&B analysis detect oligomerization of Huntingtin in live cells.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 651a, 3387-Pos.Aggregation of misfolded proteins is a hallmark of several neurodegenerative diseases such as Huntington's disease (HD). HD is caused by a mutation of Huntingtin caused by an elongation of a polyglutamine (polyQ) sequence in the protein. Here we describe the application of the recently developed Number and molecular Brightness method (N&B) to monitor the aggregation process of Huntingtin exon1 (Httex1). N&B measures the molecular brightness of the protein aggregates in the entire cell non-invasively based on the fluctuation dynamics at each pixel of an image. This analysis provides a map of aggregation with pixel resolution.
We observed the behavior of Httex1-97QP-EGFP this is a construct with 97 polyQ repeats corresponding to Juvenile onset of the disease.
We preformed experiments in ST14A cells transfected with Httex1-97QP-EGFP. We establish that the process of nucleation leading to inclusion formation has four phases: i) Initially only monomers are present; ii) Following an increase in protein concentration ... [truncated at 150 words]
Lanzanò L, Fwu PT, Giral H, Levi M, Gratton E.
Dynamic imaging and fluctuation spectroscopy on single microvilli in opossum kidney cells by the modulation tracking method.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 751a, 3905-Pos.Regulation of renal tubular inorganic phosphate (Pi) transport occurs via the proximal tubular apical brush border membrane (BBM) sodium gradient-dependent Pi (NaPi) cotransport proteins. Distinct families of NaPi cotransporters show differential regulation under dietary and hormonal stimuli, but the way this is accomplished, for instance through localization in distinct BBM micro- or nano-domains and/or preferential interaction with different PDZ proteins, is not yet understood.
Crucial information could come from the application of single molecule fluctuation correlation spectroscopies on the BBM of living cultured Opossum Kidney (OK) cells expressing NaPi co-transporters with different GFP constructs. The BBM is composed of many microvilli, several micron long structures with a diameter of about 100nm. The microvilli show a relatively fast motion (in the seconds time scale) that makes the use of fluctuation spectroscopy difficult.
None of the current nano-resolution optical methods seems capable of measuring the clustering dynamics of proteins on the surface of rapidly ... [truncated at 150 words]
Fwu PT, Lanzanò L, Gratton E.
Modulation particle tracking.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 179a, 929-Pos.In this study, we present a novel optical imaging method that makes use of high precision particle tracking of fluorescent particles to obtain images of nanometer size structures in live cells. Particle tracking not only provides the trajectory of the center of mass but also the particle orientation and size can now be observed, in vivo and real time with the nanometer resolution. This method helps in further understanding of the dynamics of the small particles in biological systems, which was hard to achieve by the current optical techniques. The method is based in rapidly modulating the position of the laser beam around small structures on the order of 100nm in size. When the laser spot oscillates in the direction toward the particle surface, the fluorescence of the particle is modulated. The modulation, which is the ratio of the alternating part to the average fluorescence intensity, is a function of ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
Scanning laser image correlation (SLIC) measurements in Zebra Fish larvae.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 580a-581a, 3003-Pos.Scanning Laser Image Correlation (SLIC) is a technique to measure the flow of small particles and to observe local flow patterns over an area. SLIC can be applied to situations including blood cells flowing through blood vessels or tracer particles flowing through microfluidic channels. The main advantage of SLIC over other flow measurement techniques is that SLIC can be scaled to measure flow in areas ranging from microns to centimeters wide. To accomplish this, an image is first acquired through laser scanning and analyzed with number and brightness analysis (N&B) to identify regions of flow. This is used as a guide to manually (or potentially automatically) select a pattern within the image, such as a line along the center of a channel, that is then scanned repeatedly with the laser beam. Since the entire image is not scanned in each measurement SLIC measurements can be obtained quickly and efficiently. The ... [truncated at 150 words]
-
Fluctuation analysis with the spinning disk confocal microscope.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 182a, 942-Pos.Analysis of the fluctuations in time and space of confocal images has the potential to provide information about molecular diffusion and molecular interactions directly in live cells. Fluctuation image analysis has been commonly done in the laser scanning microscope. In the slow regime, when the fluctuations are slower than the frame rate, the time correlation between the same pixel in different frames of an image stack provides all the information about diffusion and brightness. In the fast regime, for example for molecules diffusing in the cytoplasm, the frame rate is too slow to follow the fluctuations due to diffusion. In the raster scan confocal microscope, these fluctuations are detectable because of the correlation of the intensity with the next pixel in the same line or in the next line. In fluctuation spectroscopy an important parameters is the sampling time that must be shorter that the time of the decay of ... [truncated at 150 words]
Stringari C, Digman MA, Donovan P, Gratton E.
Multiple components mapping of live tissue by phasor analysis of fluorescence lifetime imaging.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 214a, 1116-Plat.In fluorescence lifetime microscopy (FLIM) of live tissues a major issue is the assignment of autofluorescence to specific molecular components and their interactions within the physiological context. Here we use the phasor approach to fluorescence lifetime imaging to analyze complex decays in a live tissue. The tissues used were seminiferous tubules from the testes of wild type mice or mice expressing GFP from an Oct4 transgene. Lifetime images were acquired in the time domain and analytically transformed in the phasor representation. By examination of the clustering of the phasors we identified different molecular components: auto fluorescence, GFP, collagen and retinol. Each chemical species was identified and categorized by its specific location in the phasor plot. This phasor fingerprint reduces the importance of knowing
the exact lifetime distribution of the fluorophores and emphasizes the contribution of the species to the signal. To better identify specific tissue components we also used spectral imaging ... [truncated at 150 words]
Xu S, Cerussi A, Gratton E.
Self-referencing differential spectroscopy analysis in breast translational research.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 749a, 3895-Pos.A self-referencing differential spectroscopy analysis approach has been developed for broadband near-infrared (NIR 650-1000 nm) absorption spectra to reveal intrinsic optical breast cancer biomarkers. Through the application of this method that accounts for inter-patient variability using the normal tissue as an internal control, we have characterized the metabolic differences between malignant and normal tissues that result from subtle alterations in molecular disposition.
From a pilot study of 15 cancer patients performed in 2007, absorption signatures, not arising from the individual abundance in the four major chromophores (lipid, oxy-hemoglobin, deoxy-hemoglobin and water), have been demonstrated to successfully differentiate the normal and malignant tissues. Based on the data acquired from a NIR Diffuse Optical Spectroscopy Imaging instrument, specific spectral signatures containing specific NIR absorption bands are located in regions at about 760, 930, and 980 nm indicative of lipid biomarkers or water in abnormal state. The shape of the fingerprint spectra, namely specific ... [truncated at 150 words]
Sánchez SA, Bakás L, Gratton E, Herlax V.
Alpha hemolysin induces an increase of erythrocytes calcium: a fluorescence lifetime imaging microscopy study.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 578a, 2991-Pos.α--hemolysin (HlyA) from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin), a series of proteins that share genetic and structural features. HlyA is an important virulence factor in E. coli extraintestinal infections, such as meningitis, septicemia and urinary infections. High concentrations of the toxin causes the lysis of several cells as erythrocytes, granulocytes, monocytes, endothelial cells and renal epithelial of different species and low concentrations induces the production of cytokines and apoptosis. Eriptosis, the apoptosis process in erythrocytes, can be induced by several toxins and the increase in calcium concentration inside the cell is being postulated as the trigger of this process. In this context, we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA; calcium concentration was monitored following the changes in lifetime of the calcium indicator Green 1 using fluorescence lifetime imaging microscopy (FLIM). ... [truncated at 150 words]
Sengupta P, Sánchez SA, Scarlata SF.
Caveolin-1 boosts clustering of mu (µ) Opioid receptors in the plasma membrane.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1), 2563-Pos.Caveolin-1 (Cav1), is a structural protein component of many mammalian cell plasma membrane and is known to be involved in lipid and protein sorting, receptor desensitization, receptor trafficking, cell migration and many other cellular events. Here we determine if stable expression of Cav1 in cells alters the receptor organization prototype on the membrane. We use two different cell lines for this study: Fisher Rat Thyroid (FRTwt) cells that do not express detectable level of Cav1 and a sister line that is stably transfected with canine Cav1 protein (FRTcav). We express µ opioid receptors (MOR) tagged with either YFP (MOR-YFP) or CFP (MOR-CFP) in cells for different experiments. Förster resonance energy transfer (FRET) measurement between MOR-CFP and Gai-YFP in FRTwt and FRTcav cells shows receptor sequestration in the presence of Cav1. We find that diffusion of MOR-YFP in plasma membrane of FRTcav cells is slower compared to FRTwt cells by scanning ... [truncated at 150 words]
Digman MA, Fu CC, Ossato G, Lee LP, Gratton E, Khine M.
Biometallic nano-structures: a thousand-fold fluorescence enhancement with nanopetals.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 746a, 3882-Pos.Although, metal nanoparticles have been shown to increase emission fluorescence of molecules due to the propagation of electron density waves, i.e. surface plasmons, at the interface between the metal and substrate, biometallic nanostructures yield a higher emission enhancement and can be suitable for measuring cell surfaces at the nano scale. We have developed a method to attain bimetallic structures on the surface of memory polymers in order to achieve sharp bi-layered uniaxial and biaxial nanopetals. The sharp edges of the nanopetals exhibit remarkable increase of emission intensity of fluorescent molecules. We observe several thousand fold increase in intensity at the edges or “hotspots” of both uniaxial and biaxial nanopetals. The fluorescence intensities observed at the hotspots are brief bursts of intensity as the molecules diffuse through the structures. These bursts are below the resolution limit of our optics and possibly be due to single molecular emission. The intensity of the ... [truncated at 150 words]
-
Spatial correlation of speckle fluctuations reveals thickness and features of the ocular surface tear film.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 407a, 2099-Pos.Here we present Fluctuation Analysis of Spatial Image Correlation (FASIC), a non-invasive method for evaluating the complex dynamics of the tear film surface by spatial correlation analysis. Tear film stability and its interaction with the corneal surface play an important role in maintaining ocular surface integrity and quality of vision. Dry Eye Syndrome (DES) refers to abnormalities of tear film secretion and/or stability diagnosed by conventional methods such as the Schirmer test and tear break-up time (TBUT). Several different physical methods have been developed to measure non-invasively the structure and function of the tear film including high-speed videokeratography and dynamic wavefront aberrometry. Interferometry and optical coherence tomography are amongst new proposed methods to measure tear film thickness that have remained in research phase.
With FASIC, a series of images are obtained using a laser illumination and a cMOS camera. The spatial correlation is calculated for every frame. A sinusoidal background due ... [truncated at 150 words]
Hellriegel C, Caiolfa VR, Sidenius N, Gratton E, Zamai M.
Receptor-ligand interactions in the plasma membrane of live cells resolved in space and time by N&B analysis.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 750a, 3898-Pos.In this presentation we show how we push the Number and Brightness analysis (N&B) to the limits of applicability. We demonstrate that by N&B we can observe how a GFP labeled membrane receptor (namely uPAR) dimerizes upon ligand binding in live cells. We show how we obtain real time, spatially and temporally resolved images of the molecular reorganization of uPAR in the cell membrane. These results are backed by extensive simulations, and by well-defined live cell calibration experiments (using monomeric and dimeric GFP-uPAR constructs ).
N&B quantifies the amplitudes of fluorescence intensity fluctuations as individual fluorescent species diffuse in and out of a pixel in a series of images. The basic idea is that the amplitude fluctuations of a diffusing molecule labeled with two dyes (e.g. a dimer, or a bound ligand-receptor pair) will be twice as large as the amplitudes of a molecule with only one dye (i.e. a monomer, ... [truncated at 150 words]
Salvemini IL, Gratton E, Moens PDJ.
Single point FCS on a commercial confocal laser scanning microscope with analog detectors.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 180a, 935-Pos.Fluorescence Correlation Spectroscopy is a technique invented in the early 1970s to measure diffusion coefficient, chemical reaction rates and photo physical processes. It is a common belief that in order to obtain single point FCS data, one needs either a sophisticated FCS instrument with photon counting detectors or avalanche photon detectors or an instrument custom made for this type of experiments. Here we show that we can obtain single point FCS data on a commercial confocal laser scanning microscope without any modifications (Nikon C1). We successfully measured the diffusion coefficient and the concentration of Rhodamine B in solution for concentrations ranging from 5 nM to 280 nM. We also determined the diffusion coefficient of two different labeled lipid analogs (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate and BODIPY TMR phosphatidylinositol (4,5) bisphosphate) incorporated in the membrane of giant unilamellar vesicles. The results obtained for these lipid analogs are in good agreement with previously published data. ... [truncated at 150 words]
-
The pair-correlation approach to FCS.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 761a, 3958-Plat.Molecular diffusion and transport processes are fundamental in physical, chemical, biochemical and biological systems. Current approaches to measure molecular transport in cells and tissues based on perturbation methods like fluorescence recovery after photobleaching are invasive, fluctuation correlation methods are local and single particle tracking requires the observation of isolated particles for relatively long periods of time. We propose to detect molecular transport by measuring the time cross-correlation of fluctuations at a pair of locations in the sample. When the points are further than two times the size of the point spread function, the maximum of the correlation is proportional to the average time a molecule takes to move from a specific location to another. We demonstrate the method with simulations, using beads in solution and by measuring the diffusion of molecules in cellular membranes. The spatial pair cross-correlation method detects barriers to diffusion and heterogeneity of diffusion because the time ... [truncated at 150 words]
Caracciolo G, Callipo L, de Sanctis SC, Cavaliere C, Pozzi D, Caruso G, Digman MA, Sánchez SA, Gratton E.
Surface adsorption of protein corona controls the cell internalization mechanism of multicomponent lipoplexes in serum.
54th Annual Meeting of the Biophysical Society. February 20-24, 2010. San Francisco, California.
Biophys J. 2010; 98(3, Suppl 1): 722a, 3752-Pos.Designer multicomponent lipoplexes have recently emerged as especially promising transfection candidates, since they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Here, we show, for the first time, that after internalization binary complexes of lower transfection potency remain in compact perinuclear endosomes, while multicomponent systems have intrinsic endosomal rupture properties that allow plasmid DNA to escape from endosomes with extremely high efficiency. Endosomal rupture results in an extraordinarily homogeneous distribution of unbound plasmid DNA throughout the cytoplasm and in the nucleus. Serum has often been reported as a barrier to efficient lipid-mediated transfection. Here we found that the transfection efficiency of multicomponent lipoplexes increases in serum. To provide insight into the mechanism of lipoplex-serum interaction, several state-of-the-art methodologies have been applied. The nanostructure of lipoplexes was found to be serum-resistant as revealed by high resolution synchrotron small angle X-ray scattering, while ... [truncated at 150 words]
-
In vivo imaging of single-molecule translocation through nuclear pore complexes by pair correlation functions.
49th Annual Meeting of the American Society for Cell Biology. December 5-9, 2009. San Diego, California.
Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoproteins across the double-membrane nuclear envelope. We recently introduced a method based on pair correlation functions (pCF) which measure the time the same molecule takes to migrate from one location to another within the cell (1). The spatial and temporal correlation among two arbitrary points in the cell can provide a map of molecular transport, and also highlight the presence of barriers to diffusion with very high time resolution (in the microsecond scale) and spatial resolution (limited by diffraction).
Here we report the use of this method to directly monitor a model protein substrate undergoing transport through NPCs in living cells, a biological problem in which SPT has given results that cannot be confirmed by traditional FCS measurements because of the lack of spatial resolution. Our substrate is composed by a GFP linked to a functional nuclear localization sequence (NLS) ... [truncated at 150 words]
Digman MA, Lanzanò L, Gratton E.
Modulation tracking of cellular adhesions in 3D collagen matrices.
49th Annual Meeting of the American Society for Cell Biology. December 5-9, 2009. San Diego, California.
Cell mobility, proliferation, and cell adhesions are fundamental for cancer metastasis, wound healing, stem cell differentiation, and development. The dynamics of protein interactions and cell migrations have been extensively studied in the 2D environment. We have developed methods applicable to 2D raster scan images (RICS and scanning FCS) that have given information about the formation of complexes responsible for cell adhesion and migration. It is now established that cell movement in 3D matrices and in tissues have different dynamics. In 3D the search of the adhesions site with the extracellular matrix is a daunting problem. Cells make only few transient contacts and the entire cell moves in 3D going out of focus in conventional confocal or 2-photon excitation microscopy. There is no simple way to use the raster scan fluctuations techniques in the 3D environment that were successful in determining protein complexes and interaction in 2D. We have developed an ... [truncated at 150 words]
Choi CK, Bachir A, Zareno J, Digman MA, Gratton E, Horwitz AR.
Fluorescence fluctuation analyses reveal that FAK-Paxillin and α5 Integrin-Talin form complexes within adhesions.
49th Annual Meeting of the American Society for Cell Biology. December 5-9, 2009. San Diego, California.
Transient, localized protein interactions are central to adhesion formation, turnover, and signaling during cell migration. In highly motile cells, initial contact with the extracellular matrix is established via nascent adhesions in the lamellipodium. These adhesions function as signaling hubs that regulate protrusion and adhesion and also serve as traction points for migration. Paxillin and its putative binding partner FAK have emerged as critical regulators of migration. Using dual-color total internal reflection fluorescence (TIRF) and confocal imaging modalities, we measured the simultaneous fluctuations of paxillin and FAK fluorescence intensity and used cross-variance and cross-correlation techniques to detect their interactions and kinetics in living cells at high spatiotemporal resolution. The TIRF-based cross-variance analysis shows that paxillin and FAK reside and exchange as multimeric complexes in nascent adhesions. A phosphomimetic mutant of paxillin (Y31E, Y118E), which increases protrusion and adhesion assembly and turnover, significantly increases the complex size. In contrast, a non-phosphorylatable paxillin ... [truncated at 150 words]
Ossato G, Digman MA, Aiken C, Marsh JL, Gratton E.
N&B analysis of Huntingtin aggregation in live cells.
NIH NCRR/NIBIB P41 Principal Investigator Meeting. Bethesda, Maryland. October 2009.
Aggregation of misfolded proteins is a hallmark of several neurodegenerative diseases such as Huntington's disease. Here we describe the utility of the recently developed Number and molecular Brightness method (N&B) to monitor the aggregation process of Hungtintin exon 1 (Httex1). N&B measures the molecular brightness of the protein aggregates in the entire cell non-invasively based on the fluctuation dynamics at each pixel of an image. Dynamic imaging by the N&B method allowed detection of monomers, oligomers and inclusions in different regions of the cell simultaneously at different time points. We observed the behavior of Httex1-EGFP, with polyglutamine (polyQ) repeats of different lengths (Httex1-97QP-EGFP, Httex1-46QP-EGFP, Httex1-25QP-EGFP in transfected COS-7 cells. We found that Httex1 is present throughout the cell and that the non-pathogenic protein (Httex1-25QP-EGFP) does not aggregate while the pathogenic proteins (Httex1-46Q-EGFP and Httex1-97QP-EGFP) form inclusions. We establish that the process of nucleation leading to inclusion formation has four phases: ... [truncated at 150 words]
Gratton E, Lanzanò L, Fwu PT.
Imaging nanometer structures using modulation tracking (opening lecture).
International Symposium - Analysis of Biomolecular Machines in the Nanometer Range. September 20-23, 2009. Jena, Germany.
We describe a new modality of imaging nanostructures using modulation tracking based on optical feedback. The method uses a variant of circular tracking that we have developed some years ago for tracking particles and single molecules in cells. Since the method does not use the diffraction properties of light, we are not breaking the diffraction limit. We will show nanometer images of microvilli in living cells. The microvilli have a diameter of approximately 100 nm and length of several microns. The method allows the observation of patches of fluorescently labeled protein on the microvilli surface and the diffusion of fluorescent proteins along the microvilli membranes.
Cerussi AE, Tanamai W, Hsiang D, Chung S, Kukreti S, Mehta R, Gratton E, Tromberg BJ.
Breast cancer applications of optical imaging biomarkers measured with diffuse optical spectroscopic imaging.
AAPM 51st Annual Meeting, July 26-30, 2009, Anaheim, California.
Optical imaging biomarkers measured by Diffuse Optical Spectroscopic Imaging (DOSI) provide unique physiological profiles of tissue biochemistry that may be useful in breast cancer for detecting/characterizing lesions and evaluating tumor response to therapy. Imaging technologies have long played an important role in oncology, covering early detection, therapeutic planning, and remission management. Imaging biomarkers, such as tumor volume, are being considered as surrogate endpoints in place of traditional endpoints such as morbidity and mortality. Yet there is a need and a desire to see more. Spectral information content is often added to increase information content, and thus improve tumor detection and classification. For example, dual energy mammography can improve the visibility of microcalcifications. The capabilities of Magnetic Resonance Imaging (MRI) can also be augmented through the inclusion of spectroscopy (MRS). MRS applied to breast cancer has yielded some promising results, namely via the detection of choline, which may distinguish between benign ... [truncated at 150 words]
-
Measuring diffusion by spatial-cross-correlation.
7th EBSA European Biophysics Congress, July 11-15 2009, Genova, Italy.
Eur Biophys J. 2009; 38(Suppl 1): S38, O-14.Fluorescence correlation spectroscopy (FCS) has emerged as a very powerful method to study the motions of proteins both in the interior and exterior of the cell. It provides information at the single molecular level by averaging the behavior of many molecules thus achieving very good statistics. Single particle tracking (SPT) is also a highly sensitive technique to measure particle movement. However, the FCS method suffers in spatial resolution while the SPT technique only allows for the tracking of isolated molecules. Here we propose a change of paradigm in which using spatial pair cross-correlation functions we can overcome this limitation. Our method measures the time a particle takes to go from one location to another by correlating the intensity fluctuations at specific points on a grid independently on how many particles are in the imaging field. Therefore we can trace the average path of the particles. For example, our method could ... [truncated at 150 words]
-
Determining stoichiometry of molecular complexes in live cells.
7th EBSA European Biophysics Congress, July 11-15 2009, Genova, Italy.
Eur Biophys J. 2009; 38(Suppl 1): S47, O-47.In this presentation, we report a method for determining both the presence and the stoichiometry of protein complexes at pixel resolution and apply it to disassembling focal adhesions. The method is derived from fluorescence fluctuation methods that have single molecule sensitivity and is based on our previously described N&B (Number and Brightness) method that measures the number and brightness (aggregation state) of fluorescent molecules in every pixel of a confocal microscope image. The new method exploits the correlation of fluorescence amplitude fluctuations for two colors and detects the presence of molecular complexes and their stoichiometry. While the original N&B method was developed for one color, i.e., a single molecular species, the new method, ccN&B, extends the analysis to two colors and introduces the concept of cross-variance. This method is similar in concept to the two-color PCH analysis. However, the covariance based ccN&B method also generates pixel resolution maps of protein ... [truncated at 150 words]
Vetri V, Ossato G, Militello V, Digman MA, Leone M, Gratton E.
Oligomerization of Concanavalin A in live cells detected by fluctuation analysis.
7th EBSA European Biophysics Congress, July 11-15 2009, Genova, Italy.
Eur Biophys J. 2009; 38(Suppl 1): S137, P-393.We report an experimental study on ConcanavalinA (ConA) aggregation in live cells. In vitro, close to physiological temperature, ConA readily forms fibrils involving secondary structure changes leading to β-aggregate structures. The effect of ConA on cell cultures and formation of protein aggregates were measured by confocal fluorescence microscopy. In particular, we monitored protein aggregation in live cells by means N&B analysis, Cross-N&B and RICS. N&B showed the aggregation kinetic and the progressive formation of ConA oligomers at cell surface. This suggests that, at cell membrane where local concentration is higher, nucleation sites for aggregation are provided. In parallel, the morphology of the cells changes indicating the progressive cell compaction and death. Aggregation and binding of small aggregates to the cell surface were assessed by RICS: it is possible to distinguish regions where small aggregates are diffusing and regions where they are bound to the cell. Oligomers formation may stimulate non-specific ... [truncated at 150 words]
Caracciolo G, Caminiti R, Pozzi D, Digman MA, Gratton E, Sánchez SA, Marchini C, Montani M, Amici A, Amenitsch H.
Efficient escape from endosomes determines the superior efficiency of multicomponent lipoplexes.
7th EBSA European Biophysics Congress, July 11-15 2009, Genova, Italy.
Eur Biophys J. 2009; 38(Suppl 1): S85, P-199.Designer multicomponent lipoplexes have recently emerged as especially promising transfection candidates, since they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes [1-3]. Here, we show, for the first time, that after internalization binary complexes of lower transfection potency remain in compact perinuclear endosomes, while multicomponent systems have intrinsic endosomal rupture properties that allow plasmid DNA to escape from endosomes with extremely high efficiency [4]. Endosomal rupture results in an extraordinarily homogeneous distribution of unbound plasmid DNA throughout the cytoplasm and in the nucleus [4].
References:
[1] Caracciolo, G. et al. Langmuir 2005, 21, 11582.
[2] Caracciolo, G. et al. Appl. Phys. Lett. 2006, 89, 233903.
[3] Caracciolo, G. et al. Langmuir 2007, 23, 4498.
[4] Caracciolo, G. et al. J. Phys. Chem. B 2009, DOI:10.1021/jp811423r.
Michalos A, Gupta R, Olopade CO, Picchiett DL, Hungs M, Gratton E.
Cerebrovascular responses in OSA patients during CPAP therapy measured in the frontal lobes of the brain bilaterally, simultaneously, and non-invasively using frequency-domain near-infrared spectroscopy.
International Conference of the American Thoracic Society. May 15–20, 2009. San Diego, CA.
Am J Respir Crit Care Med. 2009; 179, A3554.OSA is associated with severe cardio/cerebrovascular morbidity. Our goal is the development and introduction of Frequency−Domain Near−Infrared Spectroscopy (FD−NIRS) in Clinical Diagnostics and Sleep Medicine to identify patients at risk. FD−NIRS allows non−invasive, continuous, lengthy, and real−time measurements of cerebral tissue oxygenation and hemodynamics by measuring absolute hemoglobin (oxygenated, deoxygenated, and total) concentrations. We performed FD−NIRS concomitantly to conventional polysomnography in 135 subjects. Cerebrovascular autoregulatory responses were evaluated with an Absolute NIRS Brain Oximeter (OxiplexTS, ISS, Champaign, IL) in the frontal lobes bilaterally. After rigorous NIRS screening criteria, 40 controls and 40 OSA patients were considered. The reported findings are from a specific Severe OSA Cohort (N=5 OSA, age range 49−55, 4 males/1 female, AHI 55−90) within the total OSA group, where the subjects serve as their own controls. We are reporting quantitative measurements of cerebral tissue hemodynamic variables during the initial 3−6 hours of diagnostic PSG and subsequently, during ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
Spatial temporal image correlation spectroscopy (STICS) for flow analysis with application for blood flow mapping.
3rd IUPAP International Conference on Women in Physics. October 8-10, 2008. Seoul, South Korea.
AIP Conf Proc. 2009; 1119: 239.It is important for surgeons to be able to measure blood flow in exposed arterioles during surgery. We report our progress in the development of an optical technique that will measure blood flow in surgically exposed blood vessels and enable previously difficult measurements. By monitoring optical fluctuations, the optical technique, based on Spatial Temporal Image Correlation (STICS), will directly measure the velocity of micron-scale particles—such as red blood cells. It will complement existing technology and provide qualitative measurements that were not previously possible. It relies on the concept that blood, when viewed on a small enough scale, is an inhomogeneous substance. Individual blood cells passing between a near-infrared light source and a detector will cause fluctuations in the transmitted optical signal. The speed, direction, and flow pattern of blood cells can be determined from these optical fluctuations. We present a series of computer simulations and experiments on phantom and animal ... [truncated at 150 words]
East A, Rossow MJ, Matulin WW, Gratton E.
Embryonic zebrafish (danio reio) as a model for the investigation of blood flow measurements using scanning laser image correlation (SLIC).
34th Annual West Coast Biological Sciences Undergraduate Research Conference, San Diego, California 2009.
Zebrafish (Danio Reio) function as a model organism for vertebrate biology, physiology and human pathology due to their transparent embryos, rapid external fertilization and development, as well as the simplicity of their system. Zebrafish can also function as a model for in depth investigation of blood flow. In light of their critical role in biomedical research, we chose the Zebrafish to establish the effectiveness of the Scanning Laser Image Correlation (SLIC) technique for blood flow measurement. SLIC works by scanning a laser beam along the blood vessels and detecting and analyzing reflected light. In this method, the blood cells act as reflective particles, and detailed flow information can be obtained through statistical analysis of the optical signal. SLIC can be used in future cardiovascular studies for both Zebrafish and other animal models. In the experiments presented here, we demonstrate SLIC to be an effective measure of flow. ... [truncated at 150 words]
Celli A, Sánchez SA, Behne MJ, Hazlett TL, Gratton E, Mauro T.
Detection of calcium gradients in live ex vivo human epidermis using a novel approach based on phasor analysis of two-photon excitation Fluorescence Lifetime Imaging (FLIM).
J Invest Dermatol. 2009; 129(Suppl 1s): S73, 437.Ionic gradients are found across a variety of tissues, and control vital functions in skin. It has been
difficult to accurately measure whole tissue ionic gradients in vivo, as previous methods have been
ill-suited to address problems of tissue thickness, concentration artifacts, and calibration across
inhomogeneous tissue. Here we show that a novel, minimally invasive, experimental and analytic
imaging approach accurately quantifies and localizes the free Ca2+ gradient in ex vivo epidermis
and dermis, and allows simultaneous analysis of epidermal morphology and Ca2+ concentrations
in the same samples. This method is based on the combination of two-photon excitation fluorescence
lifetime imaging (FLIM) and a novel data analysis technique based on the phasor representation
of the FLIM data (Digman et al 2008). Skin samples from adult patients were incubated with
the fluorescent calcium sensitive dye Calcium Green 5N (CG5N) whose lifetime is sensitive to variation
in calcium concentrations. The use of two-photon excitation provides high axial and lateral
spatial resolution, deep tissue ... [truncated at 150 words]
Tanner K, Ferris DR, Lanzanò L, Mandefro B, Mantulin WW, Gardiner DM, Rugg EL, Gratton E.
Image correlation spectroscopy reveals global dynamics of wound healing.
53rd Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2009.
Biophys J. 2009; 96(3, Suppl 1): 42a, 217-PosB96.We apply techniques based on correlation spectroscopy to quantify cell migration during wound re-epithelialization in an axolotl wound healing model. We show that cell hypertrophy (about 37% in volume) is present from the time of injury and continues throughout re-epithelialization. Our combined imaging techniques (transmission and laser scanning fluorescence) microscopy and analysis algorithms ( Image Correlation Spectroscopy and Spatio-Temporal Image Correlation Spectroscopy) allow us to determine this complex sequence of events from the point of injury until the re-epithelialization is complete. Using non-invasive optical sectioning, we determine that the basal keratinocytes migrate into the wound bed faster than the suprabasal keratinocytes. Additionally, Image Correlation Spectroscopy (ICS) reveals cell hypertrophy as there is an increase in width of the spatial autocorrelation function as a function. Using camera based transmission microscopy, we observe that the enlarged cells produce a point of dislocation that foreshadows and dictates the initial direction of the migrating ... [truncated at 150 words]
-
Analysis of diffusion and binding in cells using the RICS approach.
53rd Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2009.
Biophys J. 2009; 96(3, Suppl 1): 43a, 221-PosB100.The movement of macromolecules in cells is assumed to occur either through active transport or by diffusion. However the determination of the diffusion coefficients in cells using fluctuation methods or FRAP frequently give diffusion coefficient that are orders of magnitude smaller than the diffusion coefficients measured for the same macromolecule in solution. It is assumed that the cell internal viscosity is partially responsible for this decrease in the apparent diffusion. When the apparent diffusion is too slow to be due to cytoplasm viscosity, it is assumed that weak binding of the macromolecules to immobile or quasi immobile structures is taking place. In this work we derive equations for fitting of the RICS (Raster-scan Image Correlations Spectroscopy) data in cells to a model that includes transient binding to immobile structures and we show that under some conditions, the spatio-temporal correlation provided by the RICS approach can distinguish the process of diffusion ... [truncated at 150 words]
Ossato G, Digman MA, Aiken C, Marsh JL, Gratton E.
Protein aggregation in live cells: N&B analysis of Huntingtin.
53rd Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2009.
Biophys J. 2009; 96(3, Suppl 1): 88a, 455-PosB334.The aggregation of the huntingtin (Htt) protein is thought to be responsible for Huntington’s disease. One problem is the detection of the different size of aggregates and the stages of aggregation directly in live cells. Recently we develop a method (N&B) to detect the size of aggregates and the number of particles in every pixel of a confocal image. N&B can be performed on the entire cells simultaneously so it is possible to follow the kinetics of aggregation. This method is based on the variance of intensity fluctuations of particles as they diffuse through the excitation volume. We performed experiments in transfected COS7 using different lengths of the polyglutamine sequence (Httex1 97QP-GFP, Httex1 46QP-GFP and Httex1 25QP-GFP). We can determine the presence of units, small aggregates and inclusion bodies in different parts of cell and their time evolution. We observed the presence of Htt throughout the cell and that oligomer ... [truncated at 150 words]
Vetri V, Ossato G, Militello V, Digman MA, Leone M, Gratton E.
Detecting protein aggregation on cells surface: Concanavalin A oligomers formation.
53rd Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2009.
Biophys J. 2009; 96(3, Suppl 1): 88a, 454-PosB333.A number of neurodegenerative diseases involve protein aggregation and amyloid formation. Recently evidence has emerged indicating small-transient prefibrillar oligomers as the primary pathogenic agents. Noteworthy, strict analogies exist between the behaviour of cells in culture treated with misfolded non-pathogenic proteins and in pathologic conditions, this instance together with the observation that the oligomers and fibrils are characterised by common structural features suggest that common mechanisms for cytotoxicity could exists and have to be perused in common interactions involved in aggregation.
We here report an experimental study on ConcanavalinA (ConA) aggregation and its effects on cells. In vitro, close to physiological temperature, this protein readily forms fibrils involving secondary structure changes leading to b-aggregate structures. The effect of a ConA on cell cultures was tested and the formation of protein aggregates in these samples was studied by confocal fluorescence microscopy. We used the N&B analysis method to monitor ConA aggregation in live ... [truncated at 150 words]
Digman MA, Gratton E, Wiseman PW, Horwitz AR.
Multicolor fluctuation spectroscopy in cells: obtaining the stoichiometry of molecular complexes.
53rd Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2009.
Biophys J. 2009; 96(3, Suppl 1): 208a, 1076-Plat.We have developed a method that is capable of measuring the stoichiometry of these molecular complexes and mapping dynamic processes in living cells such as molecular interactions and binding to specific locations. The method is based on measuring simultaneously fluctuations of the fluorescence intensity at two image channels, each detecting a different kind of protein. The statistical analysis of correlated fluctuations is performed by measuring the duration of correlated fluctuations and the covariance of the amplitude of the fluctuations giving the relative intensity of the complex in two separate channels. We apply the cross-variance method to determine the stoichiometry of complexes containing paxillin and vinculin or FAK (focal adhesion kinase) in disassembling adhesions in mouse embryo fibroblasts expressing FAK, vinculin and paxillin tagged with EGFP and mCherry. One important finding of our studies is that we were unable to detect complexes of these proteins in the cytoplasm far from the ... [truncated at 150 words]
-
Obtaining quantitative information on the cell-induced deformation of collagen with digital holographic microscopy.
53rd Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2009.
Biophys J. 2009; 96(3, Suppl 1): 297a, 1512-PosB356.Principles of holography are applied to study the dynamics of cells and their extracellular matrix with digital hologram microscopy (DHM). The goal of this study is to measure the deformation of collagen matrix induced by cell migration. DHM has appeared as a unique tool to study the displacement at the nano-scale by resolving differences in refractive index. DHM is capable of conducting quantitative size and depth measurements in 3-D. A digital holographic microscope, in transmission, is designed and built to record two-dimensional holograms on a CMOS camera. The digitally recorded holograms are computationally reconstructed using the angular spectrum method (ASM) providing a better signal to noise ratio in comparison with the traditional Fresnel approximation method. The ASM method also outputs the phase image that is used to perform quantitative phase-contrast analysis. The phase images represent the optical pathlength disturbance caused by the sample. These images are unwrapped by applying the ... [truncated at 150 words]
-
Laurdan GP fluctuations in membranes of intact erythrocytes.
53rd Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2009.
Biophys J. 2009; 96(3, Suppl 1): 151a, 779-PosB658.Lipids in the cell membranes are believed to be organized in micro-domains, known as rafts. If they in fact exist, rafts may provide important boundaries for the organization and sequestering of membrane proteins. The prevailing theory holds that membrane rafts are as small structures (10-200 nm), heterogeneous in size, highly dynamic and sterol- and sphingolipid-enriched domains that compartmentalize cellular processes.
The existence of these small domains is still under debate despite a great deal of work in the area. The most commonly used method to detect rafts is their resistance to solubilization by the nonionic detergent Triton X-100 and sensitivity to cholesterol depletion. These measurements are indirect and potentially open to alternative interpretations. Directly visualizing rafts in living cells has been a difficult task because they are extremely small. Their existence still remains controversial.
Here we report the use of a new methodology were two powerful microscopic techniques are applied simultaneously. The ... [truncated at 150 words]
Cutrale F, Digman MA, Gratton E.
Paxillin focal adhesions, localization and implication: insight from photo-activated localization microscopy.
53rd Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2009.
Biophys J. 2009; 96(3, Suppl 1): 637a, 3284-PosB331.Photo-Activated Localization Microscopy (P.A.L.M.) as described by E. Betzig (2006) optically resolves selected subsets of photo-activatable fluorescent probes within cells at mean separations of less than 25 nanometers through serial photo-activation and subsequent photobleaching of numerous sparse subsets of photo-activated fluorescent protein molecules.
The position information from all subsets is then assembled into a super-resolution image, in which individual fluorescent molecules are isolated at high molecular densities. In this work COS-7 and ST14A tdEos-Paxillin transfected cells were used. We observed some features that limit the versatility of PALM, both in this setup and in its present version. It takes actually hours to go through the cycles of photo-activation and image acquisition, to collect all of data needed and to generate a single high-resolution image limiting the use to fixed specimens which precludes PALM.’s use for imaging of live cells. More important is the loss of data. Depending on the spatial concentration ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
Line scanning flow measurements.
53rd Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2009.
Biophys J. 2009; 96(3, Suppl 1): 314a, 1601-PosB445.We are developing a new technique to measure flow of micron scale particles using laser scanning. This technique will also detect complex flow patterns, identify stationary particles and determine particle size. In this method, a laser beam is raster scanned over an area containing a flowing liquid. Particles in the liquid scatter the laser. Detailed information about the flow can be obtained from analyzing the fluctuations in this scattered radiation.
Detailed flow information, such as can be provided by this technique, is valuable in medical applications. Blood cells can serve as the particles that scatter lights and the laser scanning can be applied to surgically exposed blood vessels in a patient or in a animal model. The information available with this method can help study or monitor conditions such as sickle cell anemia in which abnormal blood cells do not move smoothly through blood vessels or become stuck. It can also ... [truncated at 150 words]
Ramella N, Tricerri MA, Sánchez SA, Ferreira ST, Rimoldi OJ.
Amyloid-like aggregation of a human Apolipoprotein A-I variant.
53rd Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2009.
Biophys J. 2009; 96(3, Suppl 1): 89a, 458-PosB337.Amyloidosis are characterized by extra cellular deposits of anomalous fibrilar proteins. Human apolipoprotein A-I (apoA-I) is not normally involved within these pathologies. However, one case of severe amyloidosis associated with atherosclerosis was observed when apoA-I shows a deletion of a lysine residue in a central region of the protein (apoA-I Lys107-0). In order to get insight on the local cellular environment that promotes this anomalous aggregation, we studied the folding of the deletion mutant, as compared with wild type apoA-I (Wt). Analysis of chemical denaturation and by using hydrostatic pressure show that apoA-I Lys107-0 is more unstable and has a stronger tendency to form β sheet structure as incubation time increases, specially at acidic pH. Under these conditions, mutant denaturation is less cooperative, suggesting intermediate states folding. In order to confirm that these states prone protein aggregation, we followed protein folding by two-Photon Fluorescence Correlation Spectroscopy. Our results clearly show ... [truncated at 150 words]
Digman MA, Wiseman PW, Horwitz AR, Gratton E.
Mapping focal adhesion protein complexes in living cells from laser scanning confocal image sequences: the ccRICS method.
48th Annual Meeting of the American Society for Cell Biology, San Francisco, California. December 13-17, 2008.
We have developed a method for measuring molecular complex interactions based on single molecule fluorescence fluctuations from laser scanning confocal images. By cross-correlating fluctuations of EGFP tagged focal adhesion kinase (FAK) or vinculin to mcherry tagged paxillin, a map of the spatial distribution of these protein-protein complexes and their diffusion and/or binding properties can be calculated. The exact interactions of binding and unbinding process are revealed in an image. This method is based on the raster image correlation spectroscopy (RICS) approach, which relies on the fact that successive pixels are measured at different times. The spatial correlation of the image contains information about dynamic processes occurring over a large time range, from the microseconds to seconds. Our results indicate that the assembly and disassembly processes at adhesions during cellular retraction are unique. FAK, vinculin and paxillin do not reside in preassembled complexes throughout the cytoplasm. They do however, dissociate in ... [truncated at 150 words]
Kwok S, Lee CY, Sánchez SA, Hazlett TL, Gratton E, Hayashi Y.
Genetically encoded probe for fluorescence lifetime imaging of CaMKII activity.
38th Annual Meeting of the Society-for-Neuroscience. Washington, DC, USA. November 15 -19, 2008.
Neuroscience Meeting Planner. 2008; Online, 737.5/L4.Ca2+/calmodulin-dependent protein kinase II (CaMKII) is highly enriched in excitatory synapses in the central nervous system and is critically involved in synaptic plasticity, learning, and memory. However, the precise temporal and spatial regulation of CaMKII activity in living cells has not been well described, due to lack of a specific method. Our previous work employed fluorescence resonance energy transfer (FRET) technique, which optically measures the association or conformational change of molecules, to monitor CaMKII activity changes. Among several different methods to detect FRET, the fluorescence lifetime imaging (FLIM) provides a robust contrasting mechanism that identifies the local environment of the fluorophore and quantifies the relative concentration of various species that coexist. A probe for FLIM has a distinct set of requirements in efficient FRET detection when compared to that of the ratiometric method. In this study, we attempted to generate an optical probe for fluorescence lifetime imaging of CaMKII activity ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
An optical tool to investigate cerebral aneurysms.
2008 Society of Women Engineers (SWE) National Conference. November 6-8, 2008. Baltimore, Maryland.
Cerebral aneurysms - bulges in a weakened section of blood vessel wall - are serious medical conditions that kill thousands of people each year in the United States alone. Frequently, there are no symptoms associated with aneurysms. However, they are dangerous because they can rupture, causing internal bleeding, stroke, and death.
Improved understanding of aneurysms and aneurysm treatments is necessary to save lives. Currently the treatment of aneurysms is inhibited by limited information on the fluid dynamic properties of aneurysms. While doctors have several options for treating cerebral aneurysms, without understanding how blood flows in treated area it is difficult to predict whether an aneurysm will rupture, difficult to determine which treatment will be the most effective, and difficult to evaluate the success of the treatment.
Near infrared spatial temporal image correlation spectroscopy (NIR-STICS), a technique we have developed, can provide information about blood flow in aneurysms in a medically relevant ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
Spatial temporal image correlation spectroscopy for detailed flow analysis.
2008 Biomedical Engineering Society (BMES) Annual Fall Meeting. October 2-4, 2008. St. Louis, Missouri.
Digman MA, Wiseman PW, Choi CK, Horwitz AR, Gratton E.
Obtaining the stoichiometry of molecular complexes at adhesions using the N&B cross-variance method.
Frontiers in Cell Migration. National Institutes of Health, Bethesda, Maryland, September 16-18, 2008.
We have developed a method that is capable of measuring the stoichiometry of molecular complexes and mapping dynamic processes in living cells. The method is based on measuring simultaneously fluctuations of the fluorescence intensity at two image channels, each detecting a different kind of protein. This is an extension of the number and brightness (N&B) analysis in which we use the use of the ratio between the variance and the average intensity to obtain the brightness of molecules. In the N&B cross-variance method we measure the cross-variance among the intensity fluctuations in two channels. We apply the cross-variance method to determine the stoichiometry of complexes containing paxillin and vinculin or FAK (focal adhesion kinase) in disassembling adhesions in mouse embryo fibroblasts expressing FAK, vinculin and paxillin tagged with EGFP and mCherry. One important finding of our studies is that we were unable to detect complexes of these proteins in the ... [truncated at 150 words]
Digman MA, Wiseman PW, Horwitz AR, Gratton E.
Mapping protein complexes in living cells from laser scanning confocal image sequences: the ccRICS method.
Frontiers in Cell Migration. National Institutes of Health, Bethesda, Maryland, September 16-18, 2008.
We propose a new method for detecting molecular complexes based on the analysis of single molecule fluorescence fluctuations from laser scanning confocal images. The method is based on the RICS (Raster scan Image Correlation) approach. Since in a raster scanned image successive pixels are measured at different times, the spatial correlation of the image contains information about dynamic processes occurring over a large time range, from the microseconds to seconds. The correlation of intensity fluctuations measured simultaneously in two channels detects protein complexes that carry two molecules of different colors. This information is obtained from the entire image. A map of the spatial distribution of protein complexes in the cell and their diffusion and/or binding properties can be constructed. Using this cross-correlation raster image spectroscopy (ccRICS) method, specific locations in the cell can be visualized where dynamics of binding and unbinding of fluorescent protein complexes occur. This fluctuation imaging method ... [truncated at 150 words]
-
Latest data from 3D single particle tracking.
7th International Weber Symposium. Kauai, Hawaii. June 6-12, 2008.
3D Particle tracking is used to characterize the motion of a particle (labeled protein, bead, organelle, etc...). At no extra cost, these trajectories also map substrates (membranes, surfaces, fibers) with high-resolution (2-20 nm) when the particles move in close proximity to those structures. The possibility to record intensity, spectrum and lifetime of the particle's emission - simultaneously while tracking - opens the possibility to characterize the interactions between particle and the substrate even further. In this contribution will be show the latest examples that illustrate these capabilities of the tracking method.
-
Exploiting fluctuations to determine protein aggregation and stoichiometry in cells.
7th International Weber Symposium. Kauai, Hawaii. June 6-12, 2008.
The study of protein interactions in live cells is a major challenge in cell biology. We have developed a method for detecting and quantifying when and where two different proteins interact during complex cellular processes. Our method detects these complexes and also provides the fraction of molecules that reside in complexes as well as their rates of diffusion or binding kinetics. In this analysis, we compute the cross-correlation of temporal fluctuations in the fluorescence intensity from molecules labeled with two different fluorescence tags. The novelty of the method is that the cross-correlation is obtained simultaneously across an entire image and thereby produces a detailed, cellular map of protein interactions. This method is robust and applicable to images obtained on commercial laser scanning confocal microscopes and therefore is of general interest cell biologists. Unlike FRET, the technique is not constrained by the distance of ... [truncated at 150 words]
Ossato G, Frasnelli E, Lanzanò L, Digman MA, Marsh JL, Gratton E.
Imaging Htt protein aggregation in cells by fluctuation analysis.
7th International Weber Symposium. Kauai, Hawaii. June 6-12, 2008.
The aggregation of the huntingtin protein is thought to influence Huntington’s disease pathogenesis. We performed experiments in transfected COS 7 cells using human Htt exon 1 with varying lengths of polyglutamine fused to GFP (Httex1 97QP-GFP, Httex1 46Qp-GFP and Httex1 25QP-GFP). Expression of the Htt protein with different glutamine lengths results in the formation of visible fibrils in the cytosol and in the nucleus of COS7 cells. The macroscopic formation of large aggregates depends on is different for the different glutamine sequence lengths. Using N&B fluctuation spectroscopy analysis performed simultaneously on the entire cells, we followed the kinetics of aggregate formation. In this analysis the size of the aggregates is deduced from the brightness (B) map. This map shows different aggregate sizes in the cytosol and in the nucleus. In a first phase, large aggregates and fibrils appear in the cytosol followed by ... [truncated at 150 words]
Katayama Y, Burkacky O, Lange S, Bräuchle C, Jansen RP, Gratton E, Lamb DC.
On the trail of single particles: 3D single particle tracking in live cells.
7th International Weber Symposium. Kauai, Hawaii. June 6-12, 2008.
Imagine tracking a tourist in Rome using GPS. From the resulting trajectory, you can gather information over what mode of transportation this person prefers, what their daily routine is and what interests she or he has. In the last decade, a revolution has occurred in fluorescence microscopy, making it possible to follow individual molecules and particles with nanometer accuracy. We can gather similar information by following individual biomolecules within living cells using single particle tracking. Intracellular mRNA localization is a common mechanism for creating asymmetric distributions of proteins in live cells. The mRNAs are transported to the translocation site and the proteins are produced “on site” rather than having to be transported through the cell. Using yeast as a model system, we have investigated how different mRNA species are transported to their final destination. We have developed a novel labeling strategy that allows the labeling two different mRNAs in ... [truncated at 150 words]
-
Laurdan GP fluctuations in biological membranes.
7th International Weber Symposium. Kauai, Hawaii. June 6-12, 2008.
It has been proposed that lipids in cell membranes are organized in micro-domains, rafts, which may provide important boundaries for the organization and sequestering of membrane proteins. Membrane rafts are conceived as small structures (10–200 nm), heterogeneous in size, highly dynamic and sterol- and sphingolipid-enriched domains that compartmentalize cellular processes. Despite a large body of work, doubts persist about the existence of these small domains in natural membranes. The most commonly used method for rafts detection, is their resistance to solubilization by the nonionic detergent Triton X-100 and sensitivity to cholesterol depletion. These measurements are indirect and potentially open to alternative interpretations. Given their size, the direct visualization of rafts in living cells has been a difficult task and their existence still remains controversial. Here we report the use of a new methodology were two powerful microscopic techniques are applied simultaneously. The first one, Laurdan Generalized ... [truncated at 150 words]
Choi CK, Vicente-Manzanares M, Zareno J, Brown CM, Mogilner A, Digman MA, Kolin DL, Wiseman PW, Gratton E, Horwitz AR.
The assembly, maturation, and disassembly of adhesions in migrating cells.
7th International Weber Symposium. Kauai, Hawaii. June 6-12, 2008.
Cell migration is driven by actin polymerization and the formation and turnover of adhesions at the cell front and the disassembly of adhesions and retraction at the cell rear. Using dual label imaging and high resolution TIRF microscopy, we have shown that nascent adhesions assemble and become stable within the lamellipodium of migrating cells. The assembly is myosin II-independent, requires actin polymerization, and most components enter simultaneously. As the back of the lamellipodium moves past the nascent adhesions, they either disassemble as the protrusion continues to advance or begin to grow and elongate as the protrusion pauses. Adhesion maturation is guided by an α-actinin-actin template and the crosslinking properties of myosin IIA. We have developed a toolbox of fluctuation methods for studying adhesion formation and disassembly at high spatial and temporal resolution during migration. Measurements of the dynamics and brightness of adhesion components, using correlation microscopy and number & ... [truncated at 150 words]
Gunther G, Toro CA, Zanocco A, Rifo M, Gratton E, Sánchez SA.
Interaction of human erythrocyte with MMS detergent: changes in membrane fluidity reported by Laurdan.
7th International Weber Symposium. Kauai, Hawaii. June 6-12, 2008.
LAURDAN is a membrane probe that gives information about the water content in the bilayer that is related with the membrane fluidity. The spatial resolution necessary for cellular work is given by the use of 2-photon microscopy and dual channel detection. In this report we present the changes observed in fluidity of the lipid bilayer when it interacts with detergents using 2-photon Laurdan GP imaging. The solubilization of biological membranes by detergents has been used as the main method for the isolation and purification of membrane proteins and other constituents and no much information in the literature related with the fluidity changes during the process is found. We present here results of the fluidity changes in the solubilization process of artificial and natural systems produced by the sucrose monoester of myristic acid, b-D-Fructofuranosyl-6-O-myristyl-a-D-Glucopyranoside (MMS). In POPC liposomes, the solubilization process is accompanied by changes in the size of ... [truncated at 150 words]
-
The SimFCS environment: from simulation to data acquisition and analysis.
7th International Weber Symposium. Kauai, Hawaii. June 6-12, 2008.
SimFCS is a complex program for simulation, data acquisition, data analysis and modeling of fluorescence data. In simulation mode, SimFCS can provide FCS, scanning FCS, RICS N&B and particle tracking data to test models and functions. In data acquisition, SimFCS support several of the popular data acquisition cards based on National Instrument drivers and also from other manufactures. In the data acquisition mode, SimFCS can also move the scanner in x, y and z coordinates along predetermined patterns. The scan pattern can be changes on the fly for real time particle tracking in 3D. In the analysis mode, several modules are available for the analysis of FCS data, a comprehensive toolbox for scanning FCS with a large number of libraries. For image analysis, most of the image correlation methods have been implemented including RICS, tICS, STICS and N&B. In the particle tracking mode, ... [truncated at 150 words]
Digman MA, Ossato G, Frasnelli E, Buonaventura G, Aiken C, Marsh JL, Gratton E.
Huntingtin (Htt) oligomer formation in live cells.
7th International Weber Symposium. Kauai, Hawaii. June 6-12, 2008.
The process of protein multimerization can elicit crucial signaling cellular events to ensure the survival of the cell or lead to devastating effects such as progressive degenerative diseases. Current methods to measure aggregation of proteins are few and limited to few pairs of proteins for examples using FRET, Homo-FRET or limited to measuring one point at a time in live cells such as in photon counting histogram analysis. We have developed a novel method, i.e., the Number and Molecular Brightness (N&B) method, based on moment analysis to map the number and size of aggregates in live cells. The N&B analysis can distinguish with pixel resolution, the aggregation state of proteins by calculating the average intensity and the variance of the molecular fluctuations. We use this approach to study the aggregation of the huntingtin protein (Httex1 97QP-eGFP, Httex1 46Qp-eGFP and Httex1 25QP-eGFP) as a function of time in living cells. Our ... [truncated at 150 words]
-
Quantitative analysis of the dynamics of cell and the extracellular matrix with digital holographic microscopy.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 797-Pos/B642.Principles of digital holography are applied to study the dynamics of cell and its extracellular matrix. Digital holographic microscopy (DHM) has appeared as a unique tool to study the shape and deformation at the nano-scale by resolving differences in refractive index. A digital holographic microscope in transmission mode is designed and built to record two-dimensional holograms on a CCD camera. The digitally recorded holograms are numerically reconstructed based on the angular spectrum method (ASM) providing a better signal to noise ratio in comparison with the traditional Fresnel method. The ASM method also outputs the phase image that is used for quantitative phase-contrast analysis. The phase images are unwrapped using the Flynn’s discontinuity algorithm to account for the two-pi ambiguity. The importance of conducting quantitative phase analysis rises up when one needs to reveal the optical thickness profile of a transparent specimen with sub-wavelength accuracy. Digital holography not only offers a ... [truncated at 150 words]
Colyer RA, Lee CY, Gratton E.
A novel fluorescence lifetime imaging system that optimizes photon efficiency.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 807-Pos/B652.Fluorescence lifetime imaging (FLIM) is a powerful microscopy technique for providing contrast of biological and other systems by differences in molecular species or their environments. However, the cost of equipment and the complexity of data analysis have limited the application of FLIM. We present a mathematical model and physical implementation for a low cost Digital Frequency Domain FLIM (DFD-FLIM) system which can provide lifetime resolution with quality comparable to time-correlated single photon counting methods. Our implementation provides data natively in the form of phasors. Based on the mathematical model, we present an error analysis which shows the precise parameters for maximizing the quality of lifetime acquisition, as well as data to support this conclusion. The hardware and software of the proposed DFD-FLIM method simplifies the process of data acquisition for FLIM, presents a new interface for data display and interpretation, and optimizes the accuracy of lifetime determination.
NIH, PHS 5 P41 ... [truncated at 150 words]
Digman MA, Horwitz AR, Gratton E.
N&B fluctuation analysis of Paxillin reveals mechanisms of adhesion assembly and disassembly.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 1588-Pos/B564.The number of particles (N) and brightness (B) analysis is used to measure the aggregation state of a paxillin-EGFP construct in CHO-K1 cells. This analysis is based on the intensity fluctuations measured at each pixel of an image sequence and has been developed for both photon counting and analog detectors. While the N&B analysis is complementary to the RICS (Raster-scan Image Correlation Spectroscopy) analysis developed few years ago, it provides pixel resolution information about the number and size of protein aggregates from relatively few images of live cells independently of the diffusion of the molecule. Our analysis shows that paxillin exists as a monomer in the cytosol and forms relatively large aggregates, the size of which can depend on the particular adhesion. In large adhesions, paxillin is relatively immobile and exchanges slowly with the surrounding population. The N&B analysis of growing adhesions show that monomers of paxillin add to the ... [truncated at 150 words]
Gratton E, Digman MA, Rück A.
Simultaneous FLIM measurement of the decay of the donor and acceptor in a FRET pair.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 1601-Pos/B577.We have developed a method based on the phasor approach to evaluate globally the decay of the donor and of the acceptor in a FRET pair in images of cell expressing a construct of EGFP and mRFP. Data where acquired with a spectral detector in the photon counting mode using the Becker and Hikl module. In the phasor plot, the donor and the acceptor phasors move along specific trajectories depending on the FRET efficiency. The superposition of these two contributions results in phasors aligned in a straight segment. Therefore, the measurements at different emission wavelengths should give phasors along this segment. The experimental position of the segment in the phasor plot and its slope provide a unique solution in terms of the efficiency of transfer, amount of donor without acceptor, amount of direct excitation of the acceptor and background contribution. This solution could in principle be obtained with a global ... [truncated at 150 words]
Toro CA, Sánchez SA, Gratton E, Zanocco A, Lemp E, Gunther G.
Effect of cholesterol in the solubilization process by β-D-Fructofuranosyl-6-O-myristyl-α-D-Glucopyranoside (MMS).
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 323-Pos/B156.The solubilization of liposomes in the presence of amphiphillic compounds (the process involving transformation from lamellar structures to mixed micelles) has been usually described with the three stage model. In the first stage, at surfactant concentrations below its cmc, the presence of surfactant molecules in the vesicle or liposome induces several changes on its properties (mainly size increase observable turbidity or by light scattering), when the bilayer is saturated, the presence of mixed structures is observed (micelles formed by lipid and surfactant) in coexistence with lamellar structures (open or closed bilayers). A decrease in turbidity is observed upon further increase in surfactant concentrations, consistent with the complete solubilization of bilayers (remaining only mixed micelles in the system).
We studied the effect of the presence of cholesterol on the solubilization process by the sucrose monoester of myristic acid, beta-D-Fructofuranosyl-6-O-myristyl-alfa-D-Glucopyranoside (MMS) in different liposomes. We used LAURDAN (a membrane probe that gives information ... [truncated at 150 words]
-
3D particle tracking in 2-photon microscopy using orbital scanning expanded.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 809-Pos/B654.The 3D spatial position of a particle can be determined by scanning the excitation volume of a 2-photon microscope in a three-dimensional orbit around the particle and by subsequently analyzing the fluorescence intensity profile along the orbit. [1] If the analysis is computed very rapidly in comparison to the orbit time, it is possible to use it in a feedback loop to constantly monitor the particle’s position and re-center the orbit at the particle, in the case the particle moves. The individual feedback steps reconstitute the particle’s 3D spatial trajectory. Trajectories of organelles in vivo, for example, have been obtained in this way with a spatial and temporal resolution in the order of 10 nm and 32 ms respectively. [2]
In this presentation we turn to some important capabilities inherent to the described orbital scan technique, which provide additional information, obtained from the tracked particle simultaneously with the tracking. Firstly, it ... [truncated at 150 words]
Moore TI, Hazlett TL, Gratton E, Yi TM.
Membrane diffusion measured using fluorescence correlation spectroscopy in polarized S. cerevisiae.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 2891/B194.The cellular membrane not only serves as the interface and barrier between the extracellular environment and intracellular biochemical processes, but also a dynamic organelle where proteins are embedded, recruited and organized, in many cases free to diffuse around the cell surface creating a homogeneous protein concentration. During polarization this uniformity is broken and localization of some proteins changes resulting in their localization becoming polarized. To explore the role of diffusion and how changes of a protein's diffusion rate may be involved in polarization, Fluorescence Correlation Spectroscopy (FCS) was utilized to measure membrane diffusion in the budding yeast, S. cerevisiae, during mating projection formation. Slow diffusion rates were determined for cytosolic and membrane bound (EGFPmem) EGFP which were consistent with previously reported values obtained using FRAP [1]. α-cells were exposed to varying concentrations of α-factor (0, 10, 100, 1000 nM) and localization of EGFPmem and the α-factor receptor (Ste2-EGFP) after the ... [truncated at 150 words]
Ossato G, Frasnelli E, Lanzanò L, Digman MA, Marsh JL, Gratton E.
Imaging HTT protein aggregation in cells by fluctuation analysis.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 233-Pos/B65.The aggregation of the huntingtin protein is thought to influence Huntington’s disease pathogenesis. We performed experiments in transfected COS 7 cells using human Htt exon 1 with varying lengths of polyglutamine fused to GFP (Httex1 97QP-GFP, Httex1 46Qp-GFP, Httex1 25QP-GFP). Expression of the Htt protein with different glutamine lengths results in the formation of visible fibrils in cytosol and in the nucleus. The macroscopic formation of large aggregates depends on the glutamine sequence lengths. Using N&B fluctuation spectroscopy analysis performed simultaneously on the entire cell, we followed the kinetics of aggregate formation. In this analysis the size of the aggregates is deduced from the brightness (B) map. This map shows different aggregate sizes in the cytosol and in the nucleus. In a first phase, large aggregates and fibrils appear in the cytosol followed by aggregation in the nucleus. After several hours, the apparent size of aggregates in the nucleus surpasses ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
Blood flow mapping through spatial temporal image correlation.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 1691-Pos/B667.We are continuing to develop an optical technique that can be used as the basis of a device to measure blood flow in the brain during neurosurgery. This technique is based on detecting and analyzing optical fluctuations and will directly measure the velocity of micron-scale particles_such as red blood cells. It will complement existing technology in that it can be used on arterioles less than 1mm in diameter: a category of blood vessels in which it has been difficult to obtain quantitative flow measurements. The ability to detect the direction as well as the magnitude of flow and map these parameters over an area also sets this technique apart.
The ability to measure blood flow is important during neurosurgery and can be crucial for patient survival. Applications of blood flow measurement include detecting residual flow after aneurysm clipping and determining that flow has been reestablished after temporary blood vessel clamping. Surgical ... [truncated at 150 words]
Sánchez SA, Tricerri MA, Gomes AMO, de Oliveira AC, Levi V, Gratton E.
Changes in membrane water content after Cholesterol removal revealed by Laurdan generalized polarization microscopy.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 389-Pos/B222.The cell membrane is composed by lipids, proteins, and carbohydrates. The amphipathic nature of lipid molecules allows them to pack tightly while maintaining a high degree of lateral mobility. In addition, lipids are organized into microdomains (e.g., rafts) that may provide important boundaries that organize integral membrane proteins. Cholesterol is a small lipid molecule that nestles among the hydrophobic tails of the phospholipids in the interior of the membrane. It is know to make the membrane more fluid and at the same time it is indicated as the main constituent of lipid rafts. The decrease in fluidity of the membrane after removing cholesterol from the membranes is normally used as an indication for the involvement of lipid raft in the process under study. In artificial systems depending on composition of the bilayer, the presence of cholesterol may increase or decrease their fluidity (measured as water content). In this report, we ... [truncated at 150 words]
Tanner K, Lanzanò L, Gratton E.
Cell migration in collagen matrices: assessment of local stiffness by fluctuation spectroscopy.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 1595-Pos/B571.We studied the migration of individual tumor cells, derived from the human glioma cell line ACBT that were implanted into a 3D gel matrix of collagen type I. Depending on the density of the matrix, cells can migrate in this 3D tissue model. The properties of the matrix are crucial for the migration of the cells. The cells and the collagen fiber network where imaged using a confocal microscope in the reflection mode so that the sample could be observed for a long period of time without bleaching. We developed a method based on image correlation spectroscopy and on local measurement of thermal fluctuations to determine the average size and the local stiffness of the collagen fibers. Intensity fluctuations were generated by the flickering of the reflections from the fibers. The reflected intensity from a single point in the fiber fluctuates due to fiber position fluctuations. We found a correlation ... [truncated at 150 words]
-
Number and brightness analysis for EMCCD cameras.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 788-Pos/B633.Recently, techniques for the determination of particle number and brightness (N&B analysis) have been developed for confocal microscopy. We extend this technique to brightfield and TIRF microscopies with high sensitivity EMCCD cameras. The high signal to noise ratio and speed of these systems provides unique opportunities to follow changes in molecular brightness and concentration within a dynamic system over time as well as the spatial localization of these events. Nevertheless, these systems are also affected by saturation and nonlinearity effects which must be avoided in order to accurately measure changes in brightness. As with other analog systems, stability is a key factor and must be accounted for in the measurement of particle fluctuations. Despite these drawbacks, we show that the method can accurately measure particle brightnesses over several orders of magnitude independently of bleaching and other concentration gradients. In addition, this method provides a sensitive measure of immobile and mobile ... [truncated at 150 words]
Zamai M, Caiolfa VR, Barreiro O, Colyer RA, Digman MA, Sidenius N, Sánchez-Madrid F, Gratton E.
FRET phasor-FLIM analysis of homotypic and heterotypic non-covalent interactions of membrane receptors in living cells.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 805-Pos/B650.It is commonly believed that the best approach to measure FRET in cells is through the measurement of the fluorescence lifetime quenching of the donor as done in FLIM. However, FLIM is still restricted to few laboratories because of the cost of the instrumentation and the difficulty of data analysis. Several assumptions are introduced in the analysis such as the use of average lifetimes, as many fluorescent proteins and the autofluorescence show multi-exponential decays in cells. The phasor approach is introduced to simplify the way data are analyzed in FLIM. Changing the data representation from the classical time delay histogram to the phasor representation provides a global view of the fluorescence decay at each pixel of an image. In the phasor representation we easily recognize the presence of different molecular species in a pixel or the occurrence of FRET also when the cellular background is relevant. We show the application ... [truncated at 150 words]
Perfetto S, Redigolo M, Gratton E.
Development of an instrument for measuring extremely low concentrations of particles.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 1617-Pos/B593.Improving the quality of screening for single structures, such as bacteria, viruses, among others, has become an important priority in Biotechnology and Medicine. There is a need for a very simple, reliable, inexpensive and valid tool. This current work addresses this need. The goal of this study was to develop and test an instrument capable to detect and analyze extremely low concentrations of different types of structures in solution for use in clinical and biotechnological applications. The detection of the particles is performed using a fluorescence signal generally obtained with antibody labeling.
Our prototype consists of a sample holder for a cylindrical cuvette that can be rotated and vertically translated while having a large volume of solution excited by a laser at the wavelength of 532nm. The excitation focus is centered about 200 µm from the wall of the cuvette inside the sample. A microscope with confocal detection scans the sample ... [truncated at 150 words]
Bruno L, Echarte MM, Gratton E, Sánchez SA, Levi V.
Switching microtubule molecular motors during melanosome transport in Xenopus Laevis melanophores.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 3213-Pos/B516.Xenopus laevis melanophores are one of the cellular systems commonly used to study the function of molecular motors in vivo. An unexpected but common observation in trajectories of single organelles moving along microtubules is that the organelle frequently revert its direction. This observation was explained by the switching of the motor responsible for the transport but the mechanism by which motors with a given polarity turns on and motors with opposed polarity turns off is not known.
In this work, we explore the mechanism of in vivo coordination of microtubule motors by using a fast and precise tracking method. We analyze the interval in the trajectories where reversals of directions are observed and postulate a model to explain the reversals in living cells.
Supported by Agencia de Promocion Cientifica y Tecnologica PICT 31975. LB and VL are members of the CIC CONICET, Argentina.
Barreiro O, Zamai M, Tejera E, López-Romero P, Gratton E, Caiolfa VR, Sánchez-Madrid F.
Nanoscale molecular interactions and dynamics of endothelial adhesive platforms (EAP) in primary living cells.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 2983.01-Pos/B286.01.Tetraspanins are ubiquitous low molecular weight proteins that span the plasma membrane four times and are able to organize themselves by homo- and hetero-oligomerization. These molecules can also associate laterally at the plasma membrane with a plethora of integral membrane receptors, modulating their functions. Tetraspanins also associate intracellularly with a number of cytoplasmic signaling mediators. Thus, the proposed existence of tetraspanin-enriched microdomains is based on the ability of tetraspanins to simultaneously interact among themselves and with a wide range of molecules, organizing discrete dynamic plasma membrane compartments. We used EGFP and mRFP1-tagged chimeras of two tetraspanins, CD9 and CD151, and two partners (VCAM-1 and ICAM-1 adhesion receptors) to characterize in primary endothelial living cells diffusion properties, nanoscale organization and specific intra-domain molecular interactions within these specific tetraspanin-enriched microdomains. These microdomains containing endothelial adhesion receptors are termed endothelial adhesive platforms (EAP). FRET between protein pairs was analyzed by the novel phasor-FLIM ... [truncated at 150 words]
Katayama Y, Burkacky O, Meyer M, Bräuchle C, Gratton E, Lamb DC.
A novel 3D confocal tracking microscope: combining the advantages of orbital tracking and wide-field imaging.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 812-Pos/B657.Complex transport mechanisms in live cells are not restricted to a single plane. Therefore, it is of great interest to explore dynamics in 3D. To this purpose, we have developed a tracking microscope based on laser scanning confocal microscopy to follow fluorescent particles in 3D. For x-y positioning, we used the previously developed orbital tracking approach of E.Gratton[1], where the laser is scanned or orbited about the particle. By analyzing the modulation of the fluorescence intensity, the x-y position of the particle is determined and, via a real-time feedback routine, the orbit of the laser is re-centered on the new position of the particle. We have enhanced and extended the capabilities of orbital tracking by incorporating a new approach for determining the z position. Two detection channels are used, where the confocal pinholes have been shifted to detection regions slightly above and below the image plane. The z-position of the ... [truncated at 150 words]
Sotomayor CP, Cuevas FJ, Aguilar LF, Soto MA, Sánchez SA.
Influence of cholesterol on the lateral mobility of Na+/K-ATPase reconstituted on giant unilamelar vesicles.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 1925-Pos/B44.Na+/K+-ATPase is an enzyme present in the majority of the cellular membranes and has a fundamental role in a number of physiological process. The idea that the physical state and the physicochemical properties of the lipidic matrix influence the dynamic and function of integral proteins is well accepted. The existence of segregated lipid domains and the possibility that enzymes will differentially distribute among them add to the effect of the lipid matrix on the protein function an extra degree of complexity. Our previous work showed that minute changes in cholesterol concentration produce abrupt changes in the membrane organization, increasing interdomain surfaces. In addition, we proposed that cholesterol induces a change in hydration at the protein-lipid interface. These changes induce small changes in the pig kidney Na+/K+-ATPase’s structure and dynamics, acting to fine-tune the enzyme.
In this work, we present preliminary data on the role of cholesterol on the mobility of the ... [truncated at 150 words]
Herlax VS, Mate S, Sánchez SA, Tricerri MA, Bakas LS.
The interaction of alpha-hemolysin with specific microdomains at sublytic concentration.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 2067-Pos/B182.a-Hemolysin (HlyA) is a pore forming toxin secreted by pathogenic strains of Escherichia coli. The toxin is synthesized as a protoxin, pro-HlyA, which is matured in the cytosol to the active form by acylation at two internal lysines. This toxin with a wide target cell specificity is considered to be the prototype of a family of toxins called RTX (repeat in toxin), a series of protein toxins that share genetic and structural features.
In this report, we studied the interaction of HlyA with lipid membranes using as experimental system sheep erythrocytes, one of the most studied target cell for this type of studies. A variety of pathogens, toxins and acylated proteins interact with microdomains enriched in cholesterol and sphingolipids (lipid rafts), therefore we studied the association of HlyA with these microdomains on sheep erythrocytes. Our experiments in functional properties of the toxin such as oligomerization and hemolytic activity studied by FRET ... [truncated at 150 words]
Sanabria H, Digman MA, Gratton E, Waxham MN.
Limiting Calmodulin revealed by image correlation spectroscopy.
52nd Annual Meeting of the Biophysical Society, Long Beach, California, 2008.
Biophys J. 2008; 94(2, Suppl 1), 92-Plat.Protein-protein interactions depend on the ability of proteins to move from one place to another in search of their respective targets. The passive transport of signaling molecules is potentially hindered by obstacles or by its amount of available (mobile) pool. Calmodulin (CaM) a Ca2+ sensor protein searches among a wide number of targets, posing the question on whether there is enough non-bound CaM that can move freely through the cytoplasm to activate all of its targets. To address this question, we have used Raster Image Correlation Spectroscopy (RICS) to study the spatial distribution and mobility of CaM inside cells at rest and following elevation of intracellular Ca2+. RICS can be performed on data collected with a standard confocal microscope and allows one to quantify the availability and diffusivity of a tagged protein from fluorescent fluctuations over space and time. Diffusion coefficients from a few hundred µm2/s to less that 1 ... [truncated at 150 words]
Kukreti S, Cerussi AE, Tanamai VW, Tromberg BJ, Mehta R, Hsiang D, Gratton E.
Intrinsic near-infrared spectroscopic biomarkers applied for evaluation of final pathological response to neaoadjuvant chemotherapy.
30th Annual San Antonio Breast Cancer Symposium, San Antonio, Texas, December 13-16, 2007.
Breast Cancer Res Treat. 2007; 106(Suppl 1): S218, 5036.Background: Near-infrared (NIR) optical methods provide a noninvasive view of tissue based on compositional and functional characterization. In the wavelength region of 650-1000nm, NIR light is sensitive to the four major absorbing components in breast tissue, namely, oxyhemoglobin, deoxyhemoglobin, bulk lipid and water. We have already shown that cancer tissue and cancer tissue undergoing neoadjuvant chemotherapy display changes in these functional parameters. However, it is debatable whether these are specific “markers” of cancer. Recently it was shown that double-differential analysis of breast tissue absorption spectra from cancer-containing tissue reveals specific tumor components (STC) present only in tumor tissue. The STC absorption signature is an intrinsic biomarker characterized by specific NIR absorption bands. Here we apply the double-differential analysis to tissue after chemotherapy. In this pilot study we ask the following question: Can the STC biomarker be used to non-invasively evaluate the final pathological response in cancer tissue after administration of ... [truncated at 150 words]
Choi JH, Wolf M, Toronov VY, Wolf U, Michalos A, Gratton E.
Spatio-temporal analysis of the cerebral spontaneous oscillations.
37th Annual Meeting of the Society for Neuroscience, San Diego, California.
A frequency-domain near-infrared spectroscopy (fdNIRS) using multi-distance is a noninvasive optical technique to measure the absolute concentration of hemoglobin in tissue and the cerebral oxygen saturation. We applied multiple fdNIRS sensors simultaneously on adult human forehead to measure the cerebral hemodynamics at multiple depths in the head including brain tissues during resting period. Spectral analysis showed pulsation, respiration, and slow oscillation in the spontaneously fluctuating cerebral hemodynamic signals. The slow oscillation has a frequency around 0.1Hz and has amplitude comparable to other systemic rhythms. In this study, we investigated the physical origin and dynamical characteristics of this slow oscillation and its physiological functions. We first determined the dynamical characteristics of the slow oscillation using random data analysis techniques such as phase portrait and fractal dimension. We then examined the spatial and temporal coherence of the slow oscillation using correlation methods and pattern analysis. The dynamic coupling between slow oscillation in ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
A new way to measure blood flow.
2007 Society of Women Engineers (SWE) National Conference. October 25-27, 2007. Nashville, Tennessee.
-
The phasor approach to FLIM analysis of FRET in live cells.
Inserm 180. Protein Interaction Networks: from bioinformatics to cellular dynamics. October 4-5, 2007. La Londe-les-Maures, France.
Changing the data representation from the classical time delay histogram to the phasor space provides a global view of the fluorescence decay at each pixel of an image. In the phasor representation we can easily recognize the presence of different molecular species in a pixel or the occurrence of FRET. The analysis of the FLIM data in the phasor space is done observing clustering of pixels values in specific regions of the phasor plot rather than by fitting the fluorescence decay using exponentials. The analysis is instantaneous since is not based on calculations or non-linear fitting. The phasor approach has the potential to revolutionize the way data are analyzed in FLIM, paving the way for the analysis of large data sets, isolating rare events in cells and, in general, making the FLIM technique more accessible to the non expert in spectroscopy and data analysis.
-
In vivo cell dynamics by spatio-temporal correlation techniques.
Inserm 180. Protein Interaction Networks: from bioinformatics to cellular dynamics. October 4-5, 2007. La Londe-les-Maures, France.
Single point fluctuation correlation spectroscopy (FCS) is an established technique to study diffusion and chemical equilibria in solution. It has limitations when applied to the cell. Major difficulties of this technique are that the movements of the cells or of cellular components are difficult to separate and filter out from the molecular dynamics. Scanning FCS, in which the laser beam is moved in a circular orbit provides the fluctuation amplitude and dynamics at many point simultaneously and it can be used to detect the cell movement. Image correlation spectroscopy has the potential to provide detailed maps of the dynamics in a cell, but it suffers from the limitation that the frame rate is relatively slow. We have developed an approach that exploits the intrinsic time structure of a confocal raster scan image to determine spatiotemporal correlations at several time scales simultaneously. Using this approach, termed RICS (Raster-scan Image Correlation Spectroscopy), ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
Spatial temporal image correlation spectroscopy analysis for fluid velocity measurement.
2007 Biomedical Engineering Society (BMES) Annual Fall Meeting. September 26-29, 2007. Los Angeles, CA.
We are developing a new optical technique for measuring the fluid velocity of micron-scale particles and for generating a map of these velocities over an area of one square centimeter. Our goal is to develop this technique into a technology to measure blood flow in exposed blood vessels during surgery. It is important for surgeons to be able to measure the rate of blood flow in order to ensure that the surrounding tissue is receiving sufficient oxygen. There are currently a number of techniques available to detect and measure blood flow but each has certain limitations and there is a need for improved technology.
Our technique is based on the analysis of intensity fluctuations in an optical signal, reflected or fluorescent, generated by flowing particles. Spatial temporal image correlation analysis (STICS)—a technique borrowed from fluorescence imaging in cells—is able to extract the velocities of these particles from a complicated, noisy ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
Flow measurement using correlation analysis of optical signals.
4th Annual NTROI Retreat, Newport Beach, California, June 28-29, 2007.
Blood flow measurement is an important area of technological development. It is critical for surgeons to be able to measure blood flow in exposed blood vessels during surgery. Accurate, fast blood flow measurements allow the surgeon to detect regions of tissue that are receiving insufficient blood—and therefore insufficient oxygen, correct the problem and determine if blood flow has been reestablished. There are a number of existing techniques to measure blood flow but each has its limitations and can only be applied to particular situations. For example, the ability to provide a map of velocities in small (~100μm) blood vessels over a region one square centimeter in size is lacking from most current technology. We are developing a technique that will measure the fluid velocity of flowing particles and can provide a map of blood flow in small, exposed blood vessels.
The technique we have developed takes advantage of ... [truncated at 150 words]
D'Amico E, Perfetto S, Ossato G, Frasnelli E, Arnesano C, Mantulin WW, Gratton E.
The non-contact optical breast scanner: optics and data acquisition development.
4th Annual NTROI Retreat, Newport Beach, California, June 28-29, 2007.
The goal of this project is to design and manufacture a non-contact laser breast scanner (LBS) that will improve speed of image acquisition, spatial resolution, spectral information-all within the context of improved patient comfort. The non-contact LBS (NC-LBS) will operate in both the frequency domain and also in the broad band spectral regime, so that it maintains design solidarity with the current NITRO protocols. However, the NC-LBS introduces a number of technical innovations not present in the current version of the LBS. The design of the NC-LBS includes the following components: automatic positioning system, light source (super continuum white light laser), non-contact scanning optics and mechanics, detection system and the data collection/analysis software. Last year we presented this conceptual design. We presented theoretical and experimental data establishing the optimal configuration of the illumination and detection optics for the non-contact scanner. From this research, the optical probe design included a ... [truncated at 150 words]
Kukreti S, Cerussi AE, Tanamai VW, Tromberg BJ, Gratton E.
Intrinsic near-infrared spectroscopic biomarkers of breast tumors.
4th Annual NTROI Retreat, Newport Beach, California, June 28-29, 2007.
Kukreti S, Cerussi AE, Tanamai VW, Tromberg BJ, Gratton E.
Intrinsic markers of breast tumors.
NIH NCRR/NIBIB P41 Principal Investigator Meeting. Bethesda, Maryland. 19-20 June 2007.
We have developed a new methodology based on high resolution spectroscopy in thick tissues that provide clinicians with a “noninvasive view” of the molecular changes due to the presence of a lesion. Preliminary results from 24 lesions suggest that this “non-invasive” approach detects tumors and discriminates different type of lesions. This technology is based on the frequency-domain spectroscopy of tick tissues that we pioneered at the LFD. In thick tissues, light in the near infrared (700 to 1100 nm) scatters multiple times and is absorbed by specific tissues chromophores such as hemoglobin, water and lipids. The amount of light that reaches a given distance from the point of illumination depends both on the scattering and absorption in the tissue. To obtain the absorption spectrum free from the scattering contribution we must have a method that independently determines at teach wavelength these two parameters. In the frequency domain spectroscopy approach we ... [truncated at 150 words]
-
Measuring the deformation of collagen matrix in response to cell migration detected with digital holographic microscopy.
2nd Frontiers in Biomedical Devices Conference, Irvine, California, 2007.
Polzonetti CM, Mantulin WW, Meissner S, Wigal TL.
Hemodynamic variability in the frontal lobes of Attention-Deficit Hyperactivity Disorder children during baseline and Attention Network Test.
62nd Annual Convention of the Society of Biological Psychiatry, San Diego, California, 2007.
Background: Children with Attention Deficit Hyperactivity Disorder (ADHD) have been reported to have functional problems in the frontal lobes, but the basis of their attentional deficit remains controversial. The Attention Network Test (ANT) has suggested the existence of a neural network of brain structures involved in attention. We studied the properties of blood-oxygen perfusion and the vaso-response shape in the frontal areas with frequency domain Near Infrared Spectroscopy (fd-NIRS).
Methods: We measured 9 children with ADHD (7.8±0.92 years; 2 females), and 24 controls (23.1±4.8 years, 15 females). Multivariate statistical analysis described the tendency of: hemoglobin concentrations (oxy-, deoxy- total-hemoglobin and tissue oxygen saturation: [HbO2], [HHb], [tHb], StO2), polarization indexes (P_[tHb] and P_ StO2), and washout anti-phase (Hilbert transform). Measurements included: baseline and cognitive testing with ANT.
Results: Absolute values showed significant differences in ADHD and controls, due to measurement condition and medication. Only StO2 showed higher concentrations for the children with ADHD. ... [truncated at 150 words]
Kukreti S, Cerussi AE, Tromberg BJ, Gratton E.
Near-infrared tumor spectral signature enhances optical mammography.
ASCI/AAP Joint Meeting, Chicago, Illinois, April 2007.
Near-infrared spectroscopy provides a non-invasive approach to obtain functional information. Traditional analysis methods of absorption spectra have provided quantitative information on total hemoglobin (oxyhemoglobin and deoxyhemoglobin), bulk lipid, and water content. While tumors display changes in these parameters, these biomarker are not unique “signatures” of cancer. Normal tissues also contain the same tissue absorbers. A novel approach to spectral analysis has revealed a unique spectral signature of cancer. These signatures are spatially localized to the tumor containing regions of the breast and not in surrounding normal tissue or the contra-lateral normal breast tissue. The laser breast scanner which uses the technique of Steady State Frequency Domain Photon Migration was used to recover non-invasively the absorption and scattering spectra from 650-1000 nm in the breast tissues of patients. This was a pilot study of 15 patients total, 10 with stage 2 or 3 cancers, and 5 normal (no known lesion) cases. ... [truncated at 150 words]
Tanner K, Tang S, Gratton E.
Dynamics of cell migration for cells embedded in collagen using a multimodal platform of optical coherence tomography, multi-photon excitation and second harmonic generation.
APS March Meeting, Denver, Colorado, March 5-9, 2007.
We developed Raster Image Correlation Spectroscopy (RICS) to analyze the dynamics of cell migration from data obtained on a confocal multi-photon microscope. We assembled a microscope that can simultaneously measure the scattering signal from optical coherence tomography (OCT), multi-photon excited emission (TPEF) and second harmonic signals (SHG) with comparable spatial resolution and the same time resolution. We present data here showing the combined 3-D images of the cells embedded in a collagen matrix. The OCT signal adds fine structural information of the cellular morphology and collagen which is enhanced by the SHG image. The RICS analysis of the TPEF signal gives the dynamics of the GFP-style proteins. We show that the cell morphology and the distribution of cell organelles are different in the collagen matrix than what is observed in cells growing on flat surfaces. Using the three modalities of cell imaging we could reach a more realistic interpretation of ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
An optical technique for fluid velocity measurement applicable to intra-operative blood flow.
51st Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2007.
Biophys J. 2007; Suppl: 332a, 1565-Pos/B618.During surgery, especially neurosurgery, it is important for doctors to be able to determine if blood flow is compromised in a vessel due to some obstruction or a vasospasm. It is also useful for doctors to be able to measure to rate of blood flow in order to ensure that the surrounding tissue is properly oxygenated and not ischemic. There currently exist a number of technologies for measuring blood flow but each of them has certain limitations and there is a need for improved technology.
We are developing a new optical correlation method for detecting and measuring the velocity of small (~1mm) particles transported in a flowing liquid. This technique is applicable to blood flow measurements since blood is a fluid (plasma) containing small particles (blood cells). The basis of this technique is the analysis of intensity fluctuations in the detected optical signal caused by particle flow. We have tested this ... [truncated at 150 words]
Tanner K, Tang S, Gratton E.
Dynamics of cell migration for cells embedded in collagen using a multimodal platform of optical coherence tomography, multi-photon excitation and second harmonic generation.
51st Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2007.
Biophys J. 2007; Suppl: 517a, 2467-Pos/B682.We developed Raster Image Correlation Spectroscopy (RICS) to analyze the dynamics of cell migration from data obtained on a confocal multi-photon microscope. We assembled a microscope that can simultaneously measure the scattering signal from optical coherence tomography (OCT), multi-photon excited emission (TPEF) and second harmonic signals (SHG). The 3 imaging modalities have comparable spatial resolution and the same time resolution. Using this microscope we examined the dynamics of different cellular structures and fluorescent-scattering particles.. We present data here showing the combined 3-D images of the cells embedded in a collagen matrix. The OCT signal adds fine structural information of the cellular morphology and collagen which is enhanced by the SHG image. The RICS analysis of the TPEF signal gives the dynamics of the GFP -style proteins. We propose that a greater understanding of the dynamics of the cells can be obtained by combining the scattering signal and fluorescence and performing ... [truncated at 150 words]
Sánchez SA, Toro CA, Tricerri MA, Gratton E, Gunther G.
Interaction of rHDL particles with membranes containing cholesterol in different lipid packing.
51st Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2007.
Biophys J. 2007; Suppl: 428a, 2052-Pos/B270.There is strong evidence that the first steps in cholesterol efflux involve the interaction of lipid poor discoidal high density lipoproteins (pre-ß-HDL) with the cellular plasma membrane initiating the reverse cholesterol transport (RCT) process, wherein excess cholesterol is transported to the liver for catabolism. It is possible that the accessibility of sterol molecules to HDL particles depends on their location on the membrane. The extent and direction of the net movement will depend on the ratio of efflux to influx and is determined by the properties of the acceptor (HDL or other) and donor (membrane). It has been proposed that factors reducing the packing density of lipid molecules would enhance the rate of cholesterol transfer.
Here we report the effect of lipid packing on the cholesterol accessibility to the HDL particles in the process of sterol removal. The technique utilized was LAURDAN generalized polarization (GP), in cuvette and in a ... [truncated at 150 words]
Unruh JR, Digman MA, Takahashi H, Blaine JT, Breusegem SY, Barry NP, Levi M, Gratton E.
Lipid dynamics in native rat renal brush border membranes as measured by scanning fluorescence correlation spectroscopy.
51st Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2007.
Biophys J. 2007; Suppl: 155a, 722-Pos/B566.Lipid rafts are small liquid ordered membrane domains that are thought to play a significant role in the function of cellular membranes. Previous studies have suggested the presence of lipid rafts in the brush border membranes of mammalian renal proximal tubule cells. The presence of these structures has been implicated in renal phosphate wasting disorders. We have grown giant unilamellar vesicles (GUVs) from native rat proximal tubule brush border membrane fragments. In addition we have performed Optiprep detergent free density gradient fractionation of these membranes into raft-like and nonraft-like fractions. Glycosphingolipid (GM1) is a classic marker of lipid rafts and is concentrated preferentially into the raft-like Optiprep fractions. Scanning fluorescence correlation spectroscopy measurements on rat BBM GUVs stained with a dye labeled cholera toxin B (ctxB) subunit (binds to GM1) shows that GM1 exists in large slowly moving aggregates containing multiple ctxB labels. In addition, the concentration of these aggregates ... [truncated at 150 words]
Colyer RA, Lee CY, Gratton E.
Time-resolved frequency-domain fluorescence lifetime imaging microscopy in the photon-counting regime.
51st Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2007.
Biophys J. 2007; Suppl: 321a, 1511-Pos/B564.Fluorescence lifetime imaging microscopy (FLIM) is growing in usefulness as a means of distinguishing molecular species, observing environmental regions which affect the lifetime of a fluorophore, and observing fluorescence resonance energy transfer (FRET). We present a convenient technique for performing FLIM in a laser scanning microscope using the photon counting regime of the photomultiplier detector in conjunction with time-resolved frequency-domain data acquisition hardware.
In our technique we synchronize the internal clock of a field programmable gate array (FPGA) to a sampling frequency which is fractionally offset from the laser repetition frequency, and use heterodyning to evenly sample the entire repetition time of the laser. The sampling clock period is divided into time-resolved measurement windows which sample the arrival of photons in different portions of the lifetime curve with a 100% duty cycle. The heterodyning frequency domain approach allows us to evenly sample the lifetime curve with each time-resolved measurement window. The ... [truncated at 150 words]
Digman MA, Dalal RB, Brown CM, Horwitz AR, Gratton E.
Determination of aggregates size and number of proteins from TIRF, LSM, and two photon microscopy.
51st Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2007.
Biophys J. 2007; Suppl: 322a, 517-Pos/B570.We developed a technique for the measurement of average number of molecules in a pixel and molecular aggregates from fluorescence microscopy images. Based on the moment analysis, pioneered by Qian and Elson for solution measurements, the time series of the intensity at any given pixel is analyzed (1). In solution, the average number of molecules <N> in a given volume is obtained from the average (first moment) and from the variance (second moment) of the series. If the intensity fluctuations are dominated by the occupation of a particle in a given volume, <N> is proportional to 1/G(0) where G(0) is the ratio of the variance to the square of the average. The molecular brightness is obtained by dividing the average intensity at one point by <N>. The moment analysis assumes that fluctuations are caused by the occupation of the fluorescent particles in a given volume. In cells, the intensity at ... [truncated at 150 words]
Gratton E, Brown CM, Horwitz AR, Digman MA.
Velocity maps of protein aggregates near adhesion sites form from TIRF data obtained with a fast electron multiplier camera.
51st Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2007.
Biophys J. 2007; Suppl: 517a (2466-Pos/B681).Several methods have been proposed to extract information about the average velocity of particles in a region of the cell, including methods based on spatio temporal image correlation spectroscopy (STICS). In this study we compare a technique based on calculation of the space-time derivative with the STICS method. First, we show that the time derivative method provides a simple map of the regions of the cell in which adhesion are growing or disassembling. The space-time derivative analysis provides information about the direction of the motion. The detailed information about the velocity of the front of adhesion obtained from Paxillin-EGFP in live cells indicates that adhesions slide or grow in alternating periods and in synchrony with adjacent adhesions. The STICS method is mainly sensitive to slow changes, has a limited spatial resolution, but has a better capability at determining the average velocity as well as the random diffusion of particles. Instead, ... [truncated at 150 words]
Azartash K, Digman MA, Holub O, Gratton E.
Deformation of collagen matrix detected by digital holographic microscopy.
51st Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2007.
Biophys J. 2007; Suppl.
Kukreti S, Cerussi AE, Tromberg BJ, Gratton E.
Intrinsic markers revealed by a novel spectral analysis of near-IR spectra of breast tumors.
29th Annual San Antonio Breast Cancer Symposium, San Antonio, Texas, December 14-17, 2006.
Breast Cancer Res Treat. 2006; 100(Suppl 1): S44, 1019.Background: Near infrared (NIR) optical methods provide contrast based on quantitative functional changes in tissue. NIR techniques have shown to be sensitive to changes in breast physiology from disease by quantifying total hemoglobin (oxyhemoglobin and deoxyhemoglobin), oxygen saturation, fat content, and water content. Tumors display changes in these parameters; however, it is debatable whether they are specific signatures of cancer. Thus differential diagnosis (benign vs. malignant) solely based on changes in normal tissue components has been unsuccessful. The question we address is: are there unique spectral differences between the normal and tumor-containing breast tissues besides spectral differences resulting from tissue composition? To this end we developed a double differential spectroscopic method which reveals intrinsic specific spectroscopic markers of cancers which have never been accounted for in conventional spectral models.
Materials and Methods: A Diffuse Optical Spectroscopy (DOS) instrument was used to recover non-invasively the absorption and scattering spectra from 650-1000 nm ... [truncated at 150 words]
Rossow MJ, D'Amico E, Gatto R, Charbel FT, Mantulin WW, Gratton E.
Intra-operative blood flow measurement with near-infrared spectroscopy and a camera detection system.
2006 Biomedical Engineering Society (BMES) Annual Fall Meeting. October 11-14, 2006, Chicago, Illinois.
We present a technique for measuring flow that can be applied to blood flow in small, exposed blood vessels during surgery. This method makes use of the fact that blood, when viewed on a small enough scale, is an inhomogeneous substance. Individual blood cells passing between a nearinfrared (NIR) light source and a detector will cause fluctuations in the transmitted optical signal. The speed at which the blood cells are traveling can be determined from these optical fluctuations. However, the transmitted NIR signal is noisy. The dense concentration of blood cells, the low transmission of the blood vessel wall, and the non-uniform velocity profile along the cross section of the blood vessel all contribute to a complicated raw optical signal. We have implemented two techniques to overcome these problems. The first is correlation analysis to mathematically extract the average time scale of fluctuation. The second is the use of multiple ... [truncated at 150 words]
Calderon-Arnulphi M, Alaraj AM, Amin-Hanjani S, Roy SS, Mantulin WW, Michalos A, Gratton E, Charbel FT.
Use of quantitative near-infrared spectroscopy for monitoring cerebral oxygenation in patients undergoing cerebrovascular angioplasty.
56th Congress of Neurological Surgeons Annual Meeting. October 7-12, 2006. Chicago, IL.
Introduction: Monitoring brain tissue oxygenation could be of great value in the evaluation of cerebrovascular angioplasty procedures. Quantitative (frequency domain) near infrared spectroscopy (Q-NIRS) allows quantitative, continuous, non-invasive measurements of tissue Oxy- (HbO2), Deoxy- (HHb), total (tHb) hemoglobin concentrations and brain tissue oxygen saturation (SO2). Q-NIRS offers three major advantages:1) absolute values of tissue oxygenation 2) real-time measurement and 3) measurements independent from scalp tissue oxygenation (ref.1,2,3,4). This technology provides immediate feedback about changes in cerebral oxygenation related to the procedure; currently there is no other portable monitoring instrument able of providing this information.
Methods: We used Q-NIRS to monitor six patients undergoing cerebrovascular angioplasty: three internal carotid artery and three middle cerebral artery angioplasties. Q-NIRS sensors were applied to the scalp of patients bilaterally to monitor the affected and the contralateral hemispheres. Monitoring was performed before, during and after the procedure, and absolute changes in cerebral oxygenation parameters were analyzed. ... [truncated at 150 words]
-
Particle tracking in 3D: chromatin structure and dynamics.
CeNS Workshop: Emerging Nanosystems. September 25-29, 2006. Venice International University, San Servolo, Venice, Italy.
The utilization of 2-photon microscopy in the field of Cell Biology is of increasing importance because it allows imaging of living cells, including those systems where UV imaging is not possible due to photobleaching or photodamage limitations . Using 2-photon excitation, the tracking method described in this work is suitable for studying the dynamics of single particles in cells or other systems sensitive to UV light.
We propose a novel approach based on the use of a scanner to produce an effective intensity trap. As the particle moves in this trap (note that there is no force applied on the particle at the power level we are using for particle detection), the detection system continuously calculates the position of the particle in the trap. As the position of the particle is calculated with respect to the trap, the scanner position is moved to minimize the ?modulation? of the light intensity in ... [truncated at 150 words]
-
Micro domain fluctuations in membranes.
The 232nd ACS National Meeting, San Francisco, CA, September 10-14, 2006.
Abstracts of papers of the ACS. 2006; 232, 656-PHYS.Micro domains form during the phase transition in synthetic membranes composed of single phospholipids or of a mixture of phospholipids with different transition temperature. The fluctuations in size and time of micro domains have not been studied extensively. We describe novel results obtained using the Laurdan fluorescence probe that report on the local water penetration in the membrane. The system studied are giant unilamellar vesicles that are well visible under the microscope. Both the excitation and the emission spectrum of Laurdan are affected by the surrounding packing of the lipids. Using a two-photon microscope and the recently described scanning fluorescence correlation spectroscopy technique we were able to study the onset of phase separation. We also describe how the phases in the two lamellae of the membrane get coupled over time. A surprising result of our studies is the relatively large micro heterogeneity of the lipid liquid phase.
Behne MJ, Sánchez SA, Moll I, Gratton E.
Major translocation of calcium upon barrier insult: Calcium dynamics visualized by - Fluorescence lifetime imaging.
J Invest Dermatol. 2006; 126(Suppl 3): S17, 88.Calcium controls an array of key events in keratinocytes and epidermis: Localized changes in
Ca2+ concentrations and their regulation are therefore especially important to assess in epidermal
barrier homeostasis and repair, neonatal barrier establishment, in differentiation, signaling,
cell adhesion, and in various pathologic states. Yet, tissue- and cellular Ca2+ concentrations
in physiologic and diseased states are only partially known, and diffi cult to measure. Here we
report a method using Calcium Green as the calcium sensor and the phasor-plot approach to
separate raw lifetime components. This enables us to quantitatively assess and visualize dynamic
changes of Ca2+ in ex vivo biopsies of unfi xed epidermis, exploiting fl uorescence lifetime
imaging. Our fi rst results comparing undisturbed epidermis with epidermis following a barrier
insult revealed major shifts from intra- to extracellular, and, more importantly, a mobilization
of high amounts of Ca2+ shortly following barrier disruption, presumably from intracellular
stores. These results partially contradict the conventional view, where barrier insults abrogate
a Ca2+ -gradient ... [truncated at 150 words]
Lee CY, Colyer RA, Gratto E.
A novel fluorescence microscopy method for skin measurements.
International Symposium on LifeChips 2006. May 16-17, 2006. Irvine, CA, USA.
Fluorescence lifetime imaging microscopy (FLIM) is a very powerful method to detect ion concentrations in cells and tissue and for the determination of FRET. FLIM has many advantages over fluorescence intensity imaging microscopy, particularly in inhomogeneous, biological systems. However, FLIM is not commonly used because of the high cost of the FLIM hardware, the difficulty in setting up an experiment, and the intricacies of data analysis. We have developed new hardware and new analysis method that has resolved these problems. The hardware is simple and inexpensive and the method of data analysis can be used by non-specialists.
Blaine JT, Takahashi H, Barry NP, Digman MA, Lee CY, Breusegem SY, Mantulin WW, Gratton E, Levi M.
The heterogeneity of NaPi protein dynamics and NaPi cotransport activity in renal brush border membranes.
Experimental Biology 2006. San Francisco, California. April 1-5, 2006.
FASEB J. 2006; 20(Meeting Abstracts), A59.Alterations in renal proximal tubule brush border membrane (BBM) cholesterol, sphingomyelin, and glycosphingolipid content play an important role in regulating the activity of the sodium-phosphate cotransporter (NaPi). The molecular mechanisms of how alterations in lipid composition may modulate NaPi activity are not known. We have fractionated BBM prepared from rat kidney using detergent-free density gradient ultracentrifugation (OptiPrep). We have found that the NaPi protein preferentially partitions into lipid rafts. To determine the potential consequences of the partitioning of the NaPi transporter in these lipid domains we have i) measured NaPi transport activity and ii) used fluctuation correlation spectroscopy (FCS) methods to determine NaPi diffusion. Partitioning of NaPi protein into lipid rafts results in i) decreased NaPi cotransport activity and ii) decreased diffusion of NaPi protein. Similar results were obtained in BBM isolated from the superficial cortex (SC) versus the juxtamedullary cortex (JMC). The three-fold decrease in NaPi cotransport activity in ... [truncated at 150 words]
Behne MJ, Sánchez SA, Moll I, Gratton E.
Calcium-fluorescence lifetime imaging in ex-vivo skin.
Experimental Biology 2006. San Francisco, California. April 1-5, 2006.
FASEB J. 2006; 20(Meeting Abstracts), A117.Calcium controls several key events in keratinocytes and epidermis: epidermal barrier homeostasis and repair, neonatal barrier establishment, differentiation, signaling, cell adhesion, and various pathologic states. Yet, tissue- and cellular Ca2+ concentrations in physiologic and diseased states are only partially known. The very barrier properties of epidermis pose a major obstacle in determinations of the Ca2+ concentrations in intact skin. Methodology to measure and localize Ca2+ in intact and unfixed tissue has been lacking, while so far Ca2+-precipitation electron microscopy, or proton-induced x-ray emission were used. Both techniques are limited in that they can determine Ca2+ in only very small sample volumes, at or below light microscopic resolution levels, require fixed tissue and a chemical precipitation, or determine only total Calcium, irrespective of ionization or binding. So far, neither cellular and/or subcellular localization can be determined through these approaches. In cells, fluorescent dyes have been used extensively for ratiometric measurements of ... [truncated at 150 words]
-
Tracking protein-coated particles in 3D.
APS March Meeting, Baltimore, MD, March 13-17, 2006.
The utilization of 2-photon microscopy in the field of Cell Biology is of increasing importance because it allows imaging of living cells, including those systems where UV imaging is not possible due to photobleaching or photodamage limitations. We propose a novel approach using 2-photon excitation based on the use of a scanner to produce an effective "intensity trap''. As the particle moves in this trap (note that there is no force applied on the particle at the power level we are using for particle detection), the detection system continuously calculates the position of the particle in the trap. As the position of the particle is calculated with respect to the trap, the scanner position is moved to minimize the "modulation'' of the light intensity in the trap. In practice, we set the scanner to perform an orbit around the particle in about 1 millisecond. The sampling rate is chosen such ... [truncated at 150 words]
Toro CA, Zanocco A, Gunther G, Gratton E, Sánchez SA.
Detergent solubilization of lipid bilayers followed by 2-photon microscopy.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 276-Pos/B142.The solubilization of biological membranes by detergents has been used as the main method for the isolation and purification of membrane proteins and other constituents.
Recently, considerable interest has been attracted by the finding that biological membranes can be solubilized selectively. Certain membrane constituents are incorporated into small micelles, whereas others remain in the so-called detergent-resistant membrane fragments that are large enough to be separated by centrifugation. This provides a unique tool to select classes of proteins to be isolated and to study the preferences of proteins for certain membrane environments, and it will be of great value also for future large-scale studies of membrane proteins.
We report here the use of 2-photon microscopy to study the solubilization process in artificial POPC liposomes and erythrocytes. LAURDAN is a membrane probe that gives information about the water content in the bilayer that is related with the membrane fluidity. We present here preliminary results ... [truncated at 150 words]
Levi V, Serpinskaya AS, Gelfand VI, Gratton E.
Single particle tracking study of organelles transport in Xenopus Melanophores.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 647-Pos/B513.Organelle transport is essential for a variety of cellular functions. Xenopus melanophores have black pigment organelles or melanosomes which, in response to hormonal signals, disperse in the cytoplasm or aggregate in the perinuclear region. Melanosomes are moved by two microtubule motors, kinesin-II and cytoplasmic dynein and the actin motor myosin V. We explored organelle transport along microtubules and actin filaments by using a new fast-tracking routine designed for a microscope under bright field illumination which has spatial precision of 2 nm and temporal resolution of 10 ms. Melanophores were incubated with nocodazole or latrunculin B to depolymerize microtubules and actin filaments, respectively. In these conditions, the transport is driven only by myosin V or microtubule-motors. Melanosomes moving along microtubules presented velocity distributions with several peaks that could not be fit with single Gaussian functions. We postulated that the melanosome velocity depends linearly on the number of active motors. According to ... [truncated at 150 words]
-
Ultrafast analysis of fluorescence lifetime images using the phasor approach: application to FRET analysis.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 681-Pos/B547.We developed a method to analyze and visualize decay components in fluorescence lifetime imaging microscopy (FLIM) based on the representation of few moments of the decay distribution. This method is based on decomposition of the decay in Fourier components which have a simple geometrical interpretation in a phasor plot. In this plot, each decay component can be represented by a single point, irrespective of the number of exponentials needed for its description. The ensemble of all decay components in an image is shown as raw data in the phasor plot. The algebra of vectors applies to the phasor representation of the decay. Decay components due to background or other fluorescent species are subtracted using vector algebra. The resolution of images in decay components can be done by finding the vectors that add to a given measured phasor point. Using vector addition the operator explores the phasor plot and determines whether ... [truncated at 150 words]
Colyer RA, Lee CY, Gratton E.
Frequency-domain fluorescence lifetime imaging microscopy in the photon-counting regime with field-programmable gate arrays.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 684-Pos/B550.Fluorescence lifetime imaging microscopy (FLIM) is growing in usefulness as a means of distinguishing molecular species, observing environmental regions which affect the lifetime of a fluorophore, and observing fluorescence resonance energy transfer (FRET). We present a technique for performing FLIM in a laser scanning microscope using the photon counting regime of the photomultiplier detector in conjunction with a field-programmable gate array
(FPGA).
An FPGA is a programmable logic device which can be used for digital signal processing. In our technique we synchronize the internal clock of an FPGA to the laser repetition rate, and use a rapidly shifting phase delay to scan through the entire repetition time of the laser. The laser's repetition time is divided into four separate measurement windows which are incrementally phase shifted by the FPGA's digital clock manager. This technique samples the entire lifetime curve evenly with a 100% duty cycle. The data is interpreted using the heterodyning ... [truncated at 150 words]
-
Fluctuation correlation spectroscopy with colloidal gold particles.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 685-Pos/B551.Fluctuation correlation spectroscopy (FCS), or, fluorescence correlation spectroscopy, was initially inspired by experiments counting free-floating gold particles passing through the focal volume of a microscope. Gold particles are still frequently used in biological applications for their easily manipulated chemistry, but rarely are FCS techniques applied to gold beads. We discuss the difference in fluctuation experiments between fluorescent probes and plasmon resonance from gold particles.
We observed the intensity fluctuation due to plasmon resonance of diffusing spherical, colloidal gold nano-particles and compared it with the fluctuation obtained with common fluorescent probes. A 543.5nm He-Ne laser was used both to excite the fluorescent probes and to illuminate the gold particles, so that the illumination volume was identical. We changed the dichroic of the standard confocal microscopy system with a beam splitter to observe the plasmon resonance signal from the gold particles. The auto-correlation curves obtained form the fluctuation correlation experiment showed unexpected ... [truncated at 150 words]
-
Characterization of particle angular fluctuations by intensity and polarization trajectory plots.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 691-Pos/B557.The orthogonally polarized emission time series from a polarization fluorescence correlation spectroscopy (pFCS) experiment contains details on the angular motions of the individual molecular species under observation. Information on the internal motion of cellular components is of general interest and can potentially be accessed through pFCS. Angular motions for larger cellular constituents and structures range from microsecond through the millisecond time scales, depending on the process, which are within a suitable time for pFCS. Unfortunately, the presence of a significant cellular autofluorescence greatly interferes with the spatial and angular correlation of the emission from the intended target fluorophores. In an attempt to separate signal from background, the two polarized intensity time series of a pFCS experiment can be plotted against one another to yield a two-dimensional image that contains the fluorophore emission dipole position scaled by the intensity. In this trajectory plot, one can visually isolate rare (relative to background) ... [truncated at 150 words]
Digman MA, Brown CM, Horwitz AR, Mantulin WW, Gratton E.
Fast dynamics in the cell interior revealed by RICS.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 699-Pos/B565.Single point fluctuation correlation spectroscopy (FCS) is an established technique to study diffusion and chemical equilibria in solution. It has limitations when applied to the cell. Major difficulties of this technique are that the movements of the cells or of cellular components are difficult to separate and filter out from the molecular dynamics. Scanning FCS, in which the laser beam is moved in a circular orbit provides the fluctuation amplitude and dynamics at many point simultaneously and it can be used to detect the cell movement. Image correlation spectroscopy has the potential to provide detailed maps of the dynamics in a cell, but it suffers from the limitation that the frame rate is relatively slow. We have developed an approach that exploits the intrinsic time structure of a confocal raster scan image to determine spatiotemporal correlations at several time scales simultaneously. Using this approach, termed RICS (Raster-scan Image Correlation Spectroscopy), ... [truncated at 150 words]
Gomes AMO, de Oliveira AC, Levi V, Goncalves RB, Katpally U, Smith TJ, Gratton E.
New insights on transport and particle dynamics obtained from real-time 3D tracking of viral particles in live cells.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 1483-Pos/B604.We investigated early events of virus infection in live cells by using the 3D tracking technique described by Levi et al. (Biophys J. 88: 2919-28, 2005). In this work, we followed Human Rhinovirus (HRV14) particles labeled with FITC entering into living HeLa-H1 cells, with nanometer accuracy and millisecond time resolution. We separated three different stages of the particle transport, specifically related to attachment to the cell surface, transport inside the cell and confinement in a region of the cell. Disruption of microfilaments and microtubules using latrunculin B and nocodazole, respectively, showed that particle transport in the cell is affected by actin depolimerization but not by tubulin breakdown. During the tracking experiment, the fluorescence intensity from the single particle is recorded and two interesting characteristics are shown: no photobleaching during tracking (>30 min) and an oscillatory intensity pattern, suggesting fast conformational changes of the particle’s size. Self-quenching due to FRET is ... [truncated at 150 words]
Brown CM, Digman MA, Gratton E, Horwitz AR.
Measuring protein binding kinetics at submicron resolution from high speed total internal reflection fluorescence microscopy images.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 2314-Plat.Traditionally fluorescence correlation spectroscopy (FCS) and image correlation spectroscopy (ICS) have been applied to laser confocal scanning confocal or two photon microscope images. Here we develop a new temporal ICS method where we perform the correlation analysis on total internal reflection fluorescence (TIRF) images. Although FCS has been applied in combination with TIRF it has never been applied to TIRF images. Using a high speed on chip gain electron multiplied CCD camera we are able to collect data with frame rates of 10 msec or 100 ps. Collecting data for about 100 s provides time series at each pixel of about 10000 points. We calculate the time correlation at each point and present the data as a map of the G(0), the amplitude of the fluctuation and a map of the rate which are obtained from an exponential fit of the autocorrelation function (ACF) at each pixel. The G(0) map ... [truncated at 150 words]
Sánchez SA, Tricerri MA, Gomes AMO, Levi V, de Oliveira AC, Gratton E.
Reconstituted HDL particles as an alternative acceptor to remove cholesterol in vitro and in vivo.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 2439-Pos/B106.The possible role of lipid rafts in the regulation of the activity of membrane proteins and the induction of signal of physiological significance are the focus of many studies. One common way to asses the involvement of rafts in cellular process is to manipulate the cholesterol content of the membrane. One way of removing cholesterol both in vivo and in vitro is using beta-methyl cyclodextrin. These compounds have a high capacity to accept cholesterol and they are easy to use, however in some cases they can be toxic for the cells.
Here we report the use of an alternative cholesterol acceptor, reconstituted HDL particles (rHDL). rHDL particles have been used for a long time to study the reverse cholesterol transport mechanism and the role of apo A-I and HDL particles in cardiovascular diseases. Here we report their use as a cholesterol acceptor that can be use in experiments that require a ... [truncated at 150 words]
-
Lipid organization fluctuations in model membranes studied by two photon fluorescence microscopy.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 2464-Pos/B131.Two-photon fluorescence microscopy was used to study water penetration and lipid organization in model lipid membranes as a function of temperature and hydrostatic pressure. Single lipid GUVs were grown by electroformation. We used Laurdan to label the vesicles. The emission spectrum of this fluorescent probe is sensitive to the degree of water penetration into the lipid membrane. The spectral shift of LAURDAN is quantified in the GUV images by using the GP function. For the high pressure experiments, we used thin quartz capillaries as the pressure vessels. POPC and DLPC GUVs were pulled in the capillary and GP images were taken during compression and decompression cycles. For the temperature experiments GP images of DEPC, DMPC and DPPC GUVs were acquired while decreasing the temperature. For every image the average value and the pixel standard deviation of the GP distribution were calculated and plotted as a function of temperature or pressure. ... [truncated at 150 words]
-
Microsecond mixing with polarization and spectral resolution for fast reaction kinetics.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 693-Pos/B559.An instrument capable of recording fluorescence spectra and fluorescence anisotropy images has been integrated with a micro-fabricated, turbulent flow mixer to resolve microsecond kinetics of biological reactions. The turbulent mixer is able to completely mix two pure reagents within several microseconds. The resulting fluorescence data, taken along the length of the exit channel, is an image of the time evolution of the fluorescence spectra (or fluorescence anisotropy) as the reaction progresses. Here we present some example reaction times that have been measured utilizing FRET and anisotropy. Diffusion limited reactions that have been used to quantify the dead time of the mixer are also presented.
Noomnarm U, Sutin JDB, Clegg RM.
Repetitive pressure-jump for relaxation kinetics.
50th Annual Meeting of the Biophysical Society, Salt Lake City, Utah, 2006.
Biophys J. 2006; Suppl, 692-Pos/B558.Relaxation kinetics follows the time course of a chemical system changing from an initial equilibrium state to a new equilibrium state as a result of an external thermodynamic perturbation. Because the perturbed chemical systems remain close to equilibrium, the analysis of even complex reaction systems can be analyzed by linear differential equations. In addition, thermodynamic information (eg DV and DH) is directly available. On the other hand, steady-state kinetics or pre-steady state kinetics start the reaction system far from equilibrium and often bypass important intermediates. Pressure changes of 1-50 bar usually cause a free energy perturbation of chemical systems several orders of magnitude smaller than result from temperature changes in the range of 1-5 K. Another great advantage of pressure, is that perturbations smaller than a few thousand bar, in contrast to temperature changes of even a few degrees, often change the aggregation state of a macromolecular complex without disrupting ... [truncated at 150 words]
-
Looking into the brain with Lasers.
Saturday Physics Honor Program, Department of Physics, University of Illinois at Urbana-Champaign.
Parts of our body are relatively transparent to light. When red light is used for illumination, the light will pass through several centimeters of tissue. The reason why we cannot see through our body is because of multiple scattering due to cells and organelles. By reconstructing the path the light followed when passing through the tissue, it is possible to determine the shape of internal organs and their optical properties. In this talk, the physical principles that are used to reconstruct the interior of the body are explained as well as a number of applications to medicine and psychology.
Calderon-Arnulphi M, Alaraj AM, Gatto R, Nersesyan H, Mantulin WW, Michalos A, Gratton E, Charbel FT.
Bedside evaluation of cerebral autoregulation using frequency domain near infrared spectroscopy.
55th Congress of Neurological Surgeons Annual Meeting. October 8-13, 2005. Boston, MA.
Strokes, hemorrhages and impaired cerebral blood flow are a frequent cause of morbidity and mortality in the United States. In these pathological conditions equilibrium of cerebral blood flow autoregulation is lost. Currently, there is no non-invasive portable tool to screen patients with possible impaired autoregulation. We have used frequency domain near infrared spectroscopy (FD-NIRS) to monitor of 19 neurosurgical patients. FD-NIRS provides quantitative, non-invasive, continuous real time information about the brain oxygenation. Oxy- (HbO2), Deoxy- (HHb), total (tHb) hemoglobin concentrations and brain tissue oxygen saturation (SO2) are measured quantitatively. Eleven patients had superficial temporal artery to middle cerebral artery (STA-MCA) bypass for cerebral blood flow augmentation, 2 patients had carotid endartectrectomy and 6 patients had subarachnoid hemorrhages. All STA-MCA bypasses had SPECT scan showing impaired autoregulation on the affected side. FD-NIRS sensors were applied bilaterally on the scalp of patients on the affected and the contralateral side measuring cerebral oxygenation ... [truncated at 150 words]
Calderon-Arnulphi M, Gatto R, Alaraj AM, Nersesyan H, Mantulin WW, Michalos A, Gratton E, Charbel FT.
Prediction of cerebral ischemia in neurovascular surgery.
55th Congress of Neurological Surgeons Annual Meeting. October 8-13, 2005. Boston, MA.
Monitoring of brain tissue oxygenation during neurovascular surgery could be of great value to neurosurgeons. It provides real time feedback that may detect acute ischemic injury. This information is not available with current intraoperative techniques. Frequency domain near infrared spectroscopy (FD-NIRS) allows quantitative continuous, real time, non-invasive measurements of tissue Oxy- (HbO2), Deoxy- (HHb), total (tHb) hemoglobin concentrations and brain tissue oxygen saturation (SO2). FD-NIRS has two major advantages: It provides quantitative values and is independent from oxygenation of the scalp tissue. We have monitored 22 neurovascular procedures using FD-NIRS including: 11 neurovascular bypasses, 3 carotid endarterectomies, 2 aneurysm clipping, 1 AVM resection and 5 balloon occlusion tests. FD-NIRS sensors were applied bilaterally on the scalp of patients on the affected and the contralateral side. Variations of the measurement reflected tissue perfusion. One patient bleed during surgery, had brain edema and died two weeks after surgery (Figure 1); another patient ... [truncated at 150 words]
Malengo G, Zamai M, Andolfo A, Sengupta P, Sutin JD, Blasi F, Gratton E, Sidenius N, Caiolfa VR.
Ligand-induced monomer/dimer dynamics of the urokinase receptor in live cells by 2P-FLIM/FCS.
Joint 15th IUPAB and 5th EBSA International Biophysics Congress. Montpellier, France. Aug 27-Sept 1, 2005.
Euro Biophys J. 2005; 34(6): 536, O-20, P-30.The urokinase receptor (uPAR) is a multifunctional receptor, which regulates cell adhesion, migration and proliferation in normal and pathological situations. It is currently unknown how the GPI anchored uPAR transmits a variety of signals into the cell. We follow the effect of uPAR ligands on live HEK293 cells expressing functional Fuorescent chimeras of uPAR, in which EGFP or spectral variants, were inserted between the GPI-anchor and the D3 receptor domain. By combining 2-photon FLIM and Fuorescence Fuctuation spectroscopy (FCS, PCH), we show that uPAR monomers and dimers are heterogeneously distributed in membrane microdomains. Dimers increase in the cell-to-cell contacts as well as in the presence of the amino terminal fragment of the ligand uPA. In contrast, PAI-1-dependent internalization of uPAR results in a reduction of uPAR dimers exposed in the plasma membrane. These results support the hypothesis that uPAR monomer/dimer interplay can be regulated by extracellular ligands such as uPA ... [truncated at 150 words]
Czeslik C, Jackler G, Ballauff M, Gratton E, Hazlett TL.
Protein immobilization at polyelectrolyte brushes.
Joint 15th IUPAB and 5th EBSA International Biophysics Congress. Montpellier, France. Aug 27-Sept 1, 2005.
Euro Biophys J. 2005; 34(6): 669, P-348.There is a series of examples where immobilized proteins play an important role, such as solid-phase immunoassays for medical diagnostics or protein biochips that are used in protein analysis. However, proteins may undergo severe conformational changes at a solid surface which can lead to a loss of biological activity. To optimize protein adsorption, the solid surface can be modifed by a poly(acrylic acid) (PAA) brush. We used two-photon excitation Fuorescence Fuctuation spectroscopy, neutron reflectometry and CD spectroscopy to study the binding of proteins to spherical PAA brush particles and planar PAA brushes. It has been found that a PAA brush binds huge amounts of protein at low ionic strength, whereas a salt concentration of a few 100 mM in the protein solution renders the PAA brush protein resistant. Remarkably, this effect of salt appears to be independent of protein net charge. Without added salt, protein molecules penetrate deeply into the ... [truncated at 150 words]
Clegg RM, Breusegem SY, Barry NP, Holub O, Govindjee, Redford GI.
How can one choose the best method for measuring FRET in a microscope with my biological system?.
Microscopy and Microanalysis 2005. Honolulu, Hawaii, USA, July 31-August 4, 2005.
Microsc Microanal. 2005; 11(Suppl 2).Which method is the best for imaging FRET? This is a common question. It is simply
answered if you have only one possibility for measuring FRET. However, even in this
case it is best to be aware of the pitfalls, as well as the advantages, for the different
methods. It can be difficult to quantify FRET in cells because of experimental artifacts
and complexities in determining the necessary photophysical parameters. There are many
reasons for these difficulties: photobleaching, distributions of distances, varying relative
angles between the donor and acceptor, aggregation of the labeled macromolecules, lack
of information about relative concentrations of the complementary FRET pairs in
different locations, stability of the image during data acquisition, less that 100% labeling
of the donor and acceptor, spatially varying index of refraction, pH and ionic strength,
different extents of static or dynamic quenching. Every effect contributes uniquely and to
a different extent to the various methods of FRET measurements. In this lecture we will
give ... [truncated at 150 words]
Celli A, Lee CY, Gratton E.
High hydrostatic pressure induced shape and lipid phase changes on giant unilamellar vesicles studied with two-photon excitation microscopy.
6th International Weber Symposium. Kauai, Hawaii. July 22-28, 2005.
We used two-photon-fluorescence microscopy to investigate the effects of pressure on the morphology and lipid phase of Giant Unilamellar Vesicles (GUVs). GUVs of the size of few tens of microns are grown by electroformation. After detaching the vesicles from the electrodes, they are pulled into transparent quartz capillaries with an inner diameter of 50 mm and an outer diameter of 375 mm. Pressure can be applied to the vesicles inside the capillaries sealing one end and connecting the other to a high pressure pump. We used LAURDAN to label the GUVs. The emission spectrum of this fluorescent probe is sensitive to the degree of water penetration into the lipid membrane. The spectral shift of LAURDAN is quantified in the GUV images by using the GP function. After locating a vesicle in the capillary, we followed both the GP and shape changes due to high hydrostatic pressure. We observed GP and ... [truncated at 150 words]
Majumdar ZK, Hickerson R, Noller HF, Clegg RM.
Measurements of internal distance changes of the 30s ribosome using FRET with multiple donor-acceptor pairs: quantitative spectroscopic methods.
6th International Weber Symposium. Kauai, Hawaii. July 22-28, 2005.
I will present analytical and experimental procedures for determining distance changes within the 30S subunit of the Escherichia coli ribosome using Förster resonance energy transfer (FRET). Ways to contend with complexities when using FRET to measure distance changes within large multi-subunit macromolecular complexes, such as the ribosome will be discussed. Complications can arise due to non-stoichiometric labeling of donor and acceptor probes, as well as environmental effects that are specific to each conjugation site. We show how to account for changes in extinction coefficients, quenching, labeling stoichiometry and other variations in the spectroscopic properties of the dye to enable more accurate calculation of distances from FRET data. Approximations that concern the orientation of the transition moments of the two dye molecules, as well as the impact of other errors in the measurement of absolute distances, will be discussed. Thirteen dye-pair locations with different distances using 18 independent FRET pairs conjugated ... [truncated at 150 words]
Digman MA, Brown CM, Horwitz AR, Mantulin WW, Gratton E.
Diversity of structure and dynamics of focal adhesions revealed by orbital scanning FCS, line scanning FCS and raster image correlation spectroscopy.
6th International Weber Symposium. Kauai, Hawaii. July 22-28, 2005.
In our study of the role of focal adhesions (FAs) in cell migration, we mapped the dynamics of paxillin-eGFP with orbital scan, line scan and a novel technique termed raster image correlation spectroscopy (RICS) developed in our laboratory. FAs are an intricate network of proteins, including paxillin, and other cellular components that form attachment points that tether the cell or form at the leading edges of cell protruding forward during cellular migration. Many studies have shown that adhesion assembly and disassembly is guided by a highly organized system of protein interactions. However, the dynamics of this process remain unclear and may differ between stable and newly formed adhesions. In stable adhesions, our results with orbital scanning of paxillin indicate dynamic processes suggesting that adhesions are not static. We are also able to determine that paxillin does not form large aggregates but may be recruited with other proteins. In newly formed ... [truncated at 150 words]
Gomes AMO, de Oliveira AC, Levi V, Goncalves RB, Katpally U, Smith TJ, Gratton E.
Real-time 3D tracking of viral particles in live cells.
6th International Weber Symposium. Kauai, Hawaii. July 22-28, 2005.
During the last year, we developed a new technique to track particles in three dimensions using a 2-photon microscope. The next step involves developing and testing the technique for tracking fluorescent particles in living cells. As a test system, we used relatively large particles, such as viruses. Detailed mechanisms of infection remain unclear for most animal viruses probably because of experimental difficulties. Tracking single particles in living cells eliminates the need for large amounts of viral material in order to have a good signal-to-noise ratio. Here we describe the possibility to track virus particles in living cells with nanometer accuracy and millisecond time resolution. The model utilized was the Human Rhinovirus (HRV14). Rhinoviruses are small RNA viruses, members of Picornaviridae family. Virus capsids were labeled with FITC and incubated with HeLa-H1 cells at 4oC to allow attachment to the membrane. Then the temperature was raised to 37oC and the particles ... [truncated at 150 words]
-
Spatio-temporal correlations for the study of fluctuations in the cell interior.
6th International Weber Symposium. Kauai, Hawaii. July 22-28, 2005.
The use of fluorescence correlation spectroscopy has expanded very much during the last 5 years, mainly for the study of the dynamics of the cell interior. Although single point FCS is a well established technique for the study of diffusion and chemical equilibria in solution, it has several limitations when applied to the cell interior. A major difficulty is that the movements of the entire cells or of cellular components are difficult to separate and filter out from the intrinsic molecular dynamics. Several approaches has been suggested to deal with these problems. Scanning FCS, in which the laser beam is moved in a circular orbit provides the fluctuation amplitude and dynamics at several point along the orbit simultaneously and it can be used to detect the cell movement. Image correlation spectroscopy has the potential to provide detailed maps of the dynamics in a cell, but it ... [truncated at 150 words]
Hazlett TL, Barcellona ML, Digman MA, Gratton E.
Rotational dynamics from the autocorrelation of the sample polarization time series.
6th International Weber Symposium. Kauai, Hawaii. July 22-28, 2005.
The concept and application of fluorescence polarization correlation spectroscopy measurements is discussed. The ability of this technique to isolate the angular and rotational information from the translational diffusion is illustrated with both simulated and sample data. The effective range of this technique in terms of particle brightness, diffusion time and rotational correlation time are explored. In our instrument, two-photon excitation is used to define a small volume of illumination where a few molecules are present at any given instant of time. The polarized output of a Ti:Sapphire laser is used as our excitation source. The emission path contains a polarizing beamsplitter cube to separate the parallel and perpendicular components of the fluorescence that are simultaneously collected. Various samples have been examined that include dsDNA, Ribosomes, small phospholipid vesicles, and proteins in viscous media.
Inoue M, Digman MA, Breusegem SY, Mantulin WW, Gratton E, Barry NP, Levi M.
Partitioning of NaPi cotransporter in cholesterol, sphingomyelin and glycosphingolipid enriched membrane microdomains modulates NaPi protein diffusion, clustering and activity.
6th International Weber Symposium. Kauai, Hawaii. July 22-28, 2005.
In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein NaPi in spite of an increase in its apical membrane abundance. This novel posttranslational regulation of NaPi activity is mediated by the increased glycosphingolipid content of the potassium deficient apical membrane. However, the mechanisms by which these lipids modulate NaPi cotransport activity have not been determined. In the present study we determined if in potassium deficiency NaPi protein is increasingly partitioned in cholesterol, sphingomyelin, and glycosphingolipid enriched microdomains of the apical membrane and if increased presence of NaPi protein in these microdomains results in modulation of its activity. Using a detergent-free density gradient flotation technique we found that 80% of the apical membrane NaPi protein partitions into the low-density cholesterol, sphingomyelin and GM1 enriched fractions characterized as “lipid raft” fractions. In potassium deficiency a higher proportion of NaPi protein was localized ... [truncated at 150 words]
Levi V, Belmont AS, Plutz M, Gratton E.
Dynamics of chromatin in interphase cells revealed by fast-tracking in a two-photon microscope.
6th International Weber Symposium. Kauai, Hawaii. July 22-28, 2005.
Increasing evidence points to a dynamical compartmentalization of the cell nucleus, yet the mechanisms by which interphase chromatin moves and is positioned within nuclei remain unclear. Here, we study the dynamics of chromatin in vivo applying a novel particle tracking method in a two-photon microscope that provides ~10 fold higher spatial and temporal resolutions than previous measurements. We followed the motion of a chromatin sequence containing a lac operator repeat in cells stably expressing lac repressor fused with enhanced-green fluorescent protein, observing long periods of apparent constrained diffusion interrupted by relatively abrupt jumps of ~150 nm lasting 0.5-2 sec. During these jumps, the particle moved an average of 4 times faster than in the periods between jumps and in paths more rectilinear than predicted for random diffusion motion. Additionally, the jumps were sensitive to the temperature and absent after ATP depletion. These experimental results point to an energy dependent ... [truncated at 150 words]
Sánchez SA, Tricerri MA, Gratton E.
Laurdan GP, rHDL and cholesterol removal.
6th International Weber Symposium. Kauai, Hawaii. July 22-28, 2005.
Originally synthesized by Gregorio Weber, Laurdan (6-lauroyl-2-(dimethylamino) naphthalene) is used as a membrane probe because of its large excited state dipole moment, which results in its ability to report the extent of water penetration into the bilayer surface. Water penetration has been correlated with lipid packing and membrane fluidity. Laurdan generalized polarization (GP) is the parameter used to quantify water penetration and then indirectly of the membrane fluidity. GP measurements these days can be done in the microscope and give the spatial resolution necessary for membranes studies. Here, we report the use of GP measurements to quantify cholesterol removal at equilibrium and with high spatial resolution using two-photon microscopy. Measurements of GP in time and FCS in Giant Unilamellar vesicles of POPC with different percentage of cholesterol show differences in the cholesterol removal capabilities of HDL particles of different sizes. In GUVs made of DOPC/DPPC/cholesterol presenting domain coexistence we ... [truncated at 150 words]
-
Green fluorescent pasta with subnanosecond lifetime.
6th International Weber Symposium. Kauai, Hawaii. July 22-28, 2005.
Ever since the days of Galileo, Italian scientists have struggled to make Green Fluorescent Pasta (GFP) with subnanosecond lifetimes. The closest we have come to this goal was when Enrico Fermi achieved a self-sustaining chain of rigatolli on December 2, 1942 at the University of Chicago. I am proud to announce that subnanosecond GFP has finally been made in the kitchen of the Laboratory for Fluorescence Dynamics at UIUC. Preliminary results also suggest that FRET can occur between the GFP and a red marinara sauce. Calimari and mushrooms, however, appear to totally quench the fluorescence. I am presently exploring the possibility of FRET between blueberry sauce and the GFP. This work was made possible by generous support from Chef Boyardee and Papa Dels Pizzaria.
Garcia-Marcos A, Sánchez SA, Parada P, Jameson DM, Gratton E, Ballesta JPG.
Two-photon fluorescence microscopy in vivo studies of GFP-yeast ribosomal stalk proteins.
30th FEBS Congress, 2-7 July 2005, Budapest.
FEBS Journal. 2005; 272(S1): 254, C6-017P.The S. cerevisiae ribosomal stalk, an essential large subunit protuberance, is made of a 34 KD protein, P0, and four 12 KDa acidic proteins, P1a, P1b, P2a and P2bIn vitro results indicate that in purified ribosomes the four acidic proteins are present as monomers, which can form two preferential associations, P1a-P2b, P1a-P2b. Nevertheless, it has been shown that free P2 proteins can form dimers in solution, and unpublished cross-linking data suggest the existence of P2 dimers in the particles. To test the composition of the stalk inside cells, P0 and the four acidic proteins were GFP tagged, and expressed in yeast strains lacking the corresponding wild type proteins. The four single disrupted strains and all possible double disruptants were prepared. The transformed strains were studied by two-photon fluorescence microscopy and data analyzed using photon-counting-histogram (PCH) method, which allowed estimation of GFP molecules per particle. Using P0-GFP as a reference, the ... [truncated at 150 words]
Lera M, Sutin JDB, Aguilar JF, Gratton E.
Confocal profiling of spherical cavities.
Focus on Microscopy 2005, Friedrich Schiller University, Jena, Germany.
Interpretation of reflected confocal images of surface profiles is difficult since a single surface will generate two signals via both backscattering and specular reflection [1]. Although the signals are separate and appear in two different geometric locations, distinguishing the signals and the true location of the surface remains ambiguous. To resolve this ambiguity, an imaging scheme using semicircular entrance and exit pupils has been developed to create contrast between the backscattered and specular reflected light [2]. Spherical surfaces are important special cases of general interfaces. Spherical surfaces are especially useful since the geometric conditions for backscattering and specular reflection can be solved analytically. For a spherical surface, specular reflection occurs when the focus is on the surface of the cavity while backscattering occurs when the focus is at the geometric center of the sphere. In theory, the maximum intensity occurs during backscattering which leads to an artifact in the retrieved ... [truncated at 150 words]
Sutin JDB, Awtar S, Hart AJ, Slocum AH, Gratton E.
Improving the performance of the optical microscope via modern opto and optomechanical design.
Focus on Microscopy 2005, Friedrich Schiller University, Jena, Germany.
The trend in biological applications of optical microscopy has been towards greater precision and more quantitative experiments, requiring greater stability from the microscope. Furthermore, in many instances improvements in the interpretation of experiments are possible if data from several complementary methods are acquired simultaneously, requiring more flexibility in the configuration of the microscope. We are systematically improving the microscope through an integrated optical and the mechanical design. Our goals are to increase the resolution and sensitivity of experiments by improving rejection of external mechanical disturbances and minimizing of drifting of the microscope itself, especially due to thermal fluctuations. In addition, to make assembly of complicated experiments more convenient, we have developed self-aligned optical modules connected via kinematic couplers which allow rapid reconfiguration of the microscope with better than 300 nm repeatability [1].
Inhomogeneous thermal expansion of the body of the microscope is a major cause of instability during experiments. However, ... [truncated at 150 words]
Celli A, Lee CY, Gratton E.
High hydrostatic pressure induced shape and lipid phase changes on Giant Unilamellar Vesicles studied with two-photon excitation microscopy.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 1682-Pos/B668.We used two-photon-fluorescence microscopy to investigate the effects of pressure on the morphology and lipid phase of Giant Unilamellar Vesicles (GUVs).
GUVs of few tens of microns are grown by electroformation. After detaching the vesicles from the electrodes, they are pulled into quartz capillaries with inner and outer diameters of 50 mm and 375 mm. Pressure was applied to the capillary by sealing one end and connecting the other end to the pressure pump. We used LAURDAN to label the GUVs. The emission spectrum of Laurdan is sensitive to the degree of water penetration into the lipid membrane. The spectral shift of LAURDAN is quantified in the GUV images by using the GP function. After locating a vesicle in the capillary, we followed both the GP and shape changes due to high pressure. We observed GP and shape hysterysis when performing successive compression and decompression cycles. We observed that different types ... [truncated at 150 words]
Sengupta P, Digman MA, Gratton E.
Scanning image correlation spectroscopy reveals fast molecular dynamics.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 1669-Pos/B655.Image correlation spectroscopy (ICS) was originally developed to determine the average number of aggregates and the degree of aggregation of proteins in cells. Conventionally in ICS, the size and number of molecular aggregates is determined from intensity images obtained with a camera. Recently, this technique was also expanded to determine spatial and temporal correlations simultaneously by collecting a time series of images. However, the temporal information is always extracted from a series of different images in ICS and is mostly used for slowly diffusing species, i.e., particles that move in time comparable to the frame rate. Here we describe a variation of ICS using laser scanning microscopy (LSM). This new tool enables us to extract temporal information from a single image. The sequential acquisition of pixels intensities in scanning techniques inherently embeds the time information even in a single image, which is otherwise lost in the parallel acquisition in camera ... [truncated at 150 words]
Colyer RA, Sutin JDB, Chhun BB, Gratton E.
Spatial Resolution Enhancement using Non-Linear Temporal Cross-Correlation Techniques in Pump-Probe Confocal Microscopy.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 1670-Pos/B656.Pump-probe microscopy uses multiple illumination wavelengths to excite and de-excite a fluorescent sample. A pump laser beam, with a wavelength in the absorbance band of the fluorophore, excites the fluorophore into the electronic excited state as in conventional fluorescence microscopy. Unlike conventional fluorescence microscopy, a second laser beam, the probe, also illuminates the sample. The wavelength of the probe beam lies in the red edge of the fluorescence emission band and nearly instantaneously brings the excited fluorophore into the ground state via stimulated emission. In this manner, the detected fluorescence emission can be modulated spatially and temporally by the probe.
Various schemes have been developed to apply pump-probe illumination to microscopy. Using modulated or pulsed lasers, pump-probe microscopy has been used to measure ultra-fast fluorescent lifetimes and molecular rotations. Linear, temporal cross-correlation of pump and probe illumination has been found to increase the spatial resolution of the microscope, even when the ... [truncated at 150 words]
Czeslik C, Hazlett TL, Gratton E, Steitz R, von Grünberg HH, Ballauff M.
Effect of salt on the immobilization of proteins at polyelectrolyte brushes.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 1893-Pos/B5.We used two-photon excitation fluorescence fluctuation spectroscopy and neutron reflectometry to study the effect of salt concentration on the degree of protein binding to spherical and planar polyelectrolyte brushes. The binding of bovine serum albumin (BSA) to poly(acrylic acid) (PAA) brushes was characterized at neutral pH-values where both the protein and the brushes carry a negative charge. It has been found that BSA binds strongly to these brushes under electrostatic repulsion at low ionic strength. The BSA volume fraction profile, as determined from the neutron reflectivities, indicates a deep penetration of the BSA molecules into a planar PAA brush. However, when the ionic strength of the protein solution is raised to a few 100 mM, the BSA binding capacity of spherical and planar PAA brushes decreases drastically. A simple mean field approach is given that explains these experimental findings. The model predicts a large gain of free energy associated with ... [truncated at 150 words]
Levi V, Belmont AS, Plutz M, Gratton E.
Dynamics of a DNA sequence in interphase cells revealed by fast-tracking in a two-photon microscope.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 2820-Pos/B101.The structure and dynamics of DNA is thought to be essential for several aspects of the nuclear function. Previous studies show that the motion of chromatin is mainly random and constrained. However, other studies report a longer scale motion, probably related to transcription. In this work, we studied in-vivo the dynamics of a DNA sequence labeled with enhanced-green fluorescent protein by using a new fast-tracking technique in a two-photon microscope. This method presents less overall photobleaching and higher spatial and temporal resolutions than previous methods used for studying DNA dynamics. We verified that the fluorescent-tagged sequence undergoes random motion confined to a given space until it jumps to another region of constrained motion. By analyzing the velocity distribution in the trajectories, we could determine that the velocity during the jumps is higher than the average velocity. This behavior could be explained by a model which considers two states for the ... [truncated at 150 words]
Hazlett TL, Barcellona ML, Digman MA, Gratton E.
Polarized FCS: detection of rotational motions of macromolecules.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 3252-Pos/B532.The use of fluctuation spectroscopy for the measurement of relatively slow rotations of large macromolecules in solution or attached to other macromolecular structures is discussed. Both simulations and experiment are presented to show the range of rotational correlation times and diffusion times under which this technique is applicable. In particular, we explore several analytical methods to analyze the polarization fluctuation data. We found that by first constructing the polarization function and then calculating the autocorrelation of this function, we can obtain the rotational motion of the molecule with little interference from the lateral diffusion of the macromolecule. However, this is only true if the rotational diffusion is faster than the lateral diffusion time. Surprisingly, for most commonly used fluorophores the autocorrelation of the polarization function is relatively unaffected by the photon statistics. In our instrument, two-photon excitation is used to define a small volume of illumination where a few molecules ... [truncated at 150 words]
Tahari AK, Motolese G, Gratton E.
Fluorescence correlation spectroscopy in turbid media: ultrasensitive detection of bacteria, viruses, and protein aggregates.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 3218-Pos/B498.We are further developing a simple, inexpensive, and powerful alternative technique to detect and analyze, without enrichment, extremely low concentrations of cells, bacteria, viruses, and protein aggregates in turbid fluids for clinical and biotechnological applications. The anticipated applications of this technique are many. They range from the determination of the somatic cell count in milk for the dairy industry, to the enumeration and characterization of microorganisms in environmental microbiology and the food industry, and to the fast and ultrasensitive detection of protein aggregates for the diagnosis of Alzheimer’s and other neurodegenerative diseases in clinical medicine. A prototype instrument has been built and allowed the detection and quantification of particles down to a few per milliliter in short scanning times. It consists of a small microscope that has a horizontal geometry and a mechanical instrument that holds a cylindrical cuvette (1 cm in diameter) with two motors that provide a rotational ... [truncated at 150 words]
Digman MA, Inoue M, Cheng MA, Breusegem SY, Halaihel N, Sorribas V, Mantulin WW, Gratton E, Barry NP, Levi M.
Partitioning of NaPi cotransporter in cholesterol, sphingomyelin and glycosphingolipid enriched membrane domains modulates NaPi protein diffusion, clustering and activity.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 40-Plat.In dietary potassium deficiency (K-deficiency) a decrease in transport activity of the type IIa sodium/phosphate cotransporter protein NaPi occurs in spite of an increase in its apical brush border membrane (BBM) abundance. This regulation of NaPi activity is correlated to increased glycosphingolipid content of K-deficient BBM. The mechanisms by which these lipids modulate NaPi activity have not been determined. We determined if in K-deficiency NaPi is increasingly partitioned in BBM microdomains and if increased presence of NaPi in these microdomains modulates its activity. Using a detergent-free density gradient flotation technique we found that 80% of BBM NaPi partitions into low-density cholesterol, sphingomyelin and GM1-enriched “lipid raft” fractions. We combined single-point FCS, scanning two-photon FCS (S-FCS) and PCH methods to determine the lateral diffusion of membrane-bound NaPi, NaPi in cholesterol and glycosphingolipid-rich microdomains, and the number of NaPi molecules in each particle. S-FCS was used to simultaneously measure fast and slow ... [truncated at 150 words]
Digman MA, Brown CM, Horwitz AR, Sengupta P, Mantulin WW, Gratton E.
Spatio-temporal fluorescence fluctuation analysis of Paxillin-EGFP in cellular adhesions using scanning FCS, ICS and PCH.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 1821-Plat.Fluorescence methodologies based on two-photon laser excitation and confocal imaging provide detailed spatio-temporal maps of protein dynamics during cellular processes. Single point fluorescence correlation spectroscopy (FCS) provides information about protein concentration and dynamics within a very small region of the cell without providing any spatial information. We developed novel extensions of FCS to probe spatial correlations and previously inaccessible temporal windows. Scanning FCS (sFCS) and image correlation spectroscopy (ICS) of more dynamic proteins have been proposed as alternative methods to gain information bridging the temporal scales of FCS (ms-ms) and ICS (ms-s). sFCS rapidly measures many focal points, providing the same concentration and dynamic information as FCS as well as information on the spatial correlation between points along the scanning path and reveals slower dynamics. This can be done by either scanning the beam in a circular path or by using confocal imaging techniques (line scanning), and thereafter calculating correlation ... [truncated at 150 words]
Garcia-Marcos A, Sánchez SA, Parada P, Eid JS, Gratton E, Ballesta JPG, Jameson DM.
Fluorescence Correlation Spectroscopy in vivo Studies of GFP-Yeast ribosomal stalk proteins.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 1942-Pos/B54.GFP was linked to the C-terminal domains of Saccharomyces cerevisiae ribosomal stalk proteins, P0 and the acidic proteins P1a, P1b, P2a and P2b. Mutant yeast strains lacking one or all the possible combinations of two acidic proteins were constructed and the recombinant GFP fusion proteins were transformed into strains lacking the corresponding native proteins. The fluorescent proteins were incorporated into ribosomes and were studied both in vivo and in vitro using two-photon fluorescence correlation spectroscopy (FCS). The FCS data were analyzed using the photon-counting-histogram method which allowed for determination of the intrinsic “brightness” of the labeled ribosomes, which is related to the number of GFPs per particle. Using the P0-GFP construct as a reference, we were able to determine the number of P1a-GFP and P2b-GFP proteins associated with ribosomes in vivo. The results showed that only one GFP was found to be associated with each ribosome when only one labeled ... [truncated at 150 words]
Gomes AMO, de Oliveira AC, Levi V, Goncalves RB, Katpally U, Smith TJ, Gratton E.
Real time 3D tracking of virus particles in live cells.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 2579-Pos/B690.During the last year, we developed a new technique to track particles in three dimensions using a 2-photon microscope. The next step involves developing and testing the technique for tracking fluorescent particles in living cells. As a test system, we used relatively large particles, such as viruses. Detailed mechanisms of infection remain unclear for most animal viruses probably because of experimental difficulties. Tracking single particles in living cells eliminates the need for large amounts of viral material in order to have a good signal-to-noise ratio. Here we describe the possibility to track virus particles in living cells with nanometer accuracy and millisecond time resolution. The model utilized was the Human Rhinovirus (HRV14). Rhinoviruses are small RNA viruses, members of Picornaviridae family. Virus capsids were labeled with FITC and incubated with HeLa-H1 cells at 4oC to allow attachment to the membrane. Then the temperature was raised to 37oC and the particles ... [truncated at 150 words]
Malengo G, Andolfo A, Sengupta P, Liao SC, Barbieri BB, Chirico G, Gratton E, Blasi F, Sidenius N, Caiolfa VR.
The GPI-anchored Urokinase Receptor (uPAR) dynamics on the cells surface followed by fluorescence fluctuation spectroscopy.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 2891-Pos/B171.The Urokinase receptor (uPAR) is a GPI-anchored protein and all of its functions depend on its interactions with proteins and lipids present in the membrane and in the pericellular environment. Although there is much known about the uPAR system using molecular biology and biochemical techniques, nothing is known about the distribution and mobility of the receptor on the cell surface, the alterations induced locally by pericellular stimuli (e.g. uPA or vitronectin) and by cleavage of the D1 domain, which strongly affects the biological activity of uPAR. To investigate the mobility of the uPAR in living cells, we have produced clones of HEK293 stably expressing the uPAR/EGFP/GPI chimera.
2-Photon-imaging combined to fluorescence fluctuation spectroscopy measurements clearly demonstrate the heterogeneous distribution of uPAR all over the cell membrane, as expected for GPI-anchored proteins.
The dominant diffusion time component obtained by autocorrelation functions is in the range of tenth of milliseconds (D » 0.5 mm2/sec) ... [truncated at 150 words]
Sánchez SA, Tricerri MA, Gratton E.
Visualizing HDL particles removing cholesterol from artificial membranes by Laurdan GP and two-photon microscopy.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 2862-Pos/B143.The traditional protocols to study cholesterol removal are done by incubation of the acceptor with a donor containing radioactive cholesterol, and measurement of radioactivity distribution after the reaction has occurred. This type of measurements disrupt the original equilibrium and give the average cholesterol that is being removed, i.e., the cholesterol coming from all the membrane independently from the original spatial location of the sterol.
Here we report the use of a technique to quantify cholesterol removal at equilibrium and with high spatial resolution using two-photon microscopy. Laurdan is a fluorescent dye that gives information about the membrane water content which is related to fluidity. Laurdan generalized polarization (GP) is the parameter used to quantify water penetration. GP measurements in a two-photon microscope give the spatial resolution of water penetration and then indirectly of the membrane fluidity. We show changes in GP of GUVs made of POPC and 33% cholesterol with time, ... [truncated at 150 words]
-
Enhanced acceptor FRET facilitated by M-DNA.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 799-Pos/B638.FRET experiments have been carried out on duplex metal-complexed DNA molecules that are doubly labeled on the 3’-ends. Metal-complexed DNA, or M-DNA1 , has been reported to be formed in the presence of divalent cations Zn2+, Ni2+, and Co2+ associating with double-stranded DNA to form a DNA-metal complex. Increased efficiency of Förster acceptor-enhanced energy transfer over distances larger than expected for the corresponding length of the DNA molecules is observed. Different methods of measuring FRET are carried out, including FRET measurements normalized by the directly excited acceptor fluorescence, (ratio)A. This latter method avoids difficulties in interpretation that might obscure the interpretation of donor quenching. FCS and light scattering studies show the onset of co-precipitation of DNA-metal complexes and metal hydroxides. Careful controls are necessary to differentiate inter-strand FRET resulting from close proximity of labeled DNA in the aggregated state from intra-strand transfer within each individual doubly labeled DNA molecule. We ... [truncated at 150 words]
Buranachai C, Ha T, Clegg RM.
Single molecule study of dynamic characteristics of Tetramethyrhodamine-DNA.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 1705-Pos/B691.Tetramethylrhodamine exhibits multiple fluorescent isomeric binding states upon conjugation on double stranded DNA. This has been shown by several previous steady state and time-resolved fluorescence studies. We have used single molecule spectroscopy to study the dynamics of conformational changes of the dye that is attached to the end of double-stranded oligo-DNA molecules immobilized on a glass surface. Transitions between three different discrete levels of fluorescence intensity versus time were observed. The intensity distributions of the three defined states were well described by Gaussian functions. The rates of transition between fluorescence states were determined by fitting an exponential decay function to the dwell-time histogram giving transition rates ranging from 0.03 to 0.5 s-1. These unexpected long dwell times have not been observed by previous studies.
Majumdar ZK, Clegg RM, Noller HF, Hickerson RP.
Monitoring internal movements of the 30S E. Coli Ribosome with quantitative and kinetic FRET measurements.
49th Annual Meeting of the Biophysical Society, Long Beach, California, 2005.
Biophys J. 2005; Suppl, 1981-Pos/B93.We have made steady state and kinetic measurements of 30S-50S association by monitoring changes in FRET between site-specifically labeled fluorescent probes. We have developed a quantitative protocol that enables reliable measurements of distance changes that can be used to visualize internal movements of ribosome structure. We show that relative distance changes can be accurately determined, by distinguishing random from systematic error in fluorescence measurements. We show experimentally that the error in distance, due to kappa-squared uncertainty, is smaller than predicted by simple cone angle models of a single dipole transition using only dynamic averaging. We are able to correlate kinetic FRET measurements of subunit association with modeled conformational changes that ensue upon formation of the 70S complex.
Gratton E, Brown CM, Digman MA.
Fluorescence correlation spectroscopy techniques.
2004 Biophysical Society Discussions Study Book. October 28-31, Asilomar, CA.
Introduction: In this study book we discuss the basic concepts of fluctuation spectroscopy and its different implementations. We also show with examples how the FCS technique can be used to obtain information about dynamic processes in living cells. Also some suggested readings are included at the end for those interested in some of the original papers in this subject.
Fluctuation spectroscopy is a technique based on the measurement of the fluctuation of the fluorescence signal in a voxel of an image. In order for this technique to work, the signal must fluctuate, i.e., the fluorescence intensity as measured by the detector must have a characteristic time behavior. Of course, the intensity fluctuation as measured by any detector always has some noise. The characteristic spectrum of the noise is what is used to obtain information about different processes occurring in a given voxel. For example, if a given voxel is emitting a ... [truncated at 150 words]
Digman MA, Cheng MA, Levi M, Barry NP, Breusegem SY, Mantulin WW, Gratton E.
Diffusion of the NAPI cotransporter in microdomains of reconstituted brush border membranes (BBM) and K deficient BBM.
2004 Biophysical Society Discussions Study Book. October 28-31, Asilomar, CA.
We have combined single point FCS, scanning FCS and PCH methods to determine the lateral diffusion of the NaPi IIa cotransporter bound at the membrane, in cholesterol rich microdomains or “lipid rafts” particles and the number of NaPi-IIa molecule in each particle. We found that lipid rafts had an effect on protein diffusion. When the protein is in the raft it has reduced activity. Specifically, we used FCS to determine the faster diffusion processes. Scanning two-photon correlation spectroscopy (s-FCS) simultaneously measures fast and slow diffusing particles. S-FCS had the additional advantage of providing a local image which was crucial for the identification of the rafts domains. In this technique as the laser beam is scanned in an orbit over the membrane surface, an intensity ‘carpet’ visualizes relatively large domains crossing the orbit. From this data and analysis of the molecular brightness, we determined that the diffusion of the monomeric transmembrane ... [truncated at 150 words]
Hanson KM, Hayden PJ, Kubilus J, Clegg RM.
Detecting reactive oxygen species in skin using two-photon fluorescence imaging microscopy.
65th Annual Meeting of the Society for Investigative Dermatology. Providence, RI. April 28-May 1, 2004.
J Invest Dermatol. 2004; 122(3): A140, 839.Reactive oxygen species (ROS) contribute to skin photodamage including photoaging, immunomodulation, actinic keratosis and skin cancers. Because these highly-reactive derivatives of molecular oxygen are extremely short-lived and essentially non-emissive, they are difficult to detect directly. In addition, until recently with the realization of two-photon excited fluorescence (TPEF) imaging, the opaque and heterogeneous environment of the skin has inhibited detection and quantification of UV-induced ROS within the skin. We have developed a TPEF imaging method to detect the presence of ROS with 0.5 m spatial resolution and >100 m depth penetration. Dihydrorhodamine (DHR, 100 uL, 50 uM) is applied to the skin surface (Epiderm-200 (epidermis) and Epiderm-200FT (epidermis/dermis) (MatTek Corp.)) and incubated for 1 hr (37 oC, 5% CO2). DHR is non-fluorescent until it reacts with ROS and forms fluorescent rhodamine-123 (R123). Samples are imaged before and after UV irradiation (200-1600 J m2, 280-400 nm, solar simulator, Solar Light Co.). A ... [truncated at 150 words]
Behne MJ, Barry NP, Hanson KM, Gratton E, Mauro TM.
Calcium-fluorescence lifetime imaging in ex vivo skin II.
65th Annual Meeting of the Society for Investigative Dermatology. Providence, RI. April 28-May 1, 2004.
J Invest Dermatol. 2004; 122(3): A81, 482.In our recent publications, we described Fluorescence Lifetime Imaging Microscopy (FLIM) to assess pH in intact epidermis. Here, we report about our recent progress in measuring and visualizing Ca2+ in ex-vivo biopsies of unfixed epidermis. In our initial Ca-FLIM studies we used Calcium Green-5N which is sensitive in the intermediate range of concentrations expected in epidermis and were able to show overall increasing Ca2+ concentrations from basal (SB) to granular (SG) layers, confirming our prior PIXE and Calcium-precipitation results. We refined and broadened this method to cover the full range of epidermal Ca2+ concentrations, from nanomolar to millimolar values. Using sequential measurements with Calcium Green-1 for low concentrations and Rhod-5N for high concentrations, we find highest Ca-concentrations to be limited to the intracellular domain in an ER like distribution, specifically in the apical (granulosum and spinosum) layers, while in deeper (basal to dermal) layers this distinction is partially lost. In ... [truncated at 150 words]
-
Imaging tumor cells using a fast fluorescence lifetime microscope.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 3163-Pos/B415.ALA is processed by cells to form Protoporphyrin IX which is fluorescent. Cells incubated in ALA are imaged using a unique fast lifetime imager to resolve the different states and concentrations of PPIX in different regions of the cell. This information is important for photo-diagnostic and photo-treatment effectivity. Lifetime images of PPIX in cultured tumor cells are presented.
Buranachai C, Leuba S, Ordahl CP, Kun E, Clegg RM.
Histone H1 and DNA interaction study using several methods in fluorescence spectroscopy.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 3055-Pos/B307.Binding of histone H1 to DNA has several functions in addition to chromatin folding such as assisting the binding of PARP complex. Binding of histone H1 onto oligo-duplex DNA has been studied using FRET, fluorescence anisotropy, FCS and time resolve lifetime measurements. Quantitative FCS and anisotropy experiments of the histone-DNA interactions show multiple binding sites of DNA on each histone H1 molecule. The binding of DNA to histone H1 can be followed by analysis of the auto- and cross correlation curves. Quenching of fluorescence from dye attached to DNA is observed upon binding of histone H1.
-
Measuring enhanced acceptor fluorescence as a probe for electron transfer in m-DNA.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 2443-Pos/B540.Some divalent cations, e.g. Zn2+, Ni2+, and Co2+, associate with duplex DNA at approximately millimolar concentrations to form a metal complex (M-DNA) that has been proposed to facilitate electron transfer over long distances.1 Quenching of a fluorescence donor was also observed over distance much greater than a typical Förster radius. We have carried out acceptor-enhanced energy transfer experiments, among other measurements, on double-stranded M-DNA that are doubly labeled on the 5’-ends in order to investigate this structure further. We observe increased emission of the fluorescent acceptor over distances larger than expected for Förster transfer. The energy transfer measurements are normalized by the (ratio)A method, unambiguously identifying the energy transfer. The normalized enhanced acceptor fluorescence, (ratio)A, avoids complications by alternate de-excitation pathways (other than Förster transfer) that would affect the donor in the excited state and obscure the interpretation of donor quenching alone. We present the analysis of these energy transfer ... [truncated at 150 words]
Majumdar ZK, Clegg RM, Noller HF, Hickerson RP.
Visualizing ribosomal movements with Förster resonance energy transfer.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 1644-Pos/B629.We have probed distances between various locations within the Escherichia Coli 30s ribosomal subunit as well as inter-subunit distances of the 70s ribosomal complex, in a number of different functional states, by measuring energy transfer between site-specifically labeled ribosomal proteins. The FRET distance information allows preliminary modeling for visualizing conformational changes of the 30s subunit upon association with the 50s subunit. Changes in energy transfer have also been observed between pre and post-translocational states of the 70s, complexed with mRNA, tRNAs and EF-G, which reveal internal movements of the ribosome that occur during translocation. The methods of measurement will be described, and results will be discussed in relation to crystal structures and cyro-electron microscopy. Supported by the NIH, PHS 5 P41-RRO3155, UIUC, and by UCSC.
Eckhoff DA, Sutin JDB, Rogozhina EV, Stuart JN, Sweedler JV, Braun PV, Clegg RM, Gratton E.
Effects of oxidation on the photoluminescence from silicon nanoparticles.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 3156-Pos/B409.Understanding the stability of silicon nanoparticles (Si-np) in oxidative environments is important to their use as a luminescent marker in biophysical applications. Treatments with NaOH, HCl, and UV radiation lead to substantial red-shifts in the emission from ultrasmall (~1.0 nm) Si-np. Modeling of the photoluminescence (PL) spectra shows the same set of three distinct, narrow (~0.47 eV), near-Gaussian emissions with their relative strengths varying among the treatments. From the expected chemical and structural effects of the treatments and the results of computational modeling, each distinct emission is correlated with its probable chemical origin. A simple model of surface oxidation captures the general behavior and PL properties of this system. These results offer the exciting possibility of tailoring the PL properties of Si-np through control of their surface chemistry. Supported by the NIH, PHS 5 P41-RRO3155, and by UIUC.
Levi V, Ruan Q, Gratton E.
A novel method for 3-D particle tracking in biological systems.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 3152-Pos/B405.We describe a novel method to track fluorescent particles in 3-D, applicable to the study of motion of fluorescent molecules in cells or other biological systems. In this method, the laser beam of a two-photon excitation microscope moves in a circular path with radius of half the width of the point spread function (PSF). When the fluorescent particle is located within the scanning radius of the laser, the precise position of the particle in the x-y plane can be determined by its fluorescence intensity distribution along the circular scanning path. A z-nanopositioner on the objective allows us to change the laser focus at two different planes located half the width of the PSF apart. The difference of the fluorescence intensity in the two planes is used to calculate the z-position of the fluorescent particle. With a fast feedback mechanism, the position of the laser beam is directed to the center ... [truncated at 150 words]
Tahari AK, Motolese G, Gratton E.
Fluorescence correlation spectroscopy in turbid media: ultrasensitive detection of bacteria, viruses and protein aggregates.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 3151-Pos/B404.Fast and efficient screening for single particles has become increasingly important for applications in biotechnology and medicine. The particles may be single eukaryotic cells with particular properties, bacteria, viruses, protein aggregates, or even molecular entities such as peptide hormones or other oligomeric compounds. In some instances there is a need for the detection of minute quantities of particular entities, such as in the early diagnosis of a disease by the direct detection of viruses in the initial phase of infection, or the fast screening for virulent forms of bacteria or other pathogens in food stuffs and drinking water. We have developed a new fluorescence scanning technique and combined it with filtration software based on particle passage pattern recognition for the detection of sub-attomolar concentrations of fluorescently tagged particles. A prototype instrument has been built and has allowed the detection and quantification of particles down to a few per milliliter in ... [truncated at 150 words]
Sánchez SA, Tricerri MA, Gratton E.
Visualization and quantification of the rate of cholesterol removal by two-photon, dual-channel microscopy.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 2918-Pos/B170.The distribution of cholesterol in natural membranes is highly heterogeneous. When cholesterol is removed from the membrane by an acceptor, the kinetics of the process can be quantified by measuring the amount of cholesterol on the acceptor as a function of in time. However, in order to locate where the cholesterol is being extracted from, we need to visualize and quantify the donor (the membrane containing the cholesterol. The combination of dual-channel, two-photon fluorescence microscopy and the membrane fluorescent dye Laurdan, allows for the visualization and quantification (Laurdan generalized polarization (GP) value) of the cholesterol content in the membrane. We have used two cholesterol donors: GUVs made of POPC containing 20% cholesterol and two raft mixtures: POPC/Sphingomyelin/cholesterol and DOPC/DPPC/cholesterol. The first mixture, POPC (20% cholesterol), shows a homogeneous cholesterol distribution from 60 to 20ºC. The raft mixtures, however, show coexistence of domains depending on the temperature and the cholesterol content. ... [truncated at 150 words]
Cheng MA, Ruan Q, Barry NP, Breusegem SY, Halaihel N, Inoue M, Levi M, Gratton E, Mantulin WW.
Membrane dynamics in giant unilamellar vesicles (GUVs) of raft and non-raft fractions of brushborder membranes.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 1064-Pos/B49.Lipid rafts are discrete regions in the plasma membrane which are composed of lipids that exist in the liquid ordered state, and are believed to function as platforms for protein and lipid transport. These structures are reported to be present in numerous membrane systems, including brushborder membranes in kidney cells. This research aims to study the membrane dynamics in brushborder membranes and membrane fractions of rat renal proximal tubular cells. Fluorescence (Laurdan) provided direct visualization of GUVs formed through electroformation of raft and non-raft fractions of intact renal brushborder membranes. Flotation through a density gradient (Optiprep) separated the raft and non-raft fractions without the use of a detergent. Two-photon scanning microscopy of the GUVs formed from the raft fraction showed uniform fluorescence intensity images with some non-fluorescent domains of a few microns in size. Previous GUV studies of raft fractions obtained through detergent extraction yielded vesicles devoid of domains. Membrane ... [truncated at 150 words]
Lee CY, Celli A, Gratton E.
Pressure studies of giant unilamellar vesicles using two photon fluorescence microscopy.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 1020-Pos/B5.We used two-photon-fluorescence microscopy to investigate the effects of pressure on the lateral organization of lipids on the membrane of Giant Unilamellar Vesicles (GUVs). GUVs of the size of few microns were grown by electroformation onto platinum electrodes. After detaching the vesicles from the electrodes, they were sucked into transparent quartz capillaries with an inner diameter of 50 µm and an outer diameter of 360 µm. This novel high pressure cell for the microscope was recently described (J.D. Muller and E. Gratton, High pressure fluorescence correlation spectroscopy, Biophysical Journal (85) October 2003). Pressure can be applied to the vesicles inside the capillaries sealing one end and connecting the other to a high pressure pump. This system allows using high N.A. immersion objectives to observe the surface of the vesicles with high spatial resolution. It was shown that the behavior of single molecules and fluctuation experiments can be performed under high ... [truncated at 150 words]
Czeslik C, Hazlett TL, Gratton E, Ballauff M, Royer CA.
Variable protein binding to polyelectrolyte brushes.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 856-Pos/B666.We used two-photon excitation fluorescence fluctuation spectroscopy with photon counting histogram (PCH) analysis as a new tool to study the binding of globular proteins to colloidal particles in situ. The binding of two proteins, SNase and BSA, to spherical polyelectrolyte brushes (SPB) was measured as a function of protein concentration and ionic strength of the solution at pH-values where SNase and BSA are positively and negatively charged, respectively. It has been found that SNase and BSA strongly bind to the SPB. When the ionic strength of the solution is raised to 100 mM, the SPB become resistant to both proteins. These findings provide further evidence for a binding mechanism where the proteins are mainly driven to the SPB by the "counterion evaporation" force, while Coulomb interactions play a minor role. The changes in the secondary and tertiary structure of BSA induced by the interaction with the SPB has also been ... [truncated at 150 words]
Digman MA, Ruan Q, Mantulin WW, Brown CM, Horwitz AR, Gratton E.
Paxillin at focal adhesions: scanning fluorescence correlation spectroscopy.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 829-Pos/B639.Cellular adhesions are important for cell migration during development, neuronal network formation, wound healing and cancer metastasis. The attachment of integrins to the extracellular matrix generates an attachment point, or adhesion, which cells can use to generate a traction point that the cell can use in order to migrate. This adhesion causes an array of signaling processes where cytosolic factors can be targeted to the adhesion, the actin cytoskeleton can reorganize and other downstream processes important for migration are activated. Amongst focal adhesion proteins, paxillin is known to play a regulatory role as an adaptor protein. A variety of methods including image correlation spectroscopy (ICS), have been used to evaluate these types of protein interactions in order to analyze protein aggregation states. ICS is best suited for spatial and temporal measurements for detecting slowly diffusing molecules. We have used a unique experimental approach, two-photon excitation scanning fluorescence correlation spectroscopy (S-FCS), ... [truncated at 150 words]
-
Photon counting and analog data acquisition in fluorescence correlation spectroscopy: issues of sensitivity and dynamic range.
48th Annual Meeting of the Biophysical Society, Baltimore, Maryland, 2004.
Biophys J. 2004; Suppl, 826-Pos/B636.Fluorescence Correlation Spectroscopy (FCS) has enjoyed an immense amount of attention in the past five years. FCS theory, data analysis tools, and instrument design have come to the state where non-specialist scientists are making use of prescribed protocols to garner information on the diffusion coefficients and fluorescence brightness factors for their biochemical and cellular systems. FCS is still a technique undergoing development and its application to complicated research areas, such as cell physiology and multisubunit organization brings new technical challenges. One point of difficulty in these areas is the presence of a heterogeneous population of structures that can result in a wide range in brightnesses. Because of the need for high sensitivity, photon counting detection electronics are predominantly used to collect FCS data. Unfortunately, photon counting technology has a limited dynamic range when compared to analog detection. For this work we have examined the sensitivity limits and the dynamic range ... [truncated at 150 words]
Michalos A, Safonova LP, Choi JH, Barbieri BB, Gratton E.
Near-infrared brain oximetry in obstructive sleep apnea syndrome.
7th World Congress on Sleep Apnea. Helsinki, Finland, 29 June - 3 July 2003.
Sleep Medicine. 2003; 4(Suppl 1): S31-S32, 144.Objectives: Obstructive sleep apnea syndrome (OSAS) is associated with cardiovascular and cerebrovascular morbidity and mortality. Our goal is the application of Near-Infrared Spectroscopy (NIRS) for the assessment of brain tissue oxygenation and hemodynamics in OSAS subjects for the detection, diagnosis and monitoring of cerebrovascular disease.
Materials and methods: We used breath holding protocols on OSAS patients (AHI $ 20) and on healthy controls. The brain tissue NIRS parameters, such as oxy-, deoxy-, total hemoglobin concentrations, and cerebral hemoglobin oxygen saturation were monitored and assessed by a frequency-domain tissue oximeter.
Results: Our findings suggest that compromised brain microvasculature in OSAS may lead to impaired ability to react to hypoxia and consequent hypercapnia. OSAS subjects showed a reduced or absent brain vascular reactivity compared to the cerebrovascular responsiveness in the healthy controls.
Conclusions: NIRS offers the advantage of performing non-invasive, transcranial, real time measurements of changes in cerebral hemodynamics and oxygenation for the screening ... [truncated at 150 words]
Gratton E, Ruan Q, Levi V.
Scanning FCS.
Intermolecular Associations in 2D and 3D. University of Nottingham. 19-20 June 2003.
Fluctuation correlation spectroscopy has become a commonly used methodology to study molecular processes in solutions and inside cells. Cross-correlation and multichannel correlation is also being developed in several laboratories. An important aspect of correlation which has not been widely exploited is the capability to detect events which occur either simultaneously or in a given statistical sequence in different spatial locations. Image correlation spectroscopy has in principle the capability to provide both spatial and temporal correlations. However, the commonly used methods used for image acquisition are relatively slow and do not allow the measurement of fast correlations, typically in the millisecond scale which are typical of protein diffusion in the cytoplasm of live cells. In this work we describe a variant of the line technique, scanning FCS that provides millisecond time resolution in a relatively large area. This technique can provide the mobile and immobile fraction of molecules in a region ... [truncated at 150 words]
Michalos A, Safonova LP, Olopade CO, Choi JH, Mantulin WW, Huber DM, Barbieri BB, Gratton E.
Near-infrared spectroscopy for the assessment of vascular responsiveness of the brain: a screening method for cerebrovascular morbidity in obstructive sleep apnea.
17th Annual Meeting of the Association of Professional Sleep Societies (APSS). Chicago, Illinois, June 3-8, 2003.
Sleep. 2003; 26(Abstract Supplement): A390-291, 0983.R.Introduction: Obstructive Sleep Apnea Syndrome (OSAS) is an important risk factor for the development of cardiovascular morbidity and mortality. Compromised cerebral blood flow (CBF) has been postulated to account for the cerebrovascular morbidity associated with OSAS. Near-infrared Spectroscopy (NIRS), a non-invasive, portable, costeffective methodology that allows transcranial and continuous real-time measurements of brain tissue oxygenation and hemodynamics is the only modality that provides information on brain oxygenation. Hence, NIRS is an important tool for the assessment of cerebrovascular health.
Methods: A. Subjects: Twenty subjects (25-74yrs) participated in the study. Eight (29-74yrs) had OSAS (AHI>20), while twelve healthy nonsnorers (25-54yrs) constituted the control group.B.
Instrumentation and Monitoring: Arterial blood oxygen saturation (SaO2) and heart rate (HR) were monitored via pulse oximetry (N-200, Nellcor Inc., Pleasanton, CA), and the breathing rate with a respiratory strain gauge (Resp-EZ, Sleepmate, New-Life Technologies, Midlothian, VA). The NIRS parameters, such as oxy-([O2Hb]), deoxy-([HHb]), total hemoglobin ([tHb]) ... [truncated at 150 words]
-
Scanning FCS: measuring spatio-temporal correlations in the millisecond range in live cells.
Focus on Microscopy 2003. University of Genova, Italy. April 13-16 2003.
Fluctuation correlation spectroscopy has become a commonly used methodology to study molecular processes in solutions and inside cells. Cross-correlation and multichannel correlation is also being developed in several laboratories. An important aspect of correlation which has not been widely exploited is the capability to detect events which occur either simultaneously or in a given statistical sequence in different spatial locations. Image correlation spectroscopy has in principle the capability to provide both spatial and temporal correlations. However, the commonly used methods used for image acquisition are relatively slow and do not allow the measurement of fast correlations, typically in the millisecond scale which are typical of protein diffusion in the cytoplasm of live cells. In this work we describe a variant of the line technique, scanning FCS that provides millisecond time resolution in a relatively large area. This technique can provide the mobile and immobile fraction of molecules in a region ... [truncated at 150 words]
Redford GI, Gohlke C, Clegg RM.
Versatile rapid lifetime imaging.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2): 584, 2860-Pos/B459.A full-field fluorescence lifetime instrument using a homodyne frequency domain method will be presented. This instrument captures and displays real-time lifetime images at rates higher than 30 frames per second. It can be adapted to a microscope or an endoscope. Multiple frequencies are easily implemented. Immediate feedback and ease of use have been emphasized so that the device to can also be used for clinical diagnostic purposes. The instrument has been designed so that the benefits of lifetime imaging can be employed conveniently wherever standard fluorescence imaging is used. Data is shown for biological and biotechnological samples.
Majumdar ZK, Sutin JDB, Redford GI, Clegg RM.
Microsecond kinetics in a continuous flow turbulent mixer - detection with fluorescence intensity and fluorescence lifetime imaging.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 2321-Pos/B697.We have constructed a continuous-flow, turbulent mixing device that mixes two solutions to complete homogeneity in less than 12 microseconds. Novel microfabrication techniques have been employed for constructing the mixer in a glass substrate and these techniques will be discussed. The entire mixing process and kinetics can be observed in a steady state measurement with an optical microscope or other conventional optical system. We show how we use the mixing device together with lifetime and intensity imaging to monitor fast kinetic events of biological reactions.
-
Integrated magnetic manipulator and microfluidic sample chamber for parallel application of isotonic forces.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 2859-Pos/B458.Magnetic micromanipulation is used to perturb molecules and cells with uniform, position independent mechanical forces transmitted locally through magnetic beads. Position independent forces are useful for many types of assays and also for simultaneous parallel experiments. Magnetic fields are also easily used to apply torques.
Multipole electromagnets are useful for creating uniform forces in 3D since forces can be accurately controlled by the current in the coils. However, two common problems with this design are that long working distances are required and that the poles flex due to the strong fields within the manipulator. To overcome these limitations, we are using photolithography and electroforming techniques to fabricate poles of high magnetic saturation strength alloy directly in microscope coverslips. The poles transfer magnetic fields from external coils to the sample. A microfluidic sample chamber is fabricated between two of these coverslips, forming the manipulator. The distances are kept small enough ... [truncated at 150 words]
Sánchez SA, Tricerri MA, Arnulphi C, Gratton E.
Reconstituted HDL particles remove cholesterol from giant unilamellar vesicles: visualization by two-photon dual channel microscopy.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 2502-Pos/B100.It is generally accepted that human High Density Lipoproteins (HDL) and their major component apolipoprotein A-I (apo A-I), play a central role as acceptors as well as carriers of the cholesterol released from membranes. The pre-B–HDL are responsible for 37-45% of the cholesterol efflux, however, the molecular basis for their ability to remove cholesterol from the cell membrane is not yet clear. The process may be the result of several mechanisms including microsolubilization of cholesterol and phospholipids by apo A-I. In this study we use dual-channel two-photon fluorescence microscopy and the membrane fluorescent dye Laurdan, to visualize the removal of cholesterol from individual GUVs by 2 types of reconstituted HDL (rHDL) particles and by the lipid-free apo A-I. The small rHDL particle of 78Å diameter showed a higher capability to remove cholesterol than the 96Å from POPC GUVs containing 20 % cholesterol. Under the same conditions, the lipid ... [truncated at 150 words]
Celli A, Sutin JDB, Gratton E.
Microfabricated sample chambers and electrode arrays for single GUV manipulation for two-photon imaging and spectroscopy.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 2322-Pos/B698.The geometry of GUVs and the fact that their chemical composition is completely controlled makes them an optimal model for the cellular membrane. However, since GUVs are currently grown in bulk and each experiment requires a new sample preparation, comparison of results to a control sample is difficult. Furthermore, under many conditions the GUVs remain connected to the lipid reservoir needed for their formation allowing interference with the physical effects under observation. In addition, the large volume of the bulk solution and the presence of the lipid reservoir make quantitative measurements difficult. To overcome these problems, we are developing microfabricated devices to separate and maintain isolated GUVs in a method compatible with two-photon microscopy. We are using poly-dimethyl-silicone (PDMS) to create chambers to hold individual GUVs. This will allow measurement on GUVs from the same preparation under different experimental conditions. In addition we have created arrays of transparent electrodes of ... [truncated at 150 words]
Eckhoff DA, Sutin JDB, Rogozhina EV, Stuart JN, Dekker NH, Bensimon D, Sweedler JV, Nayfeh MH, Braun PV, Gratton E.
Preparation and characterization of a silicon nanoparticle / DNA conjugate.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 2324-Pos/B700.This work studies the fluorescence properties of group IV semiconductor nanoparticles (Si-np and Ge-np). The primary goal is to assess their potential for use as a fluorescent marker in biological applications. Our current emphasis is with carboxyl functionalized Si-np as they are well suted for conjugation with a wide range of biomolecules. These particles were covalently attached to an oligonucleotide, further substantiating the functionalization and labeling reactions for this system. Strong fluorescence is seen from the labeled DNA, but it is accompanied by a substantial red-shift and broadening of the emission. Experiments aimed at understanding these changes and further characterizing the Si-np/DNA conjuagate will be presented. We will also discuss our projects with Ge-np and Si-np/streptavidin linking.
Barry NP, Eid JS, Gratton E.
Digital heterodyne method for fluorescence lifetime measurement.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 2325-Pos/B701.Digital circuitry has reached clock speeds (GHz) applicable to measurement of fluorescence lifetime. In direct digital methods, a detected photon is converted into a logic pulse using a high speed discriminator. Lifetime information is extracted from the pulse train without use of intervening analogue circuitry such as a TAC. We present measurements using a digital heterodyne technique similar to the frequency domain method for lifetime measurement. The primary advantage of a digital implementation of the frequency domain lifetime method is the signal to noise improvement afforded by the use of a discriminator compared to analogue operation of a PMT. The mixing/heterodyning step takes place in a simple Flip-Flop circuit. A reference pulse train with a fixed frequency offset from the excitation frequency beats with the detected photon pulses in this circuit. As with the analogue heterodyne method, the circuitry downstream of the mixer does not have to work with nanosecond ... [truncated at 150 words]
Ruan Q, Cheng MA, Gratton E, Levi M, Mantulin WW.
Membrane-protein dynamics: spatio-temporal interactions by fluorescence correlation spectroscopy (FCS).
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 2373-Plat.Giant unilamellar vesicles (GUVs) are useful as model membrane systems to study protein-membrane (P-M) interactions. Processing sequential GUVs fluorescence images only detects slower dynamics of P-M interactions. We have developed a new FCS technique, termed scanning FCS (SFCS) that performs multiple FCS measurements simultaneously by rapidly directing the excitation laser beam in a uniform (circular) scan across the bilayer of the GUVs in a repetitive fashion. SFCS measurements on GUVs allow for detection of spatial-temporal interactions between the P-M based on the diffusion rate of the protein. In this study, GUVs were assembled from rat kidney brushborder membrane fragments, which included the integral membrane proteins. To test membrane proteins incorporation into the GUVs, antibodies against NaPi II cotransporter were labeled with Alexa 488. Fluorescence images of the GUVs in the presence of the antibody showed no apparent binding. With SFCS, the binding of the antibody to the GUVs was detected ... [truncated at 150 words]
-
Punctuated lipid mass loss during enzyme-mediated lipid hydrolysis on giant unilamellar vesicles.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 1843-Pos/B216.Giant unilamellar vesicles (GUV) have been successfully used as model membranes in a variety of lipid-lipid and lipid-protein systems, including the binding and subsequent hydrolysis of GUV membrane lipids by secreted phospholipases A2 (sPLA2). Hydrolysis of GUV lipids by sPLA2 commonly results in the steady decrease in GUV size with the eventual disappearance of the GUV altogether. However, we have found that lipid loss is not always gradual but can be distinctly punctuated with sudden GUV diameter changes as great as 14%. Clearly, this effect is the result of the competition between membrane forces that tend to stabilize the bilayer structure, and the destabilizing forces introduced by the buildup of hydrolysis products, lysolipid and free fatty acid. Concomitant with the observed lipid loss is an abrupt increase in Laurdan GP and, in one case, the apparent expulsion of an internal lipid vesicle. A GP increase is indicative of greater lipid ... [truncated at 150 words]
Digman MA, Eid JS, Gratton E, Cho W.
Fluorescence Correlation Spectroscopy (FCS) and Photon Counting Histogram (PCH) study of Annexin I membrane binding and aggregation using scanning two photon microscopy.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 192-Pos/B66.Annexins are calcium dependent, anionic phospholipid binding proteins. While some annexins promote membrane aggregation, there is much controversy as to the mechanism of annexin induced membrane aggregates. Our in vitro solution FCS data on small unilamellar vesicles indicates that annexin I forms heptamers at 40 mM calcium and 14-mers at 120 mM calcium. To better quantify the molecular aggregation state we performed FCS and PCH analysis on a stationary model membrane. Two photon scanning fluorescence microscopy was used to image and quantify calcium dependent binding of eGFP-tagged annexin I to the surface of giant unilamellar vesicles (GUVs). This method allowed the determination of the concentration and molecular brightness of the eGFP-tagged annexin I inside, outside, and on the GUVs at different calcium concentrations and for varying membrane compositions. These measurements, can quantitatively determine the binding constant of the protein as well as its aggregation state for a variety of biologically ... [truncated at 150 words]
Breusegem SY, Mackinnon AC, Barry NP, Lin X, Gratton E, Williams BD, Clegg RM.
In vivo visualization of protein-protein interactions in C. Elegans muscle attachment structures by FRET microscopy.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 105-Plat.Dense bodies and M-lines are highly ordered structures that attach actin, respectively myosin filaments in the body wall muscle cells of C. elegans to the basal lamina, hypodermis and cuticle. Using genetic techniques many proteins that localize at dense bodies and M-lines have been identified. To determine which proteins interact with each other in adult muscle attachments, we applied fluorescence resonance energy transfer (FRET) microscopy in living transgenic animals in which the proteins are fused pair-wise to the GFP-mutants CFP (cyan) and YFP (yellow). Occurrence of FRET between the CFP- and YFP-labels indicates a direct intermolecular interaction between the host proteins. FRET was measured by both steady-state and time-resolved two-photon excitation microscopy methods. We show that PAT-4, the C. elegans homolog of integrin-linked kinase, forms multimers and binds to PAT-6, a homolog of actopaxin, confirming results obtained from genetic and yeast two-hybrid studies. We discuss the advantages and disadvantages of ... [truncated at 150 words]
Cheng MA, Bagatolli LA, Zajicek HK, Halaihel N, Levi M, Gratton E, Mantulin WW.
A two photon microscopy study of raft membranes from rat kidney proximal tubular cells reconstituted into Giant Unilamellar Vesicles (GUVs).
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 914-Pos/B166.Rafts are discrete regions in the plasma membrane that are believed to function as platforms for protein and lipid transport. This research aims to study the structure of rat renal proximal tubular cell membranes using techniques used in characterizing artificial membranes. The study involves the direct visualization of GUVs formed from raft fractions of integral rat kidney brush border membranes obtained through detergent extraction with Triton X-100 at 4C. Imaging of GUVs obtained from detergent resistant membranes (DR) labeled with the fluorescent probe Laurdan using two-photon scanning microscopy resulted in fluorescence intensity images showing an even distribution of Laurdan at physiological temperatures. No domain formation was observed in the DR membranes, whereas membrane domains were clearly present in intact brush border membranes. Membrane fluidity was characterized in terms of Laurdan Generalized Polarization (GP) function and was observed to be in a fairly rigid phase across the temperature range in which ... [truncated at 150 words]
Eid JS, Sutin JDB, Gratton E.
Two-photon excitation point spread function measurements and modeling.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 1383-Pos/B637.Two-photon techniques are very important for quantitative microscopy due to the intrinsic three dimensional localization of the excitation point spread function (PSF). Quantitative two-photon measurements strongly depend on the exact shape of the PSF. The shape of the two-photon PSF depends on several factors including: excitation beam profile, aberrations in the excitation optical path, and aberrations caused by sample inhomogeneities.
We are performing a series of experiments and models to explore the relationship between these factors and their effects on quantitative measurements. Specifically, the PSF is measured and modeled in the presence and absence of various spatial filters. The photon counting histogram (PCH) is used to correlate a brightness parameter for various beam profiles. Parameters recovered from fluorescence correlation spectroscopy (FCS) are compared with the measured PSF and modeled parameters. In addition we are modeling the effect of different apodizations of the objective’s back aperture on the PSF. We will discuss ... [truncated at 150 words]
Tahari AK, Motolese G, Gratton E.
Fluorescence correlation spectroscopy in turbid media: detection of somatic cells and bacteria in bodily fluids.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 1400-Pos/B654.We are continuing in our quest to find a simpler and cheaper alternative to detect and quantify cells, bacteria, and even viruses in bodily fluids for clinical and biotechnological applications. We built a dedicated confocal FCS instrument which consists of a home-built microscope that focuses about 200 microns inside a cylindrical cuvette. The cuvette is rotated and moved vertically up and down to effectively scan a large volume enabling us to measure very small concentrations of fluorescently-labeled particles. The diffusion time of the large structures in the volume of observation is negligible with respect to the time of observation of a single volume. We have been able to measure the somatic cell count in milk and bacteria down to a few thousand per ml. We are working on digital data processing and filtering techniques to achieve better sensitivity and higher dynamic range and to compensate for the ... [truncated at 150 words]
Tan E, Nahas MK, Wilson TJ, Clegg RM, Lilley DMJ, Ha T.
Dynamics of four-way junction enhances the active site formation of hairpin ribozyme.
47th Annual Meeting of the Biophysical Society, San Antonio, Texas, 2003.
Biophys J. 2003; 84(2), 888-Pos/B140.The hairpin ribozyme catalyzes cleaving reaction when distant domains of the molecule interact and form the active site. The 4-way-junction structure of the native ribozyme enhances the active site formation 1000 fold compared to the minimal form, how this is achieved has been unclear. Using single molecule spectroscopy, we have discovered that the isolated form of this junction is highly dynamic displaying rapid switching behavior between different conformation. Comparison to the single molecule dynamics of hairpin ribozyme in various states of mutation indicate that the ribozyme inherits and exploits the conformer switching behavior of junction structure.
Gratton E, Levi V, Ruan Q.
3-D particle tracking in a two-photon microscope.
Bariloche Protein Symposium, San Carlos De Bariloche, Argentina, November 17-21, 2003.
Biocell. 2003; 27(Suppl I), L5.Particle tracking in a cell offer the possibility to determine the diffusive or non-diffusive behavior of a particle over large distances. The presence of obstacles, flow and particle interaction with the substrate is recognized by the analysis of the particle trajectory. Conventionally, particle tracking has been performed using cameras and reflecting or fluorescing particles. In addition, interferometric or reflecting techniques have been used to determine the particle position. In this work, we explore the possibility to perform particle tracking in 3-D using the 2-photon excitation microscope. We show that rapid tracking is achievable over large distances. The tracking bandwidth is essentially limited by the number of photons that can be collected during the interval of time used for the feedback of the particle position. For small movements, it is not necessary to center feedback the particle position, but the 3-D position can be determined by a simple algorithm. We present ... [truncated at 150 words]
-
Protein-lipid and protein-protein interactions: direct visualization by 2-photon microscopy.
Bariloche Protein Symposium, San Carlos De Bariloche, Argentina, November 17-21, 2003.
Biocell. 2003; 27(Suppl I), S8.We have used two-photon excitation fluorescent microscopy to study the interaction of Phospholipase A2 (PLA2) and HDL particles with lipids. The interaction of PLA2 with Giant Unilamellar Vesicles of different lipid composition show morphological and fluidity changes as a manifestation of the enzymatic activity. Our studies on the monomer-dimer equilibrium of PLA2 using micelles and Fluorescence Correlation Spectroscopy indicate a very strong dimer that will not dissociate until 10-11 M. A second system we have studied is the interaction between reconstituted particles of HDL with membranes; in this case we have used the microscopy version of Laurdan Generalized Polarization technique to study the capability of these particles to remove cholesterol from Giant Unilamellar Vesicles of different lipid composition.
Levi V, Ruan Q, Kis-Petiková K, Gratton E.
A novel method for 3-D particle tracking in biological systems.
Bariloche Protein Symposium, San Carlos De Bariloche, Argentina, November 17-21, 2003.
Biocell. 2003; 27(Suppl I), BC-P12.We describe a novel method to track fluorescent particles in 3-D, applicable to the study of motion of fluorescent molecules in cells or other biological systems. In this method, the laser beam of a two-photon excitation microscope moves in a circular path with radius of half the width of the point spread function (PSF). When the fluorescent particle is located within the scanning radius of the laser, the precise position of the particle in the x-y plane can be determined by its fluorescence intensity distribution along the circular scanning path. A z-nanopositioner on the objective allows us to change the laser focus at two different planes located half the width of the PSF apart. The difference of the fluorescence intensity in the two planes is used to calculate the z-position of the fluorescent particle. With a fast feedback mechanism, the position of the laser beam is directed to the center ... [truncated at 150 words]
Arnulphi C, Tricerri MA, Sánchez SA, Gratton E.
How does apolipoprotein A-I interact with membranes?.
Bariloche Protein Symposium, San Carlos De Bariloche, Argentina, November 17-21, 2003.
Biocell. 2003; 27(Suppl I), LI-P17.In a previous work we investigated by using calorimetric and optical techniques, the interaction of apolipoprotein A-I with model membranes of various lipid composition. The dipolar relaxation of the fluorescent probe LAURDAN allows the calculation of the General Polarization (GP). This function lets infer the lipid phase of the membranes. We observed that inclusion of Sphingomyelin (SM) molecules in bilayers of pure phosphatidilcholine (POPC) at T< 32°C induced dynamical restriction of lipid environment (increasing of GP), but yielded the contrary effect at higher temperatures. This phenomenon correlates with the formation of lipid domains observed by Two Photon Fluorescence Microscopy in the same range of temperatures. The study of lipid membranes containing cholesterol (POPC/SM/CHOL) (similar to lipid rafts) gave higher values of GP, showing a change of the lipid phase towards to a gel state. The addition of the protein to the investigated membranes showed a differential behavior depending of the ... [truncated at 150 words]
Michalos A, Safonova LP, Wolf U, Wolf M, Choi JH, Gupta R, Mantulin WW, Barbieri BB, Gratton E.
Obstructive sleep apnea: evaluation of brain oxygenation and hemodynamics by near-infrared spectroscopy.
16th Annual Meeting of the Association of Professional Sleep Societies (APSS). Seattle, Washington, June 8-13, 2002.
Sleep. 2002; 25(Abstract Supplement): A390-A391, 547.R.Obstructive sleep apnea (OSA) is a potentially lethal disease, which leads to hypoxia and hypoxemia. Chronic, recurrent hypoxia during sleep may cause brain injury. Neuropsychological and cognitive deficits, as well as cerebrovascular accidents, including fatal strokes are not uncommon (1). Conventional polysomnography does not provide information on brain oxygenation, an important parameter in subjects with preexisting cardiovascular pathology. Nearinfrared spectroscopy (NIRS), a safe, non-invasive, portable, bedside method, provides real-time transcranial measurements of changes in cerebral oxygenation and hemodynamics. These characteristics make NIRS the ideal tool to study physiological and pathological processes of the brain, in research and clinical settings (2).
Breusegem SY, Barry NP, Lin X, Gratton E, Clegg RM, Williams BD.
Protein interactions in C. Elegans muscle attachment structures studied in vivo by fluorescence resonance energy transfer (FRET).
Midwest Worm Meeting. June 28-30, 2002. Washington University in St. Louis.
Dense bodies and M-lines are highly ordered, multiprotein attachment structures that span the plasma membrane and function to link the myofilaments found in body-wall muscle cells to the underlying ECM. Assembly of these structures is essential for the viability of the organism, and recent work in a number of labs has identified several new dense body and M-line components that are essential for this process. A crucial step towards understanding the assembly process is the characterization of the many protein-protein interactions that occur within attachment structures. Biochemical analysis identified protein-protein interactions between PAT-4 and UNC-112, PAT-4 and PAT-6, and PAT-4 and UNC-97 in vitro, and currently we are attempting to measure and characterize these interactions in vivo. Toward this goal, we created transgenic animals in which different dense body and M-line attachment proteins are fused to either CFP (cyan) or YFP (yellow) and are analyzing Fluorescence Resonance Energy Transfer (FRET) ... [truncated at 150 words]
Michalos A, Safonova LP, Choi JH, Gupta R, Polzonetti CM, Calderon-Arnulphi M, Mantulin WW, Gratton E.
Near-infrared spectroscopy for diagnosis and screening of cerebrovascular morbidity in obstructive sleep apnea syndrome.
Shedding new Light on Disease: Optical Diagnostics for the new Millennium. Reims, France, June 23-27, 2002.
Hanson KM, Holub O, Gohlke C, Barry NP, Behne MJ, Gratton E, Clegg RM.
Imaging in skin and plants: using photons and fluorescence lifetimes to find the molecules and quantify the information.
8th Biophysical Society Annual Meeting and Frontier Biophysics Symposium, Taiwan, ROC, 2002.
Due to sophisticated technological advances and innovative applications of new technology, numerous optical imaging techniques are being developed that are highly informative, quantitative, flexible and which can be applied directly to biological and biophysical studies and to medical diagnostics. Fluorescence imaging is one of the fastest growing areas. Many measurements that were previously limited to cuvette type studies are now being incorporated into fluorescence microscopes and into medical diagnostics (such as the endoscope). This lecture will describe two techniques 1) “Fluorescence Lifetime-resolved Imaging” (FLI - where every pixel of the image contains the lifetime-resolved information) and its application to several biological areas, and 2) two-photon scanning imaging applied to skin diagnostic measurements. A brief general description of the various methods in our laboratory will be presented. The reason for making fluorescence lifetime measurements in images will be discussed. The necessity for real-time imaging is stressed, and the performance characteristics of ... [truncated at 150 words]
Kamalesh M, Paunescu LA, Gratton E, Mantulin WW, Sawada SG.
Cerebral and peripheral tissue oxygenation changes during dobutamine infusion measured by near-infrared spectroscopy.
J Am Soc Echocardiogr. 2002; 15(5): 515, P3-23.Behne MJ, Barry NP, Hanson KM, Meyer JW, Crumrine D, Clegg RM, Gratton E, Holleran WM, Elias PM, Mauro TM.
NHE1 regulates stratum corneum acidification and permeability barrier homeostasis: identification of acidic microenvironments with FLIM.
Biomedical Topical Meeting, Miami Beach, Florida, 2002.
Detecting impeded blood flow and locating the clot causing it is a major challenge in neurosurgery. We propose an instrument that uses near-infrared spectroscopy to simultaneously detect clots and measure blood flow.
Eckhoff DA, Sutin JDB, Rogozhina EV, Braun PV, Gratton E.
pH dependent fluorescence in carboxylate-derivative silicon nanocrystals.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 427b.Our work with silicon nanocrystals focuses on their fluorescence properties with the intent of using them as markers in biological systems. We are currently working with derivatized nanocrystals containing a -COOH terminus well suited for reaction with a wide range of biomolecules. In our preparation it appears that at least two different species exist, each with distinct excitation and emission spectra. One population has a max excitation ~290nm with emission ~350-375nm. The second population has excitation peaks ~315 and 365nm with emission ~425-475nm. These derivatives show pH dependent fluorescence with changes affecting mainly the longer wavelength component. The derivatives are also able to form aggregates through a H-bonding network between carboxylate groups. At pH ~9 charge repulsion between the ionized carboxylate groups prevents substantial aggregate formation. Whereas at pH ~1 the neutral carboxylates lead to aggregate formation and the longer wavelength component is substantially quenched. Either this component is intrinsically ... [truncated at 150 words]
Gratton E, Kis-Petiková K.
Measuring distances between particles and molecules using scanning fluorescence correlation spectroscopy.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 427c.Measurement of distance between molecules is generally performed using fluorescence energy transfer when the distance between fluorophores is about 10 nm or less. For larger distances, in the range 10 nm to 200 nm energy transfer cannot be commonly applied. We have developed a method based on scanning fluorescence correlation spectroscopy (scanning FCS) that allows us to obtain distance information in the range 10 to 200 nm with a resolution on the order of 1-5 nm. The method uses two-color detection to separate the contribution of different fluorophores. We have applied the method to systems composed of fluorescent microspheres and to single fluorophores attached to a surface. The scanning FCS method can also be applied to obtain spatio-temporal correlations in the ms time scale. Example of applications to protein and membrane systems will be presented. The same method can also be used to track the movement of particles in 2 ... [truncated at 150 words]
Celli A, Beretta S, Gratton E.
Visualization and properties of lipid phases in giant unilamellar vesicles by two-photon fluorescence microscopy.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 428c.We studied the dynamics of phase transition between the liquid-crystalline and the gel states on GUVs using two-photon excitation microscopy. To visualize the lipid phases we used the spectral properties of LAURDAN whose emission spectrum shifts more than 40 nm between the two phases. To quantify this shift we used the GP function. GUVs made of a binary mixture show phase coexistence in a temperature range between the phase transition temperatures of the two components. We compared the properties of the gel domains in these vesicles with those of GUVs made of a single lipid component. Two photon excitation images of the top of the GUVs membrane were used to calculate the GP pixel distribution for both types of vesicles. The pure gel and liquid phases in vesicles made of a single lipid species are characterized by two specific values of the GP function. We found that the GP value ... [truncated at 150 words]
-
Detecting correlated brightness changes between two species using dual channel two photon fluctuation correlation spectroscopy.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 430a.Fluctuation correlation spectroscopy (FCS) is a non-perturbative fluorescence microscopy technique used to obtain kinetic information by analysis of the stochastic fluctuations in a molecule’s fluorescence. The two photon effect localizes the laser excitation to a three dimensional volume of a femto-liter. Using two channels allows additional information to be obtained from FCS: the cross-correlation. Different criteria to split the signal into the two channels can be used (i.e. dichroic, polarizer, beam splitter). The cross-correlation, at its most basic level, contains information that quantifies the presence of static correlation between two species (e.g. a dichroic can be used to discern if two differently colored fluorophores are physically associated with each other). In addition, kinetic information about a process causing a constant rate of change in the correlation between two species can also be gathered. We present simulations that elucidate the strengths and limitations of this method. The usefulness of this technique ... [truncated at 150 words]
Tahari AK, Motolese G, Gratton E.
The photon counting histogram in turbid media: detection of somatic cells and bacteria in body fluids.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 431c.Our purpose is to find alternative, simpler and cheaper methods to detect and analyze cells and bacteria in body fluids for clinical and biotechnological applications. The photon counting histogram (PCH) is a novel tool for extracting the measured photon counts per molecule and the average number of molecules within the observation volume. In practice, it is possible to realize diffraction limited spots of about 0.2 fl. The best sensitivity is obtained when, on the average, one molecule is contained in that volume. In principle, the volume of observation can be increased to achieve a good sensitivity, but background fluorescence and/or fluorescent impurities impose a limit to the lowest concentration that can be measured. We constructed a small-portable instrument specifically designed to measure somatic cells in milk that has high sensitivity and high dynamic range, it is easy to use and relatively inexpensive. The sample is rotated and moved vertically up ... [truncated at 150 words]
Breusegem SY, Barry NP, Mackinnon AC, Lin X, Williams BD, Gratton E, Clegg RM.
Protein interactions in C. elegans muscle attachment structures studied in vivo by fluorescence resonance energy transfer (FRET).
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 491e.We are interested in deciphering the protein interactions in the focal adhesion-like structures that attach the muscles of C. elegans to its hypodermis and cuticle. To this goal we have created transgenic animals in which the proteins are fused pair-wise to the GFPmutants CFP (cyan) and YFP (yellow). Fluorescence resonance energy transfer (FRET) between the CFP- and YFP-labels will only occur if a direct intermolecular interaction between the host proteins exists. FRET measurements are done in vivo using several microscopy techniques. Using two-photon excitation we applied lifetime imaging, ratioimaging, donor photobleaching kinetics and recovery of the donor intensity after acceptor photobleaching. Using one-photon excitation we applied the intensity-based three-filter set method. To evaluate the accuracy of our results we performed control measurements on a Ca2+-sensitive cameleon construct purified in solution and expressed in bacteria, and on a CFP-YFP fusion construct expressed in C. elegans. The advantages and disadvantages of the ... [truncated at 150 words]
Hanson KM, Barry NP, Behne MJ, Mauro TM, Gratton E, Clegg RM.
Two-photon fluorescence lifetime imaging of the skin's stratum corneum pH gradient.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 494c.Two-photon fluorescence lifetime imaging is used to measure pH within the uppermost layer (stratum corneum) of hairless mice (SKH1-hrBR) skin. Lifetime measurements are made using the fluorophore BCECF whose fluorescence lifetime (τf) is pH sensitive. Following two-photon excitation at 820 nm, τf of BCECF is 2.72 ns at pH 4.5 and 3.93 ns at pH 8.5. Images (100 mm × 100 mm) were acquired every 1.7 mm from the stratum corneum surface through the viable lower strata. Acidic microdomains (average pH 5.9) of variable size (5 mm to 25 mm in diameter) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space approaches neutrality (average pH 6.7). The number of extracellular acidic microdomains decreases with each increasing stratum corneum depth reaching a minimum at the first viable epidermal layer (stratum granulosum). The average pH increases with depth due to the decrease in the ratio of acidic ... [truncated at 150 words]
-
Correlation analysis applied to fluorescence lifetime measurement.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 501d.Typical fluorescence lifetimes lie in the range of 1 to 20 nanoseconds. The frequency domain technique with heterodyne detection has found widespread use in this application, primarily because, apart from the detector itself, fast (nanosecond) signal acquisition electronics are not required. The system still requires precise timing control between the excitation source and detector, usually achieved by phase locking. In the case of low signal levels or poor excitation source stability, averaging is used to improve error bars. This generally involves folding the acquired waveform or averaging the result of a FFT routine. An alternative method is to cross-correlate the signals from the reference detector and sample detector. This yields a waveform with the appropriate beat frequency, phase shift and demodulation characteristic of the fluorescence lifetime, without prior specification of its period. The cross-correlation operation is quadratic, compared to averaging (a linear operation). The manner in which phase noise propagates ... [truncated at 150 words]
Hazlett TL, Sánchez SA, Gratton E.
Exploring transient binding and diffusion of molecules on bilayer surfaces with two-photon fluorescence microscopy.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 546c.We are examining the utility of two-photon imaging techniques in the study of the association of molecules, small fluorophores and fluorophore-labeled proteins, with a bilayer surface. Giant unilamellar vesicles are being used as the model membrane surface and a variety of fluorescent materials are being presented to the bilayer surface via microinjector or passive diffusion from a resting, material-loaded capillary. We are presently optimizing the hardware and experimental design to improve our sensitivity and image time-resolution. Preliminary data on small uncharged fluorophores indicate a rapid initial binding step followed by diffusion across the membrane surface. The spreading of the material on the surface of the bilayer appears to be too fast to be described by simple membrane diffusion of the dye in the membrane. We are developing a diffusion model to explain the observed spreading in terms of a rapid solution-membrane partitioning which then increases membrane diffusion through the introduction ... [truncated at 150 words]
Cheng MA, Bagatolli LA, Zajicek HK, Halaihel N, Levi M, Gratton E, Mantulin WW.
Membrane dynamics in giant unilamellar vesicles (GUVs) of rat kidney brushborder and basolateral integral membrane extracts.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 549e.The plasma membrane in rat kidney proximal tubular cells exhibits polarity in terms of composition, structure and function. It is divided into two distinct regions: the brushborder (BBM) and basolateral (BLM) membranes. We aim to study the complex structure of these biological membranes by applying techniques used in characterizing artificial membrane systems. This involves the direct visualization of GUVs formed from integral BBM and BLM extracts (containing both lipid and protein components) through electroformation. GUVs labeled with the fluorescent probe LAURDAN were imaged using two-photon scanning microscopy. Fluorescence intensity images reveal the formation of micron-sized non-fluorescent domains at physiological temperatures (42-20°C). The absence of intensity in the domains is presumably due to the exclusion of LAURDAN from these regions. These domains differ greatly in shape from those observed in GUVs formed from natural lipid extracts. This implies that membrane proteins also play an important role in the organization of natural ... [truncated at 150 words]
vandeVen MJ, Holub O, Gohlke C, Govindjee, Valcke R, Ameloot M, Clegg RM.
Fast macroscopic chlorophyll fluorescence lifetime imaging of apple fruit skin.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 502a.Early detection of fruit skin storage diseases like bitterpit and storage scald is aided by macroscopic imaging of healthy and diseased tissue. Fast, whole area detection is necessary for quickly assessing fruit quality and for predicting storage potential. Macroscopic fluorescence lifetime imaging enhances contrast w.r.t. steady-state images and reduces geometrical effects. By monitoring photosystem II Chlorophyll a fluorescence it provides quick physiological information over an area not practically accessible by confocal (TPE) microscopy (Biophys. J., 80, 159a). Here we report using a fast-acquisition homodyning phase and modulation imaging fluorimeter (Biophys. J. 80, 169a, 428a) on a 1.8 cm diameter fruit skin area. Red and green/yellow sides of Malus Domesticus Borkh. x Golden Delicious, Jonagold and Granny Smith with and without surface defects were examined with a 690DF40 emission filter. A 6 μm single-mode fiber-optic directed the AOM modulated 488 nm laser light. Operating frequency was 80.652 MHz and total power ... [truncated at 150 words]
Tetin SY, Swift KM, Hazlett TL, Herron J, Matayoshi ED.
Studying Interactions of anti-fluorescein antibodies with fluorescein derivatives at the single molecule level.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 334b.Anti-fluorescein antibodies mAb 4-4-20 and 9-40 have been extensively characterized over the past 15 years in both solution and crystals and therefore serve as excellent mode systems for studying protein-ligand interactions. We have studied the interaction of these mAbs with fluorescein and several fluorescein derivatives using classical kinetics and equilibrium methods, as well as current single molecule detection (SMD) approaches such as fluorescence correlation spectroscopy accompanied by autocorrelation and intensity distribution analyses. The binding affinities of fluorescein, hydroxyphenylfluoron, methylfluoron, and fluorescein amine span a wide range of equilibrium dissociation constants, from 10^-11 to 10^-5 M, that are primarily a result of differences in their dissociation rates. Also, their fluorescence exhibits a variety of responses upon binding, from 96% quenching to 150% enhancement. These latter effects enable the discrimination of free and bound components and the determination of the associative and dissociative rate constants. We have found that the results from ... [truncated at 150 words]
Ruan Q, Cheng MA, Sánchez SA, Glaser M, Mantulin WW.
Characterization of adenylate kinase: fluorescence correlation spectroscopy (FCS) and giant unilamellar vesicles (GUV).
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 150a.Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. An isoform of AK, namely AKb, was recently identified in the plasma membrane. It shares the protein sequence with cytosolic AK except for an additional 18 amino acids at its N-terminal. It is hypothesized that these 18 residues might serve as the signal for myristoylation. We prepared green fluorescence fusion protein with both AK isoforms. Our FCS studies on these constructs in live mammalian cells revealed their interactions with the plasma membrane, possible interactions with other cellular proteins and information about their cellular environment. To further study the biological properties of AK and AKb, they were expressed and harvested in E. Coli. GUV are comparable in size to mammalian cells. They are useful model systems to study protein and membrane interactions. The purified AK and AKb showed no binding affinity to GUV (POPC). ... [truncated at 150 words]
Behne MJ, Hanson KM, Barry NP, Clegg RM, Gratton E, Holleran WM, Elias PM, Mauro TM.
Correlation of extracellular enzyme activity and pH in Stratum Corneum by fluorescence lifetime imaging.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 501a.Acidification of the stratum corneum (SC) is essential for formation and maintenance of the mammalian epidermal permeability barrier, through its activation of SC pHdependent enzymes. Glucocerebrosidase (ß-GlcCer’ase) is active extracellularly in the lower SC. Its deletion results in abnormal SC structure and function both in human disease (Gaucher’s), and in transgenic models. Since ß-GlcCer’ase lipid processing requires an acidic milieu, its in situ activity reflects local pH. Paradoxically, the conventional view of the SC-pH gradient, increasing acidity from lower to outer layers, is contrary to the observed ß-
GlcCer’ase activity. Here, we report a positive correlation of pH and enzyme activity, using a Resorufin conjugate as a synthetic substrate for ß-GlcCer’ase, and simultaneous SC-pH measurement using two photon Fluorescence Lifetime Imaging (FLIM), and the pH sensitive dye BCECF. In contrast to flat electrode measurements, which are invasive, and cannot distinguish pHe vs. phi, we find acidic microdomains in lower layers of ... [truncated at 150 words]
Majumdar ZK, Sutin JDB, Clegg RM.
A continuous flow, turbulent mixer for studying fast reaction kinetics.
46th Annual Meeting of the Biophysical Society, San Francisco, California, 2002.
Biophys J. 2002; 82(1): 483d.We have employed recently developed techniques for microelectromechanical systems (MEMS) for the construction of an ultra-fast, continuous-flow, turbulent mixer. The mixer constitutes a microfluidic system with channel dimensions of 20-80 micrometers made from PDMS (Polydimethylsiloxane elastomer). The design is tailored for making optical measurements of kinetics and the small scale is suitable for microscope or stereomicroscope observation, with the potential to consume volumes smaller than 100 microliters. This system is ideal for studying fast kinetics of biological macromolecules and chemical reactants initiated by a change in the chemical environment.
Wolf U, Wolf M, Choi JH, Paunescu LA, Michalos A, Gratton E.
Mapping of oxyhemoglobin and deoxyhemoglobin changes of the human calf during venous occlusion.
29th Annual Meeting of the International Society on Oxygen Transport to Tissue, Philadelphia, PA. August 11-15, 2001.
With a new probe and a new frequency-domain spectrometer we monitored concentration changes in oxyhemoglobin (D[O2Hb]) and deoxyhemoglobin (D[HHb]) in 22 locations simultaneously on the human calf during venous occlusion.
-
A multi-dimensional platform technology for noninvasively assessing tissue function: near infrared frequency domain spectroscopy.
2nd International Conference on Near-Field Optical Analysis: Photodynamic Therapy & Photobiology Effects. Houston, Texas, USA, May 31-June 1, 2001.
Near infrared (near IR) light penetrates deep into human tissue thereby permitting noninvasive examination of interior regions of the anatomy. Researchers have exploited this fact to advance methods in optical imaging of tissues (for example, optical biopsies for breast cancer detection) and a quantitative, functional assessment of tissue viability and tissue oxygenation (such as in peripheral vascular disease and sleep apnea). As the near IR light enters the tissue it is highly scattered and the photons follow a circuitous path, modeled as photon diffusion, before emerging from the tissue. Contrast in the collected near IR data arises from differences in the recorded optical signals corresponding to oxy- and deoxy-hemoglobin. We have devised instrumentation, which is now commercially available, that operates in the frequency domain and permits quantitative measurement of the optical properties of tissues in terms of both scattering and absorption coefficients. These measurements also provide assessment of tissue oxygenation, ... [truncated at 150 words]
Belomoin G, Barry NP, Smith A, Akcakir O, Abuhassan L, Gratton E, Nayfeh MH.
Synthesis of ultrasmall ultrabright photostable yellow luminescent Si nanoparticles.
APS March Meeting, Seattle, Washington, March 12-16, 2001.
We have synthesized ultrasmall yellow luminescent Si nanoparticles that are ultrabright under uv single photon excitation or under two-photon near infrared excitation. The brightness is comparable to that of rhodamine dye molecules. Monte Carlo quantum calculations yield a Si66 prototype structure. Previously we synthesized ultrabright blue luminescent particles (Si29 prototype). Direct imaging of single particles using transmission electron microscopy and electron diffraction measurements show atomically resolved silicon planes. A small number of the particles and dye molecules were immobilized in agarose gel or on a quartz substrate. Emission from single stationary particles is readily detectable. "Parking" the excitation beam, focused to an average intensity as high as 1MW/cm2, on stationery particles shows that they, unlike the dye molecules, are photostable and do not blink, hence paving the way for their use as fluorescent biomedical labels.
Nayfeh MH, Barry NP, Therrien J, Akcakir O, Gratton E, Belomoin G.
Second harmonic generation and stimulated blue emission in films of ultrasmall Silicon nanoparticles.
APS March Meeting, Seattle, Washington, March 12-16, 2001.
We dispersed electrochemical etched Si into a colloid of ultrabright, blue emitting, ultrasmall (1 nm diameter), hydrogen, oxygen, or biopolymer terminated nanoparticles, and reconstituted it into micro crystallites or uniform films on device quality Si, or silicon oxide. The material is excited by near-infrared two photon femtosecond process at 780 nm. The blue emission from single particles in colloids or frozen in a gel or on a quartz substrate are readily detectable with a high degree of photostability. When films are excited, strong blue emission is observed that exhibits a sharp threshold near 106 W/cm2, rising by several orders of magnitude, beyond which a low power dependence sets in. Also, we observe under some conditions of crystallization directed blue beams between faces of the micro crystallites with a high gain coefficient. The results are analyzed in terms of population inversion and stimulated emission in quantum confinement-induced Si-Si dimer phase, found ... [truncated at 150 words]
Akcakir O, Barry NP, Therrien J, Belomoin G, Nayfeh MH, Gratton E.
Optical properties of Silicon nanoparticles under 1- and 2-photon excitation.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 363a.Silicon nanoparticles are an exciting new candidate for biological labeling. The current sample preparation technique yields a concentrated population of nanoparticles excitable by UV 1-photon, and a more dilute population excitable by NIR 2-photon absorption, as determined by FCS measurements. The particle species distributions may be determined by wavelength dependent FCS provided that there is a distribution of spectroscopic properties. We have constructed an FCS instrument capable of excitation from 260 nm to the visible. A wavelength scan of the particle population based on its differing excitation properties will be presented. Supported by: NIH, PHS 5 P41 RR03155.
Artinian LR, Yu W, Gratton E, Gillette MU.
Multiphoton imaging of the glutamatergic signaling dynamics in the suprachiasmatic nucleus.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 159a-160a, 656.06-Pos.Circadian rhythms in mammals are generated and controlled by the suprachiasmatic nucleus (SCN). Signals of solar cycles are transmitted to the SCN via the monosynaptic retino-hypothalamic tract that releases neurotransmitter glutamate to activate the SCN acceptor cells. In the SCN brain slice preparations glutamate application resets the clock with a phase delay early night and phase advance late night1. Early night glutamate induces a phase delay via the release of the gaseous messenger nitric oxide1 and,
consequently, intracellular calcium release from ryanodine channels (CICR)2. We utilized the method of the multiphoton microscopy of the adult rat brain slice to visualize the effects of glutamate on the key components of the glutamate-induced phase delay signaling. Glutamate-induced calcium transients were visualized with calcium fluorescence dyes, Calcium Green and Fluo 4. Nitric oxide release was visualized with DAF fluorescence dye. The morphological organization of the SCN cells in the living brain slice was studied ... [truncated at 150 words]
Barry NP, Bower A, Konda SD, Singha A, Wiener EC, Gratton E.
Near infra-red contrast agent for tumor cells expressing the high affinity folate receptor.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 166a, 656.39-Pos.A number of tumors of epithelial origin including ovarian cancer, have been shown to express the high-affinity folate receptor, h-FR. Optical detection of such tumors requires good penetration of light into the tissue of interest and some spectroscopic/ absorptive signature to distinguish these cells from normal tissue. In this work we investigate the use of folic acid conjugated to the NIR fluorescent dye, Indocyanine green as a tumor specific contrast agent.. This gives us active targeting of cells that express h-FR in addition to good light penetration through tissue of both the excitation light and fluorescence emission of the NIR dye. With sufficient specificity of staining, this method may be used to verify removal of tumor cells during surgical procedures. We image ovarian tumor cells in a NIR laser scanning fluorescence microscope (excitation at 780nm). We demonstrate specificity of binding using in vitro grown ovarian cancer cell lines one positive ... [truncated at 150 words]
Beretta S, Celli A, Gratton E.
Fluctuation correlation spectroscopy of membrane domains.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 529a.We have developed a spectroscopic method to detect the small phase domains in giant unilamellar vesicles (GUV) made of synthetic phospholipids. In the GUV system, it is relatively straightforward to directly observe, under a microscope, regions of different phases (gel and liquid crystal) using mixtures of phospholipids with different transition temperatures when the size of the domain is large. When the size of the domains is smaller than the point-spread-function of the microscope, small domains of different phases give an average value of the spectroscopic property used to establish the existence of different domains. The analysis of the fluorescence intensity fluctuation in one pixel can provide evidence of an underlying phase structure. We labeled the GUVs composed of single phospholipids and phospholipid mixtures with Laurdan, a fluorescent probe that display different spectroscopic properties in the gel and in liquid crystalline phase. We used a relatively high concentration of Laurdan to ... [truncated at 150 words]
Breusegem SY, Barry NP, Mackinnon AC, Williams BD, Clegg RM.
Integrin interactions in C. Elegans muscle studied in vivo by fluorescence resonance energy transfer (FRET).
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 162a, 656.2-Pos.Fluorescence microscopy was used to study integrin interactions in vivo in the nematode C. elegans. Integrins are important transmembrane receptor proteins involved in cell signaling and adhesion. In C. elegans muscle, dense bodies and M-lines anchor the myofilament lattice to the cell membrane and the extracellular matrix. These highly organized structures contain integrin heterodimers as well as cytoskeletal adapter proteins and signaling molecules. They are analogous to the focal adhesion complexes found in human cells and as such they are good systems to investigate integrin’s adhesive and cell signaling functions. Fluorescence resonance energy transfer (FRET) between fusion proteins with GFP mutants of different color allows the detection of direct intermolecular interactions in vivo. Transgenic animals were created in which protein pairs in the dense bodies or M-lines are labeled with cyan fluorescent protein (CFP) as a donor and yellow fluorescent protein (YFP) as an acceptor. Several methods were then used ... [truncated at 150 words]
Celli A, Beretta S, Gratton E.
Fluctuation correlation of the generalized polarization in phospholipid giant unilamellar vesicles.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 529a.Two-photon excitation microscopy images of giant unilamellar vesicles (GUVs) have been used to study the pixel distribution of the Generalized Polarization (GP) function. The GP function report on the extent of water penetration in the membrane. For GUVs in the gel phase, the pixel GP distribution is centered at about 0.6 and it is narrow. The GP pixel distribution of GUVs in the liquid crystalline phase is centered in the region 0.0 to -0.3 depending on the temperature and is relatively broad, even for vesicles composed of a single phospholipid components. This observation is intriguing because it is expected that in the liquid crystalline phase, at relatively high temperature, the membrane should be rather homogeneous. We have studied the spatial and temporal correlation of the GP function in different sections of the GUVs. We have analyzed our results for possible artifacts arising from poor statistics. We have observed relatively slow ... [truncated at 150 words]
Cheng MA, Bagatolli LA, Zajicek HK, Halaihel N, Levi M, Gratton E, Mantulin WW.
Characterization of giant unilamellar vesicles of integral membrane extracts from rat kidney brushborder and basolateral membranes using two photon fluorescence microscopy.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 527a.Polarized epithelial cells exhibit differences in structure and composition in certain regions of the cell membrane. In this study, we characterize differences between brushborder (BBM) and basolateral (BLM) membranes from rat kidney cells in terms of membrane fluidity and topology. Previous studies using steady-state fluorescence spectroscopy suggested that these membranes exhibit coexistence of lipid domains at physiological temperatures. Giant unilamellar vesicles (GUVs) were formed from integral (containing both lipid and protein components) BBM and BLM extracts. Images of the Laurdan labeled GUVs were obtained through the sectioning capability of twophoton fluorescence microscopy. Laurdan Generalized Polarization function (GP) was calculated at different temperatures to study the membrane phase-state in these preparations. A novel result for both preparations was the direct observation of circular micron-size lipid domains on the GUV surface, supporting the hypothesis of “lipid raft” formation. GP values were consistently higher for the BBM as compared to the BLM. Furthermore, ... [truncated at 150 words]
-
Dual channel two photon fluctuation correlation spectroscopy.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 359a-360a.Fluctuation correlation spectroscopy (FCS) is a non-perturbative fluorescence microscopy technique used to obtain kinetic information by analysis of the stochastic fluctuations in a molecule’s fluorescence. The two photon effect localizes the laser excitation to a three dimensional volume of a femto-liter. Using two channels allows additional information to be obtained from FCS - the cross-correlation. Flexibility results from using different criteria to split the signal into the two channels (i.e. dichroic, polarizer, beam splitter). We have investigated more closely the meaning of the crosscorrelation curve and its relation to the autocorrelation curves. In addition technical issues involving the positioning of the Avalanche Photo Diode (APD) detectors affecting the two autocorrelation curves from the two channels and the cross-correlation curve was explored. A biological system was examined to determine the usefulness of this technique as a biological microscopy tool. Supported by the National Institutes of Health, PHS 5 P41 RRO3155.
Eckhoff DA, Barry NP, Akcakir O, Gratton E, Therrien J, Belomoin G, Nayfeh MH.
Fluorescence properties of "as grown" and oxidized silicon nanoparticles.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 363a.The recent introduction of nanocrystalline silicon particles as a new class of fluorescent markers has sparked intense excitement in the biological community and beyond. These particles have been shown to have good photostability, relatively small size (~1-2nm), strong fluorescence, and low toxicity, all of which make them excellent candidates for use in biological environments. The nanoparticles have the advantage that they can be tailored via their particle size and level of oxidation to exhibit a wide range of fluorescence characteristics. In this work, we study the excitation, emission, and absorption spectra of 3 samples differing in their level of oxidation. We observed a time evolution in the fluorescence, which appears to be correlated with the level of oxidation and occurs over a time scale of 1 to 2 months. For the strongly oxidized sample, there is little to no change over time, while for the mildly oxidized and unoxidized samples, ... [truncated at 150 words]
Gohlke C, Holub O, Clegg RM.
FliFast: Software for fast fluorescence lifetime-resolved image acquisition with concurrent analysis and visual feedback.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 169a, 656.54-Pos.Fluorescence lifetime-resolved imaging FLI provides indispensable, valuable information than is not available from steady-state fluorescence image measurements. In general, because of the complexity of the analysis compared to the display of simple intensity images, FLI measurements have required much longer times to process and display. However, many biological and medical applications using fluorescence imaging require real time acquisition, processing and display. Real-time operation is necessary for following dynamic biological events, or carrying out medical diagnostics. We have developed a real-time FLI system for a variety of imaging applications. The instrument uses rapid frequency-domain data acquisition hardware; however, here we demonstrate software that specifically enables the real-time processing and highly informative, convenient and easy-to-understand display of the lifetime-resolved fluorescence information. A menu of possibilities is provided to the operator to assist in the on-line interpretation and control of real-time experiments and rapid events. The programs have been streamlined in order to ... [truncated at 150 words]
Sánchez SA, Chen Y, Müller JD, Gratton E, Hazlett TL.
Dissociation of a dimeric PLA2 with a micellar substrate analog.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 546a.The venom phospholipases A2 (PLA2) are soluble, small molecular weight enzymes that hydrolyze the sn2-acyl ester bond of phospholipids. The PLA2 from C. atrox venom has been shown to exist as a homodimer in solution, and early reports had suggested that the dimer was the active form. Later studies indicated that the monomer was the active unit and structural data has supported this view. In this study, we use fluorescence correlation spectroscopy (FCS) on a fluorescein-labeled PLA2 to examine this issue. The state of the PLA2 monomer-dimer equilibrium was measured above and below the CMC of dodecylphosphocholine (C12-PN), micellar substrate analog by FCS and intrinsic fluorescence. We have interpreted that data in terms of three PLA2-lipid forms: (1) PLA2 dimers free in solution minimally bound with C12-PN monomers, (2) an intermediate condition where two loosely interacting PLA2 monomers are contained in a co-micelle of protein and C12-PN and (3) complete ... [truncated at 150 words]
Holub O, Seufferheld MJ, Gohlke C, Govindjee, Clegg RM.
Application of real-time fluorescence lifetime-resolved imaging in photosynthesis: studies of maize leaves (Zea Mays), small mustard leaves (Arabidopsis Thaliana), and of individual wild-type and mutant cells of the green alga Chlamydomonas Reinhardtii.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 428a, 1819-Pos.Chlorophyll (Chl) a fluorescence from leaves of Zea Mays, Arabidopsis Thaliana and from single cells of wild type (WT) and non-photochemical quenching mutants (NPQ1 and NPQ2) of C. Reinhardtii was observed with a new high-speed instrument for measuring fluorescence lifetime-resolved microscope images. Objects are imaged using the frequency domain phase and modulation technique in homodyne mode. The laser light is modulated at a high frequency. The fluorescence image is focused onto a modulated image intensifier and the phase-resolved images are captured on a fast CCD camera, sequentially transferred to a PC computer and displayed in real-time. Rapid measurements are necessary during the fluorescence transient maximum (the P-level) following initial illumination. The observed lifetime heterogeneity is in accordance with photosynthetic functionality. The NPQ1 and NPQ2 mutants (Niyogi et al., 1997) used in this work accumulate violaxanthin and zeaxanthin, respectively. A decrease in the lifetime of Chl a in NPQ2 was observed ... [truncated at 150 words]
Jacobson KA, Volovyk ZN, Thompson NL, Bagatolli LA, Gratton E, Levi M, Dietrich C.
Lipid rafts in model and biological membranes.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 31a.One key tenet of the lipid raft hypothesis is that the formation of sphingolipid and cholesterol rich domains can be driven solely by characteristic lipid-lipid interactions suggesting that visible rafts may form in model membranes composed of appropriate lipids. In fact, using fluorescence microscopy, less fluid domains with raft-like properties were found to coexist with more fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUV) formed from (1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or (2) from the natural lipids extracted from brush border membranes which are rich in sphingomyelin and cholesterol. This fluidfluid phase coexistence was characterized by domains several microns in diameter while non-raft mixtures (phospholipids-cholesterol) appeared homogeneous. Consistent with the raft hypothesis, GM1, a glycosphingolipid, was highly enriched in the more ordered domains that were resistant to Triton X100 detergent extraction. Thus, many of the tenets of the raft hypothesis can be confirmed ... [truncated at 150 words]
Kis-Petiková K, Gratton E.
A method for distance measurement between fluorescent particles in the 10-200 nm range.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 161a, 656.14-Pos.We introduce a method to measure relative distances of fluorescent particles of different color immobilized on a quartz surface in a two-photon scanning fluorescence microscope, with two channel photon counting detection. The method is sensitive in the 10-200 nm range, filling the gap between fluorescence resonant energy transfer and far field light microscopy. Instead of raster scanning of an image, excitation beam is moved periodically in a circular orbit with a radius of the size of the point spread function (300 nm), in order to achieve maximum sensitivity in the radial direction. Fluorescence intensity varies periodically with the scanning frequency (500 Hz). Fast Fourier transform of the fluorescence signal during one or more orbit gives the modulation of the first harmonic, which depends upon the radial distance of the particle from the center of scanning. The coordinates of the center of mass of particles are calculated from the modulation and ... [truncated at 150 words]
Kwon HS, Dong CY, Sutin JDB, Huang H, Gratton E, So PTC.
Optimizing the design of a magnetic micro-manipulation microscope for cellular and single molecular studies.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 160a, 656.1-Pos.We designed and optimized a magnetic micro-manipulator microscope intended for biological applications at the cellular and single molecular level. In our 8-pole design, samples mounted with magnetic particles placed at the trap center can experience 3-D uniform force or torsional stress. The poles are designed and positioned to allow a high numerical aperture, water immersion objective (NA 0.9, Zeiss Achroplan) with 1.46 mm of working distance to be used. Combined with optimized wire winding and implementation of back irons, magnetic field and gradient is localized to the sample region. By placing the sample at the center of the micro-manipulator, 3-D mechanical manipulation of the specimen can be achieved. Our system is capable of exerting force on the order of 800 pN per ferromagnetic particle 5 μm in diameter. We will present data showing the 3-D force and torque calibration. Biological applications of this magnetic micro-manipulation microscope will be discussed. In ... [truncated at 150 words]
Majumdar ZK, D’Amico ML, Clegg RM.
Fluorescent Properties of Cy5 (5-N-N’-diethyltetramethylindodicarbocyanine) covalently linked to DNA.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 483a, 2066-Pos.Fluorescent dyes covalently linked at specific sites on nucleic acids are used for determining various properties of the nucleic acids, such as their structure and stability by the use of FRET. Photophysical studies lead to a better understanding of the interactions of molecules with the chromophore. The Cy3-Cy5 pair is an optimal donor-acceptor pair for FRET. Both dyes have high extinction coefficients (150,000 and 250,000 cm^-1M^-1, respectively) and their emission spectra are well separated from most sample autofluorescence. We present an analysis of the fluorescence properties of Cy5 attached to the 5’ end of double-stranded (ds) DNA, with a C3-linker to the phosphodiester. Measurements of the fluorescence lifetime, time-resolved anisotropy and steady state measurements are used to understand the structure of the Cy5-DNA complex. Temperature and solvent effects on the fluorescence properties are studied as well. In addition to providing a better understanding of the structure of the Cy5-DNA complex, ... [truncated at 150 words]
Margeat E, Poujol N, Chen Y, Müller JD, Boulahtouf A, Gratton E, Cavailles V, Royer CA.
Fluorescence correlation and anisotropy approaches the study of nuclear receptor/coactivator complexes.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 361a.Nuclear receptors act as ligand-inducible transcription factors. Agonist binding leads to interaction with coactivator proteins, and to the assembly of the general transcription machinery. In addition to structural information, a thorough understanding of transcriptional activation by the nuclear receptors requires the characterization of the thermodynamic parameters governing these protein / protein interactions. In this work, fluorescence spectroscopy has been used for the in-depth investigation of the interaction between Estrogen Receptor (ER) and the nuclear receptor coactivator SRC1570-780 labeled with Alexa 488. In a first step, photon counting histogram analysis has been applied on fluorescence correlation data for the elucidation of the stoichiometry of the complex. We show that, even at high concentration of fluorescent coactivator, outside the single molecule limits, one can directly measure the number of molecules in the complex, leading to a 1 SRC-1 molecule per ER dimer stoichiometry. Then, using fluorescence polarization, we address the influence of ... [truncated at 150 words]
Minchenko M, Lamb DC, Durbin DM, Kosman J, de Beer M, van der Westhuyzen D, Mantulin WW.
Interaction of high density lipoprotein with the receptor SR-BI: a fluorescence microscopy study.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 540a.It has been recognized that the risk for cardiovascular disease due to atherosclerosis follows an inverse correlation with levels of high density lipoprotein (HDL). It was shown that the SR-BI receptor recognizes a variety of native HDL and reconstituted HDL (rHDL). In the current study, we used the murine SR-BI (HDL) receptor expressed in Chinese hamster ovary cells to visualize rHDL binding. Homogeneous rHDL composed of POPC, cholesterol and apolipoprotein AI (fluorescently labeled at either the protein or lipid moiety) allows one to follow binding and subsequent processing in a continuous fashion. Image sequences were acquired using a scanning two-photon fluorescence excitation microscope. These measurements indicated that fluorescent lipid analogs distribute throughout the cell and the apolipoprotein remains on the plasma membrane for at least an hour. Kinetic analysis of binding curves provided a rate constant of binding of rHDL to SR-BI (5.4*10^4 M^-1s^-1) and a dissociating rate constant of ... [truncated at 150 words]
Ragan TM, Belmont AS, So PTC, Gratton E.
Two-photon single particle tracking: investigation into chromatin movement.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 171a, 656.56-Pos.Recent research has indicated that the dynamics of chromatin movement inside of the nucleus may be much quicker than what was previously thought. For example, largescale chromatin structure reorganization induced by transcriptional activation has recently been observed. In order to study chromatin in vivo we are using a singleparticle tracking system that we have developed based around the two-photon microscope. The system is capable of tracking particles by either of two methods: The first method introduces aberrations into the optical system which break the axial symmetry of the point spread function and allows the 3D determination of the particle's position. The second method uses volumetric scanning to localize the particle. The system has an extended axial range of 100 microns and a frequency response of 15 Hz. To study the chromatin motion we have tagged regions of the chromosome by using lac repressor recognition of direct repeats of the lac ... [truncated at 150 words]
Ruan Q, Chen Y, Glaser M, Mantulin WW.
Characterization of Adenylate Kinase (AK1) its isoform AK1ß and their fusing EGFP construct in living cell.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 167a.Adenylate kinase (Myokinase E.C.2.7.4.3) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. In vertebrates, three isozymes have been characterized: AK1 is cytoplasmic; AK2 and AK3 are localized in the intermembrane space of mitochondria. Recently, another AK1 protein from murine cells was identified, namely cytosolic AK1 (C. Schneider et al). Its sequence is identical to cytosolic AK1, except for an additional 18 amino acids at the N-terminal. Our study showed that AK1 from murine cell when expressed in E. Coli, is competent to replace the biological function of the native AKe. However, AK1ß can not replace AKe's biological function in E.Coli. It is hypothesized that the 18 additional residues might serve as the signal and anchor for myristoylation. To better understand the function of these 18 additional residues, the genes of AK1ß were fused with the gene of EGFP (Green Fluorescence Protein) and incorporated into ... [truncated at 150 words]
Sánchez SA, Müller JD, Hazlett TL, Gratton E.
Protein dissociation under pressure using fluctuation correlation spectroscopy.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 556a.The dissociation of multimeric proteins under pressure has provided important information about protein-protein interactions. The volume of protein subunits is generally smaller than the volume of the native protein and an increase in pressure causes subunit dissociation. However, most of the present methods to detect dissociation are based on spectroscopic signals that are related to the native and the dissociated state. These signals do not directly report the dissociation, but rather changes in protein conformation that could depend on the actual separation of the subunits. These spectroscopic changes frequently occur over a relatively large range of pressure and part of the signal change may simply be due to the formation of dissociation intermediates. Our new approach is based on the direct measurement of the number of fluorescent elements in a given volume by fluctuation correlation spectroscopy (FCS). We designed a pressure cell that fits our FCS instrument. The cell is ... [truncated at 150 words]
Sillen A, Lamb DC, Gratton E.
FCS of molecules fluorescing in the ultra violet region.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 366a.Fluorescence correlation spectroscopy (FCS) is a technique used to measure diffusion coefficients and concentration of molecules in thermal equilibrium. A drawback of this technique is that a chromophore must be covalently attached to the molecule of interest since few molecules absorb or fluoresce in the visible and near infra red wavelength region. This is particularly true for proteins and they must be chemically linked with a fluorescent dye molecule or genetically linked to any of a number of the newly discovered fluorescence proteins. This fluorescent label can influence the properties of the protein under investigation. Since many proteins naturally fluoresce in the UV region, we have developed a fluorescence microscope for performing FCS measurements in the UV. Here we present FCS measurements on a multitryptophan containing protein. These results show that it is now possible to study proteins by their intrinsic fluorescent probes. This work was supported by NIH, PHS ... [truncated at 150 words]
Sutin JDB, Dong CY, Mahadevan L, Gratton E, So PTC.
Exonuclease motility studied by single-molecule magnetic force assays.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 569a.Single molecule force manipulation is an important tool for investigating how biochemical reactions produce mechanical effects, since mechanical properties are difficult to measure by conventional biochemical techniques. We are investigating the mechanism of mechanochemical coupling between DNA hydrolysis and enzyme translocation during DNA degradation by lambda exonuclease. We have developed a magnetic manipulation microscope for single molecule micromechanical experiments using magnetic microspheres. The principle feature of magnetic micromanipulation is that the force is inherently applied under isotonic conditions, without the need for feedback. We have coupled a 6-histidine mutant of lambda exonuclease to 4.5 micron streptavidin coated superparamagnetic beads via a biotin-NTA linker. The beads are introduced to amino-modified lambda DNA covalently attached to a coverslip via a silane linkage. Since the contour length of lambda DNA is ~16 micron, the motion of the exonuclease as it digests long sections of DNA can be determined repeatedly by the calculating the ... [truncated at 150 words]
Tahari AK, Motolese G, Gratton E.
Fluctuation correlation spectroscopy (FCS) in turbid media: detection of somatic cells and bacteria in body fluids.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 164a-165a, 656.31-Pos.We seek an alternate, simple, and cheap method to analyze cells and bacteria in body fluids for clinical and biotechnological applications; a task commonly performed using a flow cytometer, a large, expensive instrument. FCS can detect and quantify single molecules in small volume. Single molecule sensitivity is obtained when small volumes (~0.2 fL) of excitation is achieved. Best sensitivity is when one molecule is contained in that volume, i.e., for concentrations of ~10nM and the range spans ~1uM to ~10pM. Volume of observation can be increased for better sensitivity, but background fluorescence or impurities limit the lowest detectable concentration. For analysis of body fluids, sensitivity on the order of 100-100000 particle/ml is needed. Bacteria or somatic cells stained with fluorescent dyes give large fluorescence signals, but volume must be increased by §108 to have about one particle in the observation volume. Also, diffusion does not produce fast enough fluctuations. We ... [truncated at 150 words]
-
Conformational behavior of DNA-Cy3 complex.
45th Annual Meeting of the Biophysical Society, Boston, Massachusetts, 2001.
Biophys J. 2001; 80(1 Pt 2): 483a, 2065-Pos.The photophysical properties of 5-N,N’-diethyl-tetramethylindocarbocyanine (Cy3) attached to the blunt end of an 18bp deoxyoligonucleotides have previously been studied. We now extend the investigation by incorporating an extra base which intervenes the dye molecule and blunt end of the 18bp deoxyoligonucleotides. The dye molecule is attached via a C3 linker to the photodiester on the strand with extra nucleotide. Steady-state flourescence, life time and anisotropy decay are measured under various temperature and solvent conditions. The results are compared with earlier work to provide better understand of the conformational behavior of DNA-Cy3 complex.
Clegg RM, Stühmeier F, Gohlke C, Vámosi G.
Conformations and stabilities of nucleic acids using FRET.
Abstr Paper Am Chem Soc Natl Meet. 2000; 220: U239-U239 568-PHYS Part 2.Franceschini MA, Toronov VY, Filiaci ME, Wolf M, Michalos A, Gratton E, Fantini S.
Real-time video of cerebral hemodynamics in the human brain using non-invasive optical imaging.
6th Annual Meeting of the Organization for Human Brain Mapping. San Antonio, Texas, June 12-16, 2000.
NeuroImage. 2000; 11(5 Suppl 1): S454.Wolf M, Wolf U, Toronov VY, Paunescu LA, Michalos A, Franceschini MA, Fantini S, Gratton E.
Fast cerebral functional signals in the 100 ms range detected by frequency-domain near-infrared spectroscopy.
6th Annual Meeting of the Organization for Human Brain Mapping. San Antonio, Texas, June 12-16, 2000.
NeuroImage. 2000; 11(5 Suppl 1): S515.Introduction: Using non-invasive near-infrared spectroscopy (NIRS) fast changes in the range of milliseconds in the optical properties of cerebral tissue have been described, which are associated with brain activity. This signal is small and related to changes in light scattering due to alteration in the refractive index at neuronal membranes. We obtained functional maps of this fast signal in the visual cortex during visual stimulation with a new type of sensor, the n-sensor.
Holub O, Seufferheld MJ, Gohlke C, Clegg RM, Govindjee.
Real-time fluorescence lifetime-resolved images of individual cells of wild type and NPQ mutants of Chlamydomonas reinhardtii.
9th International Conference on the Cell and Molecular Biology of Chlamydamonas, Amsterdam, The Netherlands, May 21-26, 2000.
The chlorophyll a (Chl a) fluorescence from single cells of wild type (WT) and non-photochemical quenching (NPQ) mutants (NPQ1 and NPQ2) of Chlamydomonas reinhardtii was observed with a new high-speed instrument for measuring fluorescence lifetime-resolved images (see Instrumentation). The mutants NPQ1 and NPQ2 used in this work (Niyogi et al., 1997) , accumulate violaxanthin and zeaxanthin, respectively. Fluorescence transient studies (see poster by Seufferheld et al., 2000) show strong quenching of Chl a fluorescence in the NPQ2 mutant in comparison to the WT and the NPQ1 mutant. If the quenching is due to Förster transfer from Chl a to zeaxanthin, then the lifetime of Chl a in the NPQ2 mutant will decrease. The time-resolved fluorescence microscopy of the NPQ mutants can therefore be used to study the role of the Xanthophyll cycle in the non-photochemical quenching process in single cells. This work is supported by NIH PHS 5 P41-RRO3155 and ... [truncated at 150 words]
Sukhishvili SA, Chen Y, Müller JD, Schweizer KS, Gratton E, Granick S.
Two-dimensional diffusion of PEO adsorbed from dilute solution to a solid surface.
APS March Meeting, Minneapolis, MN, March 20-24, 2000.
The molecular weight (M) dependence was measured of center of mass diffusion (D) of polymer chains which are adsorbed onto the solid surface from aqueous solution. To this end we have applied fluorescence correlation spectroscopy with two-photon excitation to detect translational motions of polyethylene oxide chains which were terminally labelled with a bright and photostable water-soluble dye. Apart from the expected slowing down of polymer motions owing to adsorption, we found unusual scaling of polymer diffusion with molecular weight. Role of the nature of the substrate, degree of saturation of the adsorbed layer with the polymer chains and ionic strength of solution is discussed. At dilute surface coverage at a hydrophobic surface, we find D ~ M^-3/2
Akcakir O, Therrien J, Belomoin G, Barry NP, Müller JD, Gratton E, Nayfeh MH.
Detection of luminescent single ultra-small silicon nanoparticles using fluctuation correlation spectroscopy.
APS March Meeting, Minneapolis, MN, March 20-24, 2000.
We dispersed electrochemical etched Si into a colloid of ultra-small blue luminescent nano particles, observable with the naked eye, in room light. We use two-photon near-infrared femto second excitation at 780 nm to recorded the fluctuating time-series of the luminescence, and determine the number density, brightness, and size of diffusing fluorescent Si particles. The efficiency of the colloid is high enough such that we were able to detect single Si particles, in a focal volume of a pico cubic centimeter. The measurements yield a particle size of ~ 1nm, consistent with direct imaging by transmission electron microscopy. They also yield an excitation efficiency two to threefold larger than that of fluorescein.
Nayfeh MH, Barry NP, Therrien J, Akcakir O, Gratton E, Belomoin G.
Stimulated blue emission in reconstituted films of ultra-small silicon nanoparticles.
APS March Meeting, Minneapolis, MN, March 20-24, 2000.
We dispersed electro-chemical etched Si into a colloid of enriched blue emitting ultra-small (1 nm in diameter) nano particles, and reconstituted it into micro crystallites or uniform films on device quality Si. When the film is excited by near-infrared two photon process at 780 nm, we observe an extremely strong blue band that exhibits a sharp threshold near 106 W/cm2, rising by many orders of magnitude, beyond which a low power dependence sets in. We also observe directed blue beams between faces of the micro crystallites. The results are analyzed in terms of population inversion and stimulated emission in quantum confinement-induced Si-Si dimer phase, found only on ultra-small Si nanoparticles.
Holub O, Gohlke C, Clegg RM.
Want to know something about your fluorescent samples no lenses can resolve? Big scale scanning setup for real-time fluorescence lifetime-resolved imaging.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 1464-Pos.We present a new high-speed setup for measuring fluorescence lifetime-resolved images of larger objects. This instrument allows scanning of extended objects using the phase and modulation technique in homodyne mode. A low-resolution objective allows the laser illumination of the sample placed on a xy-scanning stage and also captures the whole fluorescent image and projects it on a modulated image intensifier. A fast CCD camera captures the incrementally phase-delayed modulated inages, which are then processed and displayed in real-time by programs on a PC computer. Light modulation (CW laser) is achieved by the use of a standing wave acousto-optical modulator. We demonstrate the functionality and use of the instrument with different examples. This work is supported by NIH PHS 5 P41-RRO3155 and NSF DBI96-0240.
Chong PLG, Bagatolli LA, Gratton E, Khan TK.
Two-photon fluorescence microscopy studies of bipolar tetraether archaebacterial liposomes.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 1052-Pos.The effects of temperature (10-65 °C) and pH (7.2 and 2.7) on Laurdan fluorescence intensity images of giant unilamellar vesicles (GUVs) composed of the polar lipid fraction E (PLFE) from thermoacidophilic archaebacteria S. acidocaldanius have been studied using two-photon excitation. The excitation generalized polarization (GP) of Laurdan fluorescence in PLFE GUVs always showed a broad distribution (> 0.3), and the GP values were low (< 0). When excited with the y-direction polarized light, Laurdan fluorescence in the center cross-section of PLFE GUVs exhibited a photoselection effect in favor of the x-direction. These results suggest that the emission dipole of Laurdan is aligned parallel to the membrane surface and the Laurdan chromophore resides in the polar head-group region of PLFE. The GP exhibited a small but abrupt change near 50 TC, which may correspond to a conformational change in the polar eadgroups of PLFE. Most interestingly, the two-photon Laurdan fluorescence ... [truncated at 150 words]
-
Direct visualization of lipid domains in free bilayers. a two photon fluorescence microscopy study.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 1051-Pos.Combining the sectioning capability of the two-photon microscope and the partition and spectral properties of LAURDAN, PRODAN and N-Rhodamine-DPPE on the solid and fluid lipid phases, temperature-induced changes on single giant unilamellar vesicles (GUVs) were studied. We obtained images of GUVs composed of phospholipid binary mixtures labeled with the different fluorescent probes using polarized laser excitation. To characterize the lipid phase-state we used the LAURDAN Generalized Polarization function (GP). At temperatures corresponding to lipid domain coexistence we observed concurrent fluid and solid domains in GUVs composed of DPPE/DPPC, DLPC/DPPC, DLPC/DSPC, DLPC/DAPC and DMPC/DSPC. In all cases the domains grew and moved on the vesicle surface as we decreased the temperature. The size of the solid domains was up to several microns (-30 ism), displaying different shapes dependent on the lipid mixture. In the case of DPPE/DPPC (7:3 mol/mol) we observed mainly dendritic shape. Instead, in the PC mixtures (1:1 mol/mol) ... [truncated at 150 words]
Müller JD, Chen Y, Eid JS, Akcakir O, Gratton E.
High-pressure fluorescence fluctation spectroscopy.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 755-Pos.Presure is an important parameter for the study of macromolecular interactions. Pressure can proteins, dissociate multimeric protein assemblies, kill bacteria, induce phase transitions in bilayer membranes and deactivate viruses and toxins. We developed a high-pressure cell to be under a microscope. The high-pressure cell consists of a fused silica capillary, which is high-pressure system. Two-photon excitation is used to generate a very small volume inside the capillary. Fluorescently labeled particles diffusing through the observation volume are measured by fluorescence correlation spectroscopy (FCS). FCS offers unique advantages to study pressure perturbed systems. Kinetic processes can be determined by autocorrelation function atthermnodynamic equilibrium. The dissociation or association of proteins followed as a function of pressure by a change in the fluctuation amplitude or the coefficients. We recently introduced another analysis method, the photon counting histogram (PCH), to characterize fluorescence fluctuations, which is complementary to the FCS approach. demonstrate the use of PCH ... [truncated at 150 words]
Hanson KM, Barry NP, Gratton E, Clegg RM.
Fluorescence lifetime imaging of pH in the stratum corneum.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 1478-Pos.Fluorescence lifetime imaging microscopy (FLIM) is being developed to explore the pH gradient in the uppermost layer of the skin's epidermis, the stratum corneum (SC). The SC provides the primary barrier between the environment and the body's interior. It is also considered to play an essential role in normal skin renewal each month (cornification). Over the extremely thin stratum corneum (-20 pm) the pH has been reported to gradually change from 4-5.6 at the skin's surface to pH 7.2 at the stratum corneum-granulosumjunction. This corresponds to an increase of up to 1000-fold in the proton concentration over the 20-40 SC cellular layers. Tape-stripping measurements have been employed to study pH changes in the SC; however, such measurements are invasive and provide little information on the location and shape of the proposed SC pH gradient. Consequently, we are developing FLIM experiments to explore non-invasively pH changes within the heterogeneous tissue. For ... [truncated at 150 words]
Breusegem SY, Loontiens FG, Clegg RM.
Binding of Hoechst 33258 and DAPI to (A/T)4 DNA sites: base-sequence specificity determined by fluorescence titrations and stopped flow kinetics.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 1804-Pos.Hoechst 33258 (a bis-benzimidazole compound) and DAPI (4',6'-diamidino-2-phenylindole) become brightly fluorescent when binding in the minor groove of DNA, preferentially at respectively 5 or 3 contiguous AT-base pairs. We determined the binding affinity at 20"C of both dyes for isolated (A/T)4-sites incorporated in a 28-nucleotide-hairpin by fluorescence titrations. Because of the high affinity the titrations had to be carried out at very low dye concentrations of -2 nM. The affinities of DAPI and Hoechst 33258 for the AATT site are similar (e.g. Hoechst 33258 association constant = 5.2*108 M') and the affinity decrease in the sequence series runs parallel for both dyes: AATT>TAAT>ATAT>TATAzTTAA for Hoechst 33258 and AATT>TAATnATAT>TATAzsTTAA for DAPI. For Hoechst 33258 the association constants for the most and the least affine site in the series differ by a factor of 190, for DAPI this difference is only a factor of 30. The binding kinetics of Hoechst 33258 were ... [truncated at 150 words]
Sutin JDB, Dong CY, So PTC, Mahadevan L, Gratton E.
Magnetic micromanipulation of single molecules of exonuclease during DNA degradation.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 1796-Pos.We have developed a magnetic manipulation microscope for single molecule micromechanical experiments using magnetic microspheres. The principal feature of magnetic micromanipulation is that the force is inherently applied under isotonic conditions, without the need for feedback. This is particularly advantageous in the low force regime where feedback is difficult. In addition, forces can be applied over large distances limited only by the size of the microscope field and multiple microspheres can be manipulated simultaneously. We are using the magnetic micromanipulator to probe the molecular dynamics of a single molecule of exonuclease as it digests DNA. A 6-histidine mutant of lambda exonuclease is coupled to a 4.5 micron streptavidin coated superparamagnetic bead via a biotin-NTA linker. The beads are introduced to amino-modified lambda DNA that is covalently attached to a coverslip via a silane linkage. Since the contour length of lambda DNA is -16 microns, the motion of the exonuclease as ... [truncated at 150 words]
Akcakir O, Barry NP, Gratton E, Therrien J, Belomoin G, Müller JD, Nayfeh MH.
Characterization of fluorescent silicon nanoparticles under two photon excitation.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 2604-Pos.Nanocrystaline particles have shown potential for use as fluorescent probes in biological systems. Such nanoparticles are characterized by high photostability and low toxicity when compared to typical fluorescent dyes. In this work we characterize the brightness of individual silicon nanoparticles using fluctuation correlation spectroscopy. By analysis of the amplitude and time duration of the fluorescence fluctuations we conclude that their brightness (under two-photon excitation) and size are comparable to fluorescein. A suspension of silicon nanoparticles in acetone was prepared from a porous silicon substrate. We use a two-photon microscope with a femtosecond Ti-sapphire laser excitation source at 780nm. The fluorescence emission (in the photon counting regime ) was detected using either an avalanche photodiode or a photomultiplier. The time sequence of the detected signal was analyzed using autocorrelation function and photon count histogram fitting software developed in our lab. This study enabled us to determine the number density of the ... [truncated at 150 words]
Kis-Petiková K, Chen Y, Müller JD, Gratton E.
Application of scanning fluorescence correlation spectroscopy for determination of particle shape.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 2603-Pos.Two-photon fluorescence correlation spectroscopy (FCS) has been proved as a powerful experimental technique for the study of fluctuations in the fluorescence signal of samples with low concentrations. From the measured autocorrelation function, the diffusion coefficient, chemical reaction rates or number of labeled particles can be determined. In the scanning FCS (S-FCS) technique the exciting beam is scanned periodically across the sample. By scanning through independent subvolumes, sensitivity of the measurement can be improved significantly. In addition, the autocorrelation function obtained from S-FCS in principle contains more information concerning the shape of diffusing molecules, when particles are of the size comparable to the laser beam size and scanning radius. Besides of time correlation, analyzed in FCS measurement, a spatial correlation at short distances can be analyzed. In the presented work, computer simulations of FCS and scanning FCS measurements of different shape and size of particles were performed. Optimal scanning parameters, as ... [truncated at 150 words]
Lamb DC, Bismuto E, Nienhaus GU, Gratton E.
Binding studies of ans to tuna apomyoglobin using fluorescence correlation spectroscopy.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 2600-Pos.ANS (1-anilino-8-naphthalene sulfonate) is often used as a hydrophobic probe because of its high sensitivity to its environment. For example, the quantum yield of ANS increases by over 200 fold upon binding to the heme pocket of apomyoglobin. The study of ANS binding to apomyoglobin has been used to investigate protein denaturation and refolding. Here, we have used the binding of ANS to apomyoglobin to probe protein dynamics using Fluctuation Correlation Spectroscopy (FCS). FCS measures the changes in fluorescence intensity arising from equilibrium thermodynamic fluctuations in the sample. Assuming a bimolecular reaction, it is possible to determine both the on and off rates of ANS binding from the FCS data and have been analyzed as a function of temperature and pH. At pH 7 the on rate is diffusion limited. At pH 3, the on rate is too fast to be attributed to diffusion, suggesting that the ligand is fluctuating ... [truncated at 150 words]
Eid JS, Müller JD, Chen Y, Gratton E.
Dual channel two photon fcs study in a cell.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 2597-Pos.Fluctuation correlation spectroscopy (FCS) is a non-perturbative fluorescence microscopy technique used to obtain kinetic information by analysis of the stochastic fluctuations in a molecule's fluorescence. A localized femto-liter volume is achieved by two photon excitation of the sample. This inherent three dimensional sectioning provides a distinct advantage when performing measurements in a cell since photo-damage and unwanted auto-fluorescence signal is kept to a minimum. In order to study biologically relevant processes the interaction between two or more molecules needs to be investigated. Dual channel FCS yields this information through the cross-correlation of the signal from two different fluorescent markers. The effect of cellular conditions on the interaction between two molecules can be studied by measuring the change in the cross-correlation signal as a function of the systematic modification of the cellular environment. Simple free dye experiments in cells, as well as experiments involving fluorescently labeled bio-molecules were performed to demonstrate ... [truncated at 150 words]
Sánchez SA, Gratton E, Bagatolli LA, Hazlett TL.
Phospholipase A2 association and hydrolysis of homogeneous- and mixed-lipid giant unilamellar vesicles.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 2444-Pos.The secreted phospholipase A2s (sPLA2) are interfacial enzymes which act at the surface of lipid bilayers. Membrane curvature, phase, charge, and packing have all been claimed to have direct influence on sPLA2-membrane interactions. To address issues pertaining to sPLA2 -membrane interactions, we have used giant unilamellar vesicles (GUVs) as model membrane systems. The GUVs have been doped with membrane probes in order to track PLA2-dependent vesicle morphology and fluidity changes. In addition, a fluorescently tagged sPLA2 (from Crotalus atrox venom) has been made and utilized to directly observe sPLA2 binding to the membrane surface. In the presence of active enzyme, vesicles of POPC and DPPC (liquid crystalline phase) displayed a steady shrinking, indicating lipid loss from the bilayer presumably due to absorption of hydrolysis products. Interestingly, DMPC vesicles displayed sPLA2-dependent morphology changes, a size decrease, and the formation of smaller vesicles inside the original GUV. Our binding data show that ... [truncated at 150 words]
Cheng MA, Bagatolli LA, Zajicek HK, Wilson PV, Levi M, Gratton E, Mantulin WW.
Characterization of giant unilamellar vesicles composed of natural lipids extracted from rat kidney brush border and basolateral cell membranes: a two photon excitation microscopic study.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 2874-Pos.In polarized epithelial cells there is a marked difference in the lipid fluidity of apical and basolateral membranes, due to major differences in cholesterol and sphingomyelin composition. The purpose of this study was to further characterize differences in lipid dynamics of these cell membranes. We prepared giant unilamellar vesicles (GUVs) by the electroformation procedure, using natural lipids extracted from rat kidney Brush Border (BBM) and Basolateral (BLM) membranes. We studied the temperature response of LAURDAN labeled individual GUVs using the sectioning capability of the two-photon excitation microscope. Fluorescence intensity images of LAURDAN labeled GUVs were used to calculate the spatial distribution of the LAURDAN Generalized Polarization function (GP) that allows discrimination between different lipid phases. At all temperatures, the center of the GP histograms is higher in the BBM with respect to BLM preparations. As the temperature was decreased, we detected formation of lipid domains on the surface of the ... [truncated at 150 words]
Dong CY, Kwon HS, Kim G, Huang H, Cragg GE, Sutin JDB, Gratton E, So PTC.
Design of a magnetic manipulation microscope and its application to torsional-dependent DNA binding kinetics.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 2632-Pos.We describe the design of a microscope system in which magnetic micro-manipulation and optical detection are combined for biological applications. Our multiple pole design allows maximum flexibility in 3-D micro-manipulation of biological samples. First, a uniform force field is generated for isotonic 3-D manipulation within the observation volume. Second, our design enables application of time-dependent torsional stress to samples of interest. In our design, attempts were made to maximize the magnitude of force and torque generated by optimizing pole geometry and material to permit usage of a high NA objective for enhanced optical resolution. Constant force in the range of 100 pN can be generated on paramagnetic beads 4.5 pm in size and torque in the range of 106 pN.pm can be produced on microspheres with magnetic moments of 5x10'2 A.m2. A force microscope with such versatility allows us to study a wide range of biological problems from force and ... [truncated at 150 words]
-
Dynamic property of sodium poly-acrylate gel particles studied using a two-photon fluorescence microscope.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 2617-Pos.The contraction of sodium poly-acrylate gel particles, average size of 70 to 100 microns, was found to be entirely dependent upon electrogenerated species generated at optically transparent tin oxide electrodes or at polycrystalline platinum electrodes. The pH-dependant dynamic fluorescent experiments correlate the timing and extent of the contraction of the gels to that of electrogenerated proton waves (pH gradient). Attempts to invoke contraction using pure electric fields (in the absence of Faradaic currents) failed to induce any volume change to the gel particle with externl fields as large as 435 volts. Composition of the electrolyte solution was found to affect the rate at which the proton wave migrated through the electrochemical cells - as expected from Nernst-Planck theories of migration. By using pH sensitive fluorescent probe and the line scan mode of the two-photon fluorescence microscope, we were able to image and study the dynamics of the gel particle as ... [truncated at 150 words]
Yu W, Parasassi T, Kuriashkina LR, Gratton E.
Oxidation-mediated proteolysis - a mechanism for initial vascular damage revealed by a multiphoton microscope.
44th Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 2000.
Biophys J. 2000; 78(1 Pt 2), 2618- Pos.It is commonly believed that oxidation is involved in the initial damage process of the inner wall of the artery that leads to atherosclerosis. However, it is still unclear what biochemical mechanisms are involved in the initiation process. We have performed a series of microscopy and spectroscopy studies using a two-photon fluorescence microscope examining the autofluorescence of viable rat aortas maintained under near physiological conditions. We found there was structural damage in the elastin/collagen fiber network within the aorta extracellular matrix after oxidative stress for as short as 30 min using oxidized lower-density-lipoprotein (oxLDL). In our study, we did not perform any sample manipulation, such as fixing, coloring or labeling, as had been typically done in previous studies that physically and chemically altered the sample. This approach allowed us performing sensitive studies and monitoring subtle changes resulting from oxidative stress. Based on our results and reports of previous related studies, ... [truncated at 150 words]
-
A glimpse of nucleic acid conformations through the eyes of fluorescence.
4th International Weber Symposium. Maui, Hawaii. June 23-27, 1999.
Fluorescence experiments have been applied more frequently in the last years for investigating many different facets of nucleic acid structures. In addition to straightforward surveillance applications (e.g. binding assays and identification of the presence of particular components), and quantitative determinations of relative amounts of nucleic acids, fluorescence is invaluable as a probe of DNA/RNA structure and dynamics. It is uniquely applicable to many different experimental situations, from low solution concentrations and single molecule experiments, to very complex structures such as seen in chromosomes and ribozymes, in solution experiments as well as in the fluorescence microscope. The exceptionally broad applicability of fluorescence techniques, coupled with the versatile opportunities offered by fluorescence for determining detailed physical information related to the molecular state of the nucleic acids, as well as providing very sensitive detection opportunities for molecular assays, makes fluorescence a uniquely invaluable tool with remarkable information content. In this seminar several general ... [truncated at 150 words]
-
Fluorescence fluctuation analysis for the recovery of binding equilibria.
4th International Weber Symposium. Maui, Hawaii. June 23-27, 1999.
Fluorescence Correlation Spectroscopy (FCS) provides essential information on two important biochemical parameters, the time constant of spontaneous fluctuations and the statistics of the amplitude fluctuations. One crucial requirement for the observation of spontaneous fluctuations is that the fluctuation occurs on a volume which is not much smaller than the observation volume. For the observation of single molecules, we need to have only few molecules in the volume of observation so that at any instant the number of molecules can fluctuate. For the observation of concentration fluctuations, for example calcium fluctuations in cell compartments, they should occur in volumes the size of 1/10 to 1/1000 of the excitation volume. In addition to number of fluctuations, kinetic parameters can be observed as well. For example, the change of the fluorescence intensity of one fluorescent molecule due either to conformational transitions or to binding of a substrate can produce fluorescence fluctuations. We can ... [truncated at 150 words]
-
The conformational dynamics of Adenylate Kinase.
4th International Weber Symposium. Maui, Hawaii. June 23-27, 1999.
Adenylate kinase (AK) from E. coli is a monomeric, single chain enzyme that functions in the regulation of the adenine nucleotide pool. AK catalyzes the conversion of AMP and ATP to ADP. It also appears to interact with other enzymes and may be important in the biosynthesis of phospholipids. The wild type AK enzyme (23.5kD) contains 214 amino acids and has a single cysteine (77) residue, but is devoid of tryptophan. Both the sequence and the crystal structure of the wild type AK are known. It is an a/b protein with 9 alpha helices and 7 beta sheets. In terms of tertiary structure, AK is generally segmented into three domains: (1 ) a core with substrate binding sites; (2) a mobile domain which closes over the ATP binding site by a hinge bending motion of large amplitude; and (3) a smaller domain which covers the AMP binding site with ... [truncated at 150 words]
Hazlett TL, Sánchez SA, Bagatolli LA, Gratton E.
Phospholipase A2 Interfacial Action on giant unilamellar vesicles.
4th International Weber Symposium. Maui, Hawaii. June 23-27, 1999.
In this work we are exploring the use of giant unilamellar vesicles (GUVs) and 2-photon fluorescence microscopy to investigate the interactions of an interfacial enzyme, secreted phospholipase A2 (sPLA2), with the membrane surface. Unlike membrane-bound proteins, the sPLA2s do not penetrate substantially into the hydrophobic membrane interior and do not contain membrane spanning regions nor extensive membrane penetrating sequences. Though quite soluble in aqueous buffers, these enzymes are thought to process across the membrane interface as well as diffusing on and off. On the membrane surface, sPLA2s actively hydrolyze the sn2 position fatty acid from membrane bound phospholipids forming free fatty acid and lysophospholipid. It is well known that the properties of the membrane surface have a critical influence on the binding and the activity of sPLA2. Membrane curvature, phase, charge, and packing have all been shown to modify sPLA2-membrane interactions. To address issues involving the association of sPLA2s and ... [truncated at 150 words]
Müller JD, Chen Y, Gratton E.
The photon counting histogram in fluorescence fluctuation spectroscopy.
4th International Weber Symposium. Maui, Hawaii. June 23-27, 1999.
The photon counting histogram (PCH) captures the intensity distribution of fluorescence fluctuation experiments. Particles that diffuse through a small observation volume give rise to fluorescence fluctuations. Two parameters, the molecular brightness and the average number of molecules in the excitation volume describe the PCH of a single species theoretically. We discuss the influence of both parameters on the shape of the photon count distribution and compare experiment with theory. The measured photon count distributions agree, within experimental error, with the theoretical PCH functions for a given intensity profile. The inherent sensitivity of PCH to the fluctuation amplitude allows us to resolve a mixture of species based on differences in the molecular brightness regardless of their diffusion coefficients. We expand the theory to cover multiple species and specifically address the resolvability of binary mixtures. To demonstrate the feasibility of PCH analysis binary dye mixtures were resolved by their histogram without any ... [truncated at 150 words]
-
Effect of temperature and lipid composition on giant unilamellar vesicles. a two photon fluorescence microscopy study.
4th International Weber Symposium. Maui, Hawaii. June 23-27, 1999.
Giant Unilamellar Vesicles (GUVs) are attractive cell size systems to study lipid-lipid and lipid-protein interactions. We used the so called "electric field method" to generate GUVs. Different phospholipids in the pure form and in binary mixtures were used to build the giant vesicles. To study the effect of temperature on the GUVs we used transmission and two-photon microscopy. We image the vesicles labeled with LAURDAN, PRODAN or N-Rhodamine-DPPE. In the case of LAURDAN we calculated the Generalized Polarization to ascertain the GUVs phase-state. We have used polarized laser excitation. In particular, the association of high LAURDAN GP with high polarization photoselection allows the attribution of different GP values to lipid domains of different dynamical properties. From the LAURDAN GP images we observed a broader phase transition in GUVs composed by pure phospholipids compared with multilamellar vesicles. Also we observed a "shape hysteresis" in GUVs composed by pure phospholipids at the ... [truncated at 150 words]
Chen Y, Müller JD, Patterson G, Piston DW, Gratton E.
Molecular heterogeneity probed by fluorescence fluctuation spectroscopy.
4th International Weber Symposium. Maui, Hawaii. June 23-27, 1999.
Fluorescence fluctuation spectroscopy is a promising technique for studying a system of multiple species as typically encountered in biological applications. Analyzing the time and amplitude characteristics of intensity fluctuations allows us to identify and characterize molecular heterogeneity. For a homogeneous species the normalized variance of the intensity fluctuations determined by fluctuation experiments is directly proportional to the number of molecules within the excitation volume. For a heterogeneous sample, however, the observed variance is determined by the number of molecules and the fractional intensity of each species in the mixture. The photon counting histogram (PCH) differentiates between species based on a difference in their molecular brightness, while the time dependent decay of the fluctuations to their equilibrium value uses kinetic information to separate species. The autocorrelation function of the intensity fluctuation characterizes these fluctuations in the time domain and yields diffusion and chemical reaction rates. Thus, fluorescence fluctuation spectroscopy presents a ... [truncated at 150 words]
Learmonth RP, Yu W, Shah DN, Gratton E.
Study of membrane fluidity modulation in microbial populations using laurdan, by cuvette fluorometry and 2-photon scanning fluorescence microscopy.
4th International Weber Symposium. Maui, Hawaii. June 23-27, 1999.
Yeast membranes are primary sites of environmental responses. Using DPH polarization fluorometry we found rapid fluidity changes related to environmental stresses and cell physiology. However cell density dependent scattering of polarised light was problematic. Consequently we measured Generalized Polarization (GP) of laurdan, devising protocols for labelling and analysis of intact yeast cells, to monitor fluidity responses to stress and physiological changes. Laurdan and DPH based results showed similar trends, although the laurdan technique was much less sensitive to scatter, reducing the need to precisely control cell density (which rises during experiments). To refine analyses from populations to individual cells, laurdan GP was measured by 2-photon scanning fluorescence microscopy, in relation to growth physiology and stress. Average fluidity decreased as cells aged (Fluorometry GP: young cells 0.45; old cells 0.6. Microscopy: GP ave. 0.25; 0.41). However GP images revealed increased average GP was due to a larger proportion of cells with ... [truncated at 150 words]
-
Fluctuation of scattering and absorption coefficients in the human brain.
American Physical Society Centennial Meeting, Atlanta, GA, March 20-26, 1999.
Gratton E, Barcellona ML, Müller JD, Chen Y.
Resolution of distributions of translational diffusion constants by fluctuation correlation spectroscopy.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).Fluctuation correlation spectroscopy may provide information about the translational diffusion of macromolecules in solution. The analysis of the intensity fluctuations using the autocorrelation function gives a parameter that can be related to the translational diffusion constant. In several experimental situations, macromolecular aggregates produce a distribution of molecular weights and correspondingly, a distribution of translational diffusion constants. We have explored several methods, which recover the distribution using a fitting procedure with prearranged shape for the molecular weight distribution. We present experimental results obtained with DNA polymers that have the capability to form relatively large molecular aggregates. We discuss the sensitivity of the methods and the limits of resolvability of molecular weight distributions.
Eid JS, Müller JD, Gratton E.
Efficient FCS (fluctuation correlation spectroscopy) data acquisition card with digital frequency domain lifetime capabilities.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).We developed a flexible data acquisition card that records time resolved photon events. The sequence of photon arrival is therefore available for analysis by any method(s), such as, but not limited to, autocorrelation, photon counting histogram, and higher order autocorrelation. The usual acquisition method for Fluctuation Correlation Spectroscopy (FCS) cards is the time mode, which measures the number of photon events per time interval. We have incorporated a new acquisition method, photon mode, which measures the number of time intervals between photon events. This new method matches the time resolution of the time mode which, for most experimental situations, is more efficient in terms of data transfer. The experimental regimes in which one mode is more advantageous than the other will be discussed. Our current card design allows for either time or photon mode data acquisition and is capable of 100 ns resolution. We also developed a simple addition to ... [truncated at 150 words]
Lamb DC, Chen Y, Müller JD, Gratton E.
FCS measurements of conformational fluctuations in proteins.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).Fluctuation Correlation Spectroscopy (FCS) has proved to be a powerful tool in many areas of Biophysics. One of the features of FCS is the ability to accurately measure the molecule number fluctuations of different fluorescent species in the picomolar to micromolar range. We have applied this technique to the measurement of protein dynamics. FCS was used to measure the fluctuation of apomyoglobin between the folded and the denatured states. The binding of 1-anilino-8-naphthalene sulfonate (ANS) to apomyoglobin was monitored as the protein was denatured using Guanidine HCL. In the folded state, the ANS can bind in the hydrophobic pocket, and the fluorescence quantum yield is one. As the protein denatures, water can quench the fluorescence and the quantum yield drops dramatically. When the fluctuations between the folded and unfolded states occurs rapidly with respect to the diffusion time of the particle through the excitation volume, FCS will only observe the ... [truncated at 150 words]
Chen Y, Müller JD, Eid JS, Tetin SY, Gratton E.
Heterogeneity on the single molecule level probed by fluorescence fluctuation spectroscopy.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).Normalized variance of intensity fluctuations for a homogeneous species determined by Fluorescence Fluctuation Spectroscopy (FFS) is directly proportional to the number of molecules within the excitation volume. For a heterogeneous sample, however, the observed variance is determined by the number of molecules and the fractional intensity of each species in the mixture. Three different analysis methods, photon counting histogram, autocorrelation, and moment analysis have been employed to estimate the variance from experimental data of single and mixed species samples. As an illustration of such a system, we studied binding of fluorescein labeled digoxin to high-affinity (Kd~250 pM) anti-digoxin antibody. The difference in variance between the completely bound and free ligand suggests heterogeneity in the bound species. In addition, two fluorescence lifetimes of 1.2 nsec and 4 nsec were determined for the bound species with fractional intensities of 20% and 80%, respectively. Combining this information with the results of the fluorescence ... [truncated at 150 words]
-
Sapphire-anvil-cell for high-pressure Fluctuation Correlation Spectroscopy (FCS).
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).We designed and built a sapphire-anvil-cell to be used with a 2-photon excitation microscope. The cell, designed for pressures up to 10kbar, fits on the stage of a standard microscope. Pressure calibration is achieved by measuring a pressure dependent shift of the fluorescent lifetime of the dye JDMN (the lifetime changes from 1 to 6ns for pressures between 0 to 10kbar). The calibrant, JDMN (set in a polymer layer) along with the sample solution is placed within a perforated gasket that is sandwiched between the two sapphire windows. By focusing onto the polymer layer, the JDMN lifetime may be measured. The FCS data is collected by simply defocusing into the solution. With a long working distance objective (1cm) a solution of ~1ml may be examined using the FCS technique, where the fluorescence is collected in the reflection mode. With this technique the number density and diffusion constant of the freely ... [truncated at 150 words]
Pan R, Mantulin WW, Gratton E.
Fluorescence cross-correlation with n-channel photon counting logic.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).A novel digital logic design, combines the versatile strengths of FCS with independent, synchronized, multiple channel recording of photon pulses or common clock pulses. Precise transport and thermodynamic phase phenomena, requiring high confidence in event detection and timing (less than 1 microsecond) can thus be studied. In Photon Counting Mode, a clock gates photon counting in the respective channels, thus allowing anisotropy and general polarization experiments. In Clock Counting Mode, photons at different wavelengths, from respective channels, gate counting of common clock pulses. At present, two channels have been designed and built. Testing of channels (calibration with standards, determination of deadtimes), experiments with polymers (DNA, etc.) in different phases, and N=4 channels is planned. Experimental data will be studied via scaling physics, photon counting and averaging, physics of phase transitions and theory of wavelets.
-
3-D Particle tracking with two-photon excitation: instrumentation and statistical analysis of results.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).We have extended the reach of single particle tracking (SPT) by implementing a 3-D SPT system on the two-photon fluorescent microscope. Our system utilizes an x-y galvanometer scanner and a piezo driven z-stage, both under feedback control. This poster analyzes the 3-D trajectories obtained for particles in both homogeneous media and inhomogeneous media, such as the fractal environment of agarose gels. We present the results of these trajectories and analyze the distribution of diffusion coefficients obtained for each axis, and provide criteria to classify the various motional modes observed. We provide Monte Carlo calculations for comparison. We also investigate what effect artifacts, such as vibrations, will have on the results. In addition, we discuss the applicability of this technique in the investigation of various biological systems by addressing the trade-off between the frequency response and the accuracy of the determination of particle position. Finally, we consider alternatives to a simple ... [truncated at 150 words]
Ruan Q, Glaser M, Mantulin WW.
The folding reactions of adenylate kinase from E. Coli.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).Adenylate kinase (AK) from E. Coli is a small monomeric enzyme (MW23.5kD) that catalyzes the phosphorylation of AMP by ATP to form two ADP molecules. We have been characterizing the structure, function, and dynamics of this enzyme and a variety of mutants for some years. Wild type AK is devoid of tryptophan (Trp). We have constructed a series of single Trp mutants (41,73,86,133,137,193) with the mutations distributed in various domains of the enzyme. These mutants have been shown to maintain activity and structure similar to the wild type enzyme. In general, the equilibrium unfolding properties of the Trp mutants vary somewhat from the wild type, reflecting local and global reporting by the Trp indicator. Labeling AK with exogenous fluorescent dyes facilitates examination of folding reactions and fluorescence resonance energy transfer (FRET) to detect domain motion. Performing the dye labeling reaction under high hydrostatic pressure has proven to be efficient in ... [truncated at 150 words]
Breusegem SY, Loontiens FG, Clegg RM.
Binding kinetics of Hoechst 33258 and some of its derivatives to DNA.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).The dye Hoechst 33258 and some of its meta-phenyl-substituted derivatives bind in the minor groove of B-DNA to sequences consisting of 4 to 5 juxtaposed A or T bases. The intense increase in dye fluorescence upon binding was used to monitor the binding equilibrium and the stopped-flow kinetics. The binding reaction was measured with polymeric DNA's (CT-DNA, poly[d(A-T)] and poly[d(A-5BrU)]), the duplex d(CGCGAATTCGCG)2, and with a dilution-resistant 28-nucleotide-hairpin in which the G-3' and C-5' ends of the duplex are connected by a tetra-T sequence. Low concentrations of the dye (1 to 5 nM) were used to prevent its stoichiometric associations on polymeric DNA. DNA concentrations were expressed as sites for the dye. The kinetics for binding of Hoechst 33258 were consistent with a single-step bimolecular association that is practically controlled by diffusion: for calf-thymus DNA, k+=1.5x109 M-1 sec-1; with the other DNA's, somewhat smaller k1 values were obtained. Kinetically defined ... [truncated at 150 words]
Pilas B, Sánchez SA, Hazlett TL, Durack G.
Measurements of cellular chloride concentrations with 6-methoxy-n-ethylquinolinium-iodide using conventional and lifetime-based flow cytometry.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).The ability of halide-specific fluorescent dye 6-methoxy-N-ethylquinolinium-iodide (MEQ) to measure intracellular chloride concentrations and changes during regulatory volume decrease in single cells was examined. In solution, MEQ fluorescence is quenched by chloride with a Stern-Volmer quenching constant of 120 M-1 and a quenching rate constant, kq, of 5x10-9M-1s-1. MEQ was then used as an intracellular chloride ion reporter group. CHO cells were loaded in vitro using a reduced MEQ derivative which is oxidized intracellularly to the chloride sensitive form. Fluorescence emission and fluorescence lifetime measurements on loaded cells were made by flow cytometry. Calibration of the intracellular MEQ was performed by varying the extracellular chloride concentration and equilibrating intracellular with extracellular chloride using nigericin and tributyltin. Stern-Volmer quenching constants of 10M-1 (fluorescence intensity) and 9.6 M-1 (fluorescence lifetime) (kq=2x10-9M-1s-1) were determined for intracellular MEQ. The intracellular chloride concentration for CHO was estimated at 55.8 ± 4.3 mM. The discrepancy between ... [truncated at 150 words]
-
A new approach to the measurement of the multiphoton excitation cross-section of a fluorophore.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).Multiphoton excitation of fluorophores has recently found widespread use across the range of fluorescence measurement techniques. Measurement of the multiphoton excitation cross section as a function of wavelength is an important parameter in the characterization of existing fluorophores and development of new indicators for these applications. We describe a technique that addresses some typically encountered problems when making this measurement. The desire for efficient excitation naturally suggests the use of a short pulse laser system (e.g., 100 femtosecond pulse duration) however the. chromatic dispersion of the laser pulse in the experimental system may lead to significant uncertainties in the temporal properties of the excitation pulse. We find that the use of a picosecond Ti:Sapphire laser, with its smaller spectral bandwidth can reduce this potential source of error. A second factor to be considered is the separation of different order effects e.g., two photon excitation and three photon excitation. In a ... [truncated at 150 words]
Yu W, Kuriashkina LR, Gratton E.
Fluorescence lifetime imaging and correlation spectroscopy for studying Gaba receptor activity in living tissue.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).A multi-photon fluorescence microscope is used as a platform for studying receptor activity in a living brain slice, traditionally studied by electrophysiological measurements. gamma-Aminobutyric acid (GABA) subtype A receptor is believed to cause hyperpolarization of neuron membrane and inhibition of neuronal excitability via influx of extra-cellular chloride (Cl-). There has been great interest in knowing both the local chloride concentration of individual neuronal cells, as well as their GABA receptor activities [Wagner et al., Nature 387, 598-603, 1997]. We used scanning two-photon, time-resolved fluorescence microscopy to quantitatively determine local chloride concentration in a viable brain slice. A femto-second pulse laser was used in combination with a pulse picker to explore harmonics of the basic frequency to measure fluorescence lifetime in the microscope using the frequency domain method. Signal arising from different fluorescence lifetime components can be either enhanced or suppressed when using different harmonics. Local fluorescence intensity signals of the ... [truncated at 150 words]
Krasnowska EK, Gratton E, Parasassi T.
Cholesterol influences PRODAN partitioning between phospholipid phases and water.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).The effect of cholesterol on the packing and polarity of bilayers has been studied by the fluorescent probe 6-propionyl-2-dimethylaminonaphthalene (PRODAN), a probe located at the membrane surface. In the gel phospholipid phase and in the absence of cholesterol, PRODAN's phospholipid:water partition coefficient has been spectroscopically determined using the fluorescence intensities at three emission wavelengths and was about 14 × 104. In the liquid-crystalline phase of the bilayer this value was about 500 × 104 (Krasnowska et al., Biophys. J., 1998, 74:1984). PRODAN fluorescence emission from the aqueous environment was monitored by the appearance of a red emission band centered at 520 nm. When cholesterol was added to phospholipid gel phase bilayer, at concentrations >3mol%, the red emission band centered at 520 nm disappeared and the PRODAN partition coefficient was similar to that determined for liquid-crystalline phase bilayers. We attributed this effect to cholesterol-induced looser interactions between adjacent phospholipid polar head ... [truncated at 150 words]
Stühmeier F, Hillisch A, Diekmann S, Clegg RM.
Fluorescence resonance energy transfer analysis of DNS-structures containing several A5 bulges and their interaction with proteins.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).Fluorescence Resonance Energy Transfer (FRET) was utilized to probe the geometry of DNA structures containing multiple A5 bulges. The bulges (unpaired nucleotides in one strand) fold back the molecule and decrease considerably the distance between the ends of the DNA; therefore, energy transfer could be detected between two dyes covalently attached to the 5'-ends of the DNA, although the dyes were separated by up to 60 to 70 consecutive bp. The molecules contain 24 bp long sequence with the binding site for the DNA binding protein CAP, which bends the helix of the DNA. The structural distortion of the DNA under the influence of CAP was monitored by measuring the change of the FRET efficiency between the dyes attached to the DNA. No direct interaction between CAP and the dyes could be detected. A quantitative comparison between the FRET data and molecular models based on the NMR structure of the ... [truncated at 150 words]
-
DNA flexibility and bending: results from brownian dynamics simulations and single-molecule experiments.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).We are investigating intrinsic and protein-induced DNA flexibility, bending, and looping by experimental and simulation methods. We are developing a Brownian dynamics simulator to model the results of single molecule tethered particle motion analysis (Sutin, et al., Biophys. J. 74: A71 (1998)) and fluctuation correlation spectroscopy (FCS) experiments. We are simulating the trajectories of DNA fragments of lengths from 10's to 100's of base pairs on the micro to millisecond time scales. The boundary conditions of the simulations are for both a DNA fragment alone in solution and for a DNA fragment tethering a microsphere to a surface. The resulting DNA trajectories are transformed to the corresponding measured experimental parameters. For tethered particle motion analysis, the distribution of displacements of the microsphere attached to the free end of the DNA fragment will be related to the underlying DNA conformation. We will also consider the information obtainable in a scanning FCS ... [truncated at 150 words]
-
"Second order" sequence specificity of DAPI-binding to AT-regions in DNA.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).DAPI binds strongly (with pronounced sequence specific affinities in the nanomolar range [Loontiens et al., 1991]) in the minor groove to AT-rich regions of DNA. We measured the binding constants to "AATT" and "TTAA"-containing oligonucleotides of different lengths in a study involving fluorescence energy transfer between DAPI molecules bound to oligonucleotides with these two sequences separated by different lengths of helix, and found a significant sequence dependence of the binding affinities. Time-resolved fluorescence measurements of DAPI bound to oligonucleotides containing only one of these binding sites showed only lifetimes between 0.2-4 ns at all dye/DNA ratios for 5'CGCGAATTCGCG3', but an additional lifetime about 10 ns appeared at larger dye/DNA ratio (where the affine sites were already occupied) when DAPI interacts with 5'CGCGTTAACGCG3' and 5'CGAATTGGCACAGC3'. This longer lifetime has also been observed with other repetitive sequences of DNA (poly d[AT] and d[GC]) and CT-DNA [Cavatorta et al., Biophys. Chem. 22 (1985)]. ... [truncated at 150 words]
Bagatolli LA, Parasassi T, Fidelio GD, Gratton E.
Effect of temperature and lipid composition on giant unilamellar vesicles. A two photon fluorescence microscopy study.
43rd Annual Meeting of the Biophysical Society, Baltimore, Maryland, 1999.
Biophys J. 1999; 76(1 Pt 2).Giant Unilamellar Vesicles (GUVs) are attractive cell size systems to study lipid-lipid and lipid-protein interactions. We used the "electric field method" to generate GUVs. Different kind of lipids, phospholipids (PLs), glycosphingolipids (GSLs) and cholesterol (Chol), were used as component of the giant vesicles. To characterize the yield, size and shape of GUVs we used transmission and two-photon microscopy. We imaged the vesicles labeled with 2-dimethylamino-6-lauroylnaphthalene (LAURDAN) and we calculated the Generalized Polarization to study the effect of temperature in single GUVs formed by different chain length phosphatidylcholines (PCs) in the pure form or mixed with GSLs or Chol. We also used polarized laser excitation. The association of high LAURDAN GP with high polarization photoselection allows the attribution of GP values to lipid domains of different dynamical properties. The formation of lipid domains was found for example at the phase transition temperature of pure PC GUVs and also in GUVs composed ... [truncated at 150 words]
Lamb DC, Müller JD, Akcakir O, Yu W, Gratton E, Carlson JW, Sligar SG, Schulte A.
Millisecond reactions in single proteins monitored with two-photon fluorescence spectroscopy.
APS March Meeting, Los Angeles, CA, March 16-20, 1998.
Because of the inherent heterogeneity of proteins and the existence of many intermediate states, the conformational changes involved in a particular reaction are obscured when measuring an ensemble of molecules. Single molecule spectroscopy allows one to investigate the degree of heterogeneity in proteins as well as the variety of reaction pathways available to the system. Two-photon fluorescence spectroscopy combines excellent background rejection with small probe volume selection, making it possible to detect single molecules. Here we present kinetics of 8-anilino-1-naphthalene sulfonic acid (ANS) binding to single apomyoglobin molecules that have been tethered to a quartz coverslip. The quantum yield of the ANS fluorescence increases by over 2 orders of magnitude when the ligand is bound to the protein. Thus, it is possible to distinguish between the ligated and unligated conformations of the protein. The rate coefficients of the reaction are directly extracted from the time sequence of binding events. Supported ... [truncated at 150 words]
-
Stereochemistry of DNA molecules by homo-energy transfer.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A288, W-Pos192.Förster Fluorescence Resonance Energy Transfer (FRET) between two identical dyes has been used for studying the stereochemistry of complex DNA molecules. The dye DAPI binds to DNA with a tight site-selective affinity, the crystal structure of dve-DNA complexes is known. Oligonucleotides were made with two binding sites, 5' AATT 3' and a 5' TTAA 3' separated by varying lengths of duplex DNA. The efficiency of energy transfer was determined from fluorescence anisotropy, which decreases as the energy is transferred between the two identical dye molecules (the intensity of fluorescence is not affected by this homo-transfer process). The two binding sites have different binding affinities: the binding affinities were determined simultaneously with the efficiency of energy transfer. The fluorescence anisotropies were determined during a continuous automatic titration of the dye to the DNA. The binding of DAPI to the double-stranded oligonucleotides was analyzed with a model consisting of three binding processes: ... [truncated at 150 words]
Schneider PC, Holub O, Clegg RM.
Real-time fluorescence lifetime-resolved images of 5-aminolevulinic acid (ALA)-induced Protoporphyrin IX (PPIX) in cells.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2), Tu-Pos250.An instrument using the phase and modulation technique in homodyne mode was developed that is capable of acquiring, processing and displaying fluorescence lifetime-resolved images in real-time (frame refresh rates up to 13 Hz). Using fluorescence lifetime-resolved imaging microscopy (FLIM) luminescence emission can be lifetime-resolved directly) at each location of a microscope image. With this method the mean lifetime is determined simultaneously at every pixel of a digital image. A SUN UltraSparc Computer with Creator 3D graphics processor is used. Programs for data acquisition. image analysis and 3D display modes have been written using AVS/express. We observed PPIX accumulation in cells - fast data acquisition is important due to the fast bleaching rate. In the biosynthetic pathway of heme. ALA is converted to PPIX. an endogenous fluorescent photosensitizer. A surplus of ALA leads to an accumulation of PPIX because of a strong heme feedback mechanism. This was studied in several cell ... [truncated at 150 words]
Zajicek HK, Yu W, Wang Il, Gratton E, Parasassi T, Levi M.
Spatial resolution of the effects of cholesterol deprivation and/or enrichment on OK cell membranes by LAURDAN GP two-photon fluorescence microscopy.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A403, Th-Pos331.To detemine the effects of alterations in cholesterol content on membrane lipid dynamics opossum kidney (OK) cells were grown either a) in a lipoprotein deficient serum (LPDS), which resulted in a decrease in cell cholesterol content, or b) in LPDS which was then supplemented with LDL, which resulted in an increase in cell cholesterol content. The cells were labeled with the membrane fluorescent probe 2-dimethylamino-6-lauroylnaphthalene (LAURDAN) and imaged by a two-photon excitation microscope. From the fluorescence intensity images, LAURDAN generalized polarization (GP) images were calculated. Both in control and in treated cells, a broad GP distribution was observed, with higher GP values in the plasma membrane, intermediate values in the cytoplasm and lower values in the nuclear membrane. In agreement with previous cuvette-based fluorescence spectroscopy studies, cholesterol deprivation resulted in an average decrease of the GP value. From the images, this decrease in GP appeared mainly localized in the plasma ... [truncated at 150 words]
Gratton E, Parasassi T, Yu W, Bagatolli LA.
Are giants unilamellar phospholipid vesicles really unilamellar? A two photon microscopy study.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A374, Th-Pos163.Unilamellar liposomes are used for several purposes such as transport studies and drug delivery. We want to utilize such liposomes composed of a single bilayer for the study of domain coexistence and for the estimation of the domain size. A variety of methods for their preparation are described in the literature. We used several of those methods and we used fluorescence microscopy and electron microscopy to assess the number of lamellae present in the vesicles following each preparation. Using some of the methods reported in the literature, we were able to obtain only a small percent of unilamellar vesicles in a population mainly composed of multilamellar vesicles. By using two-photon microscopy to image the vesicles labeled with 2-dimethylamnino-6-lauroylnaphthalene (LAURDAN), we observed that the vesicle lamellar structure also depends upon the phase state of the lipids. In some cases, we were able to assess the number of lamellae present in each ... [truncated at 150 words]
Müller JD, Akcakir O, Lamb DC, Carlson JW, Yu W, Schulte A, Sligar SG, Gratton E, Nienhaus GU.
Reaction dynamics of single protein molecules.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A182, Tu-Pos212.Studies of protein reactions at the single-molecule level allow us to observe the heterogeneous behavior of individual proteins rather than entire ensembles. This additional information can provide insight into the fundamental ideas of protein dynamics and function. Here, we focus on the kinetics of ligand binding to single apomyoglobin molecules. Two-photon fluorescence excitation spectroscopy combines excellent background rejection with small probe volume selection, both of which are essential for the detection of single molecules. We observe the reaction of freely diffusing ANS with an immobilized protein in the excitation volume. The change in quantum yield of the fluorescent ligand upon binding to the protein by more than two orders of magnitude allows us to distinguish between the bound and unbound state of the protein. The rate coefficients of the binding reaction are directly extracted from the time sequence of photon counts. Our method demonstrates the use of two-photon spectroscopy for ... [truncated at 150 words]
Bagatolli LA, Parasassi T, Fidelio GD, Gratton E.
Effect of solvent polarity and lipid chemical structure on naphthalene derivatives excitation spectra.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2), Tu-Pos324.Professor Gregorio Weber first synthesized several naphthalene derivatives for the study of the fluorescence dipolar relaxation. Among them is the 2-dimethylamino-6-lauroylnaphthalene (LAURDAN), which became a popular membrane polarity probe. Beside the emission spectral shift due to dipolar relaxation occurring in polar solvents. this probe also shows variations of the excitation spectrum. in particular the appearance of a red excitation at about 390 nm band in polar solvents. In ester phospholipids, LAURDAN excitation red band is particularly intense in the gel phase. while it displays a lower intensity in the liquid-crystalline phase of ester phospholipids. in both phases of ether phospholipids, glycosphyngolipids and sphingomyelin. The occurrence and intensity of the red excitation band also depends upon the residue in the position 2 of the naphthalene moiety. To explore this last point. excitation spectra of the 2-hydroxy and 2-methoxv-6-lauroylnaphthalene (LAURNA and LAURMEN, respectively) in solvents and in lipids were examined. W\e also ... [truncated at 150 words]
Chen Y, Müller JD, Carlson KE, Katzenellenbogen JA, Gratton E.
Molecular aggregation and ligand-receptor interaction probed at the single molecule level using two-photon microscopy.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A182, Tu-Pos214.Two-photon excitation spectroscopy has inherent 3-D resolution with excitation volumes as small as 0.1 fL, which compared to conventional fluorometers constitutes a 10^10 times reduction of the excitation volume. Via fluctuation correlation spectroscopy (FCS), the fluorescence fluctuations within the small excitation volume provide a unique way to study interesting biological phenomena. Molecular aggregates are easily identified at the molecular level, both in terms of number fluctuation and changes in the translational diffusion coefficient. These molecular characteristics are obscured once they are averaged over an assembly of millions of molecules. We apply both Photon Counting Histogram (PCH) and autocorrelation analysis to study ligand binding to estrogen receptors. These two methods complement each other and accurately recover the number of molecules of free ligand and ligand bound to the receptor. In addition, kinetic information can be obtained using these two analysis methods. Supported by the National Institutes of Health, RR03155.
Buehler C, Dong CY, So PTC, Gratton E.
Multi-photon, multi-wavelength pump-probe spectroscopy.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A182, Tu-Pos215.We report applications of pump-probe stimulated emission spectroscopy using a fluorescence microscope. Two synchronized, mode-locked titanium sapphire lasers with slightly different repetition rates are focused onto a common focal volume in a fluorescence sample using a microscope. The pump laser excites the fluorescent molecules, and the probe laser is used to either excite or induce stimulated emission from the sample. The two lasers operating at different repetition frequencies optically heterodyne the fluorescence signal to the low, cross-correlation frequency and harmonics, hence, no fast photo detector is needed to record high frequency molecular dynamics. Due to the high harmonic content of the two pulsed lasers, the frequency-domain heterodyning method samples the complete fluorescence dynamics simultaneously, which results in fast data acquisition. The unique aspect of using two mode-locked, independently tunable Ti:Sapphire lasers offers the capability of performing multi-photon, multi-wavelength studies of biochemical systems. Using this method, we present time- and spectrally-resolved ... [truncated at 150 words]
Ragan TM, So PTC, Gratton E.
3-D particle tracking using two-photon excitation: probing the cytoplasmic structure.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A183, Tu-Pos219.Particle tracking techniques have been especially fruitful in the investigation of the cellular plasma membrane. Previous results (Luby-Phelps et al. 1986) indicate that the cytoplasm, like the plasma membrane, has a structure comprised of many compartments. Particle tracking would be the ideal technique to explore these questions. Unfortunately, all the particle tracking techniques developed so far have been limited to 2 or 3-D systems with a restricted axial range and frequency response. To address this problem, we developed a 3-D particle tracking system built around a microscope that uses the inherent 3-D localization of two-photon excitation. We use an x-y scanner and piezo z-stage to quickly scan the sub-micron two-photon excitation spot over the region of interest to build a 3-D image, and, with an automatic feedback mechanism, we reposition the scan region to follow the fluorescent particle and construct a 3-D trajectory. Our system has an axial range of ... [truncated at 150 words]
-
High pressure stage for fluctuation correlation spectroscopy (FCS) microscopy.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A183, Th-Pos220.High pressures (3k-10k bar) are required to study phenomena such as the hysteresis of protein dimer dissociation curves, as well as pressure induced denaturation. Fluctuation Correlation Spectroscopy (FCS) via fluorescence microscopy can provide important information about such processes by number fluctuations and translational diffusion constants from the intensity fluctuations in the fluorescent signal. To achieve the small excitation volumes critical for this technique, 2-photon excitation is used. High numerical aperture objectives (which have short working distances) necessary for efficient signal collection force the sample window of the high-pressure cell to be very thin. We will report on the progress of the design of this high-pressure cell for microscopy use. Supported by the National Institutes of Health (RR03155).
Parasassi T, Yu W, Kuriashkina LR, Gratton E.
"In vivo" tissue microscopy imaging: autofluorescence and LAURDAN generalized polarization of rat aorta.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A184, Tu-Pos222.A rat aorta portion has been longitudinally opened and used either for autofluorescence imaging, or labeled with 2-dimethvlamino-6-laurovlnaphthalene (LAURDAN). During labeling and measurements, tissue viability was maintained by a continuous flow of EBSS medium, supplemented with glucose and sodium bicarbonate and bubbled with oxygen. The open aorta wall was imaged at various depths by two-photon fluorescence microscopy. From LAURDAN fluorescence intensity images. generalized polarization (GP) images have been calculated. The organization of aorta tissue layers was clearly shown in the various optical sections. From the lumen, the endothelium and the tunica media can be distinguished both for their typical morphology and for the different average GP values. The endothelium GP value was much higher than that of the tunica media. The tunica media GP images resealed elongated cell structures. The endothelium layer was also shown in the autofluorescence images. although with a relatively low signal. By imaging the interior of ... [truncated at 150 words]
Masters BR, Mantulin WW, Gratton E, So PTC.
Simultaneous two-photon fluorescence and reflected light confocal imaging of in vivo human skin.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A185, Tu-Pos229.In vivo human skin has been imaged with both two-photon reflected light (Raijadhyaksha et. al., 1995, J. Investigative Dermatology, 6, 946-954; Masters et. al., 1997, J. Microscopy, 185, 329-338), and two-photon fluorescence microscopy (Masters et. al., 1997, Biophys. J., 72, 2405-2412). The two forms of microscopy, two-photon fluorescence and confocal reflected light imaging, provide complementary information. Two-photon fluorescence imaging can be used to probe cellular morphology and functional states in deep tissue. The endogenous chromophore, NAD(P)H, can be relied upon to provide sufficient cytoplasmic contrast to yield information on cell shape. Further, cellular redox activity can be monitored by measuring NAD(P)H concentration. On the other hand, confocal reflected light imaging can detect certain cellular structures invisible to two-photon microscopy. Reflected light confocal microscopy maps the changes in tissue refractive index and no endogenous chromophores are required. This mechanism allows the imaging of cellular membrane boundaries as well as intracellular nucleic ... [truncated at 150 words]
Dong CY, Buehler C, So PTC, Gratton E.
Two-photon time-resolved polarization imaging inside mouse fibroblast cells.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A188, Tu-Pos248.Two photon time-resolved microscopy has proven to be a powerful technique to image biological systems. The high incident photon flux from a high peak power light source, such as a mode-locked titanium sapphire laser, can be focused to a diffraction limited spot inducing effective two-photon excitation of the chromophores. The nonlinear nature of the two photon process results in a strong localization of the excitation volume, which minimizes photo-bleaching of the specimen. We combined the 3-D sectioning effect inherent in two photon microscopy with time-resolved methods to study rotational correlation times of fluorophores in living mouse fibroblasts. Images were acquired with a microscope in a point-by-point fashion by raster scanning a mode-locked titanium sapphire laser beam across the specimen. At each sampling point, time-resolved information is obtained by analyzing the sample frequency response to the laser harmonic content. The measurement of the decay of the emission anisotropy allowed us to ... [truncated at 150 words]
NicClendon S, Yu W, Schulte A, Gratton E.
Construction of an antenna microscope for single molecule detection.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A188, Th-Pos249.detection schemes for the study of single molecules use fluorescence. requiring excitation of the target molecule. A drawback of these techniques is severely restricted observation times, due to photobleaching. XVe are constructing an optical microscope based on a different detection method which. by avoiding excitation of the molecule. overcomes the above mentioned shortcomings while still allowing good spatial resolution. The method we use is a further developement of a method first proposed by H.K. XVickramasinghe et al. [Appl. Phys. Lett. 65. 1623 (1994)] based on detecting the light scattered from an antenna tip brought close to the sample. An interferometric detection system using DIC optics is implemented on a Zeiss Axiovert inverted microscope. This work was supported bv the National Institute of Health. RR03155. and the National Science Foundation. PHY95-13217.
Yu W, Durack G, Mantulin WW, Gratton E.
Fluorescence lifetime of single cells using a flow cytometer.
32nd Annual Meeting of the Biophysical Society, Kansas City, Missouri, 1998.
Biophys J. 1998; 74(2 Pt 2): A180, Tu-Pos199.We have developed a novel technique for flow cytometry capable of measuring fluorescence lifetime from a single cell at a rate of several hundred cells per second. Our instrument is based on an existing Coulter flow cytometer, the "EPICS Elite". A lifetime measurement mode is introduced using a fast frequency-domain heterodyning technique. For our current setup, a Pockels cell is used to sinusoidally modulate an Argon ion laser at the desired frequency. The fluorescence is collected through the standard optics of the Coulter flow cytometer and detected by using a Hamamatsu R928 photomultiplier tube which is gain modulated at a frequency 500 KHz to 1 MHz greater than that of the laser modulation. The waveform of this frequency difference, the cross-correlation frequency, is digitized and Fourier transformed to obtain the phase and the modulation of the signal. By using a second reference PMT for the laser, we obtained the phase ... [truncated at 150 words]
Cerussi AE, Fantini S, Franceschini MA, Gratton E.
Frequency-domain lifetime spectroscopy in the multiple scattering regime.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), Tu-Pos423.The fluorescence lifetime provides a useful quantitative spectroscopic parameter that is sensitive to the fluorophore local environment. A distinct advantage of the fluorescence lifetime over the fluorescence intensity lies with the independence of the lifetime with respect to the fluorophore concentration. A variety of fluorophores exist that exhibit measurable lifetime changes while in the presence of physiologically relevant analytes such as Ca++ and Mg++, and as a consequence of variations in local environment conditions such as pH. There are many potential applications offered by the fluorescence lifetime's sensitivity in highly scattering environments such as tissues that are not amenable to traditional spectroscopic methods. It has recently been demonstrated that a model for fluorescence spectroscopy in multiple scattering media allows the accurate recovery of fluorescence parameters such as the lifetime. In the frequency-domain an analytic expression for the fluorescence signal has been obtained, whereas in the time-domain an analytical expression cannot ... [truncated at 150 words]
French TE, Cerussi AE, Fantini S, Franceschini MA, Gratton E.
Measurement of absolute fluorescence quantum yield in turbid media.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), Tu-Pos424.We demonstrate the measurement of the absolute fluorescence quantum yields performed inside a multiple scattering medium. A model for frequency-domain fluorescence spectroscopy in multiple scattering media that allows the accurate recovery of fluorescence parameters such as the quantum yield has recently been verified. Our work uses this model to determine the absolute quantum yield of a fluorophore that is uniformly distributed throughout a multiple scattering medium. Using this technique, it is not necessary to have a reference fluorophore of known quantum yield. The only reference compound that is necessary is one which is used to calibrate the spectral response of the detection system. This technique does not require the calibration of the excitation and emission light paths. Because of the multiply scattering of the medium (titanium dioxide particles suspended in water), the excitation and emission geometries are identical. We present the quantum yield of Rhodamine B in water measured in ... [truncated at 150 words]
Dong CY, Buehler C, So PTC, Gratton E.
Frequency-domain pump-probe stimulated emission spectroscopy using a fluorescence microscope.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), Tu-Pos422.We developed a pump-probe stimulated emission system for spectroscopic studies inside a microscope. Our pump-probe technique involves focusing two pulsed laser beams onto a fluorescent sample. One laser, the pump, is used to excite the fluorophores. The other laser, the probe, is used to induce stimulated emission from the molecules in the excited state. The repetition frequencies of the two lasers are offset by an amount small compared to the base laser repetition frequency; this results in a signal at the difference frequency between the two lasers and corresponding harmonics. There are several advantages of this technique. First, the high frequency content of pulsed lasers can be analyzed at a very low frequency. As a result, fast optical detectors are not needed to investigate ultrafast molecular phenomena. Second, optical overlapping of two laser beams is efficient inside the microscope. With relatively high numerical aperture objectives, tight focusing and effective signal ... [truncated at 150 words]
Parasassi T, Levi M, Yu W, Gratton E.
Two-photon fluorescence microscopy of LAURDAN GP domains in biological membranes using polarized laser excitation.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), Th-Pos322.Two-photon microscopy images of membranes of whole red blood cells, renal tubular cells (ORcells) and purified rat renal brush border and basolateral membranes labeled with LAURDAN show a fine texture of coexisting regions of different generalized polarization (GP) values. By measuring the relative water content of the membrane, LAURDAN GP function also monitors the lipid molecular dynamics or "fluidity". We have used polarized laser excitation. In images with well defined membrane orientation, such as the erythrocyte cell sections, the association of high GP with high polarization photoselection allows the attribution of coexisting GP values to lipid domains of different dynamical properties. Due to the presence of strong fluorescence from the cytoplasmic membranes, the interpretation of the membrane GP texture in images of renal tubular cells is more complex. After masking the cytoplasmic membranes, analysis of the images of red blood cells and of renal tubular cells lead to the conclusion ... [truncated at 150 words]
Kumar V, Wilson PV, Parasassi T, Gratton E, Levi M.
Influence of shingomyelin on lipid dynamics as detected by laurdan fluorescence.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), Th-Pos333.Sphingomyelin is a major constituent of cell membranes. In renal proximal tubular apical brush border membranes (BBM) sphingomyelin accounts for 40 to 60 mole % of membrane phospholipids. The purpose of this study was to determine the effect of sphingomyelin on lipid dynamics as determined by the Generalized Polarization of Laurdan (GP Laurdan) which reflects the relative water content of the lipid bilayer. In DOPC and in DOPC-DPPC (1:1) bilayers brain, egg, or milk sphingomyelin caused a dose-dependent blue shift in the emission spectra and an increase in the excitation GP, i.e. a decrease in lipid fluidity. The dose-dependent effect of sphingomyelin to cause a progressive increase in the GP was also seen in the presence of cholesterol ( 25 mole % CHOL) in CHOL-DOPC-DPPC bilayers. In contrast, in total lipid extract from the renal BBM, selective removal of sphingomyelin caused a red shift in the emission spectra and a ... [truncated at 150 words]
Chen Y, So PTC, Gratton E.
Study of molecular aggregation by analysis of photon histograms of single molecules.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), Tu-Pos438.Molecular aggregation is a crucial regulatory mechanism in biological systems. However, most experimental techniques require relatively high concentrations (from mM to gM). Since many interesting physiological events happen at concentrations below micromolar, we have explored the possibility to detect molecular aggregation in the low concentration range (from AM to pM) using two-photon fluorescence microscopy. Two-photon fluorescence microscopy has an inherent 3-D resolution and using high numerical aperture objectives the excitation volume can be made as small as 0.1 fl,, which only contains 0.06 molecules at 1 nM concentration. A small excitation volume is essential for detecting fluorescence fluctuations among molecules. By analyzing the histogram of photons bursts, one can obtain the degrees of aggregation from their distribution (H. Qian and E. Elson, 1990, PNAS, 87, 5479). We model our photon histograms with a simple two species system and compare experimental results to the theoretical predictions. This method of analysis is ... [truncated at 150 words]
Ragan TM, So PTC, Gratton E.
Two-photon 3-D particle tracking in cells.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), Tu-Po400.Particle tracking is a powerful method to explore various problems in cell biology. It can be used to study membrane protein and lipid diffusion, cytoplasm compartmentalization, endocytosis of extracelullar proteins and phagocytosis of antigens. In particular, particle tracking experiments have led to a better understanding of the motion and behavior of proteins in the plasma membrane. Unfortunately, most of the particle tracking techniques developed to date have used area detectors and have thus been limited to two dimensional systems. To address this limitation, we have developed a 3-D particle tracking system that uses the inherent localization of two-photon excitation. We use a feedback system to position the sub-femtoliter two photon excitation volume to maximize the fluorescence and obtain a series of images from which we construct the 3-D trajectory of the particle. In addition, the images provide information about the local environment of the particle. The axial range of our ... [truncated at 150 words]
Masters BR, So PTC, Gratton E.
Multi-photon excitation fluorescence microscopy and spectroscopy of in vivo human skin.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), Tu-Po441.Multi-photon excitation microscopy at 730 nm and 960 nm was used to image the autofluorescence of in vivo human skin from the surface to a depth of about 200 microns. The emission spectra and fluorescence lifetimes were determined. We have imaged the fluorescent cytoplasm of individual skin cells at depths of between 25 to 75 µm below the skin surface for both excitation wavelengths. Fluorescence emission spectra and fluorescent lifetimes of the tissue were obtained at single points, relative to the surface (0-50 microns) and at deeper depths (100-150 microns). The source of the fluorescence emission spectra and fluorescence lifetimes are consistent with reduced pyridine nucleotides, NAD(P)H, as the primary source of the skin autofluorescence using 730 nm excitation. With 960 nm excitation, fluorescence emission at 520 nm indicates the presence of flavoprotein contribution. A second, discrete fluorescence emission component, which starts at 425 nm is observed with 960 nm ... [truncated at 150 words]
McClendon S, Yu W, Gratton E.
Two-photon FCS using a dual channel single photon counting system.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), Tu-Pos465.Fluorescence Correlation Spectroscopy (FCS) measures the intensity fluctuation from a sample to obtain dynamic information about the system (such as diffusion rates, protein aggregation, etc). In a number of systems, we are interested in studying the dynamic behavior of two aspects of the system simultaneously. To achieve this, we have constructed a dual channel, single photon counting detection system, and implemented it in a two-photon microscope. The signal detected at two separate photomultiplier tubes can be treated as separate signals and individually autocorrelated (as in normal FCS) or cross correlated to study the relation between the two signals. Our experimental setup consists of an inverted twophoton fluorescence microscope, and a custom made dual-channel photon-counting data acquisition PC card. The sample rate (typically between 100 Hz and 100 kHz) depends upon the system being investigated. An advantage of this system over normal FCS (when it is used in the cross-correlation mode) ... [truncated at 150 words]
Buehler C, Dong CY, So PTC, Gratton E.
Pump-probe stimulated-emission microscopy.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), Tu-Pos466.Pump-probe stimulated-emission microscopy involves a multi photon process that enables fluorescence lifetime imaging with confocal -like spatial resolution and sub-nanosecond time resolution. In this novel technique, two lasers running at different amplitude modulation frequencies and different colors are focused onto a common volume in the specimen. One laser excites the biochemical system and the other causes stimulated emission. The offset in the lasers' repetition frequencies, typically a few kHz, results in a heterodyning of the high frequency fluorescence decay to the frequency difference of the two lasers. Because the stimulated emission process occurs primarily at the overlapping volume of the pump and the probe beams, a 3-D sectioning effect comparable to confocal or two-photon microscopy can be obtained by observing the fluorescence at the lasers' difference frequency. Furthermore, as an inherent feature of pump-probe method, this instrument has ultrafast time resolution without using fast photodetectors. In the case of pulsed ... [truncated at 150 words]
Yu W, Durack G, Mantulin WW, Gratton E.
Lifetime-resolved flow cytometry for cell sorting and population heterogeneity using a heterodyning frequency-domain approach.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), Tu-Pos471.Flow cytometry is a widely used technique in many branches of biological science. The added capability of measuring fluorescence lifetimes in a conventional flow cytometer allows us to study lifetime distributions among cell populations very efficiently. Cell sorting based on lifetime differences improves accuracy. We have developed a time-resolved flow cytometer using a fast, frequency domain, heterodyning technique. In our experimental setup, an Argon ion laser is sinusoidally modulated through a Pockels-cell at about 100 MHz. The cross-correlation frequency is determined by the speed which a cell passes through the laser beam. For example, a flow of 1 m/s, the cell is under the laser beam for approximately 10µs. Typical cross-correlation frequencies applied at the detectors in our instrument are on the order of 400 kHz to 800 kHz, so that several periods of the cross correlation frequency can be acquired per cell. By referencing the phase signal from a ... [truncated at 150 words]
Gratton E, Levi M, Yu W, Parasassi T.
Two-photon fluorescence microscopy of LAURDAN GP domains in multilamellar phospholipid vesicles using polarized laser excitation.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), M-Pos272.Two-photon microscopy images of multilamellar phospholipid vesicles labeled with LAURDAN show coexisting regions of different generalized polarization (GP) values. The LAURDAN GP function measures the relative water content of the membrane. We have used polarized laser excitation which selectively excites high GP domains. The histogram of the GP values in vesicles of gel, of liquid-crystalline and of equimolar mixture of the two phases is distributed. The GP distribution in the gel phase vesicles is relatively narrow, while in the liquid-crystalline phase and in the mixture of the two phases is broad. Analysis of the images lead to the conclusion that the coexisting regions of different GP values have dimensions smaller than the microscope resolution (about 200 nm). The vesicles composed of an equimolar mixture of gel and liquid-crystalline phase show coexisting domains but the "rigid" domains have lower average GP than the pure gel phase and the "fluid" domains have ... [truncated at 150 words]
-
Single protein molecule reaction by two-photon excited fluorescence.
41st Annual Meeting of the Biophysical Society, New Orleans, Louisiana, 1997.
Biophys J. 1997; 72(2 Pt 2), M-Pos378.2-Photon fluorescence excitation occurs in a small volume (-0.1 fl), it has excellent background rejection, and it can detect single molecules. The study of protein reactions at the single molecule/protein level is a promising tool to address many fundamental issues regarding protein conformation and dynamics. In particular, we are interested in the observation of proteins in different conformational substates. For example, by analyzing the kinetics of single protein ligand binding, information on the statistical distribution of conformational substates at the single protein level may be obtained. Two major technical problems confront research in this area: 1) immobilization of proteins in a matrix in which native conformations occur and the background fluorescence is sufficiently low for single molecule detection, and 2) heat induced denaturation or heat induced substates changes upon excitation of the single fluorescing protein. Progress to overcome these obstacles by suitable choice of host environment will be discussed. Supported ... [truncated at 150 words]
-
Dynamics of a single protein.
22nd Annual Lorne Conference On Protein Structure And Function. February 9-13, 1997. Lorne, Australia.
So PTC, Yu W, Ragan TM, Gratton E.
An interactive approach to two-photon microscopy.
APS March Meeting, St. Louis, Missouri, March 17-22, 1996.
Typical fluorescence microscopy is a method for passive observation. Novel two-photon microscopy has typical excitation volume on the order of 0.1 femtoliter. This ability to selectively excite a small region in a sample has opened new opportunities for interactive experimentation. Two emerging interactive two-photon techniques will be described. Single particle tracking has been applied to the study of protein diffusion and interaction. The traditional approach is limited to monitoring two dimensional motions with video rate time resolution. We have developed a 3-D two-photon particle tracking instrument. Using a real-time feedback system, we can interactively follow the position of the diffusing fluorescent particle by maximizing detected intensity. This system has been used to study the process of antigen endocytosis of macrophage. (2) There are no reliable manipulation methods for objects, such as virus particles, of sizes below 0.1 micrometer but larger than atomic dimensions. Two-photon excitation can initiate localized chemical reaction ... [truncated at 150 words]
Dong CY, So PTC, Gratton E.
Pump-probe fluorescence microscopy: a new method for time-resolved imaging with high spatial and temporal resolution.
APS March Meeting, St. Louis, Missouri, March 17-22, 1996.
We present a unique time-resolved, fluorescence microscope using the pump-probe technique. In this microscope, two laser sources with different repetition frequencies are focused onto the fluorescent sample. The wavelengths of the lasers are chosen such that one laser excites the sample, and another laser induces stimulated emission from the molecules in the excited states or excite the ground state molecules. Due to the difference in the lasers' repetition frequencies, the fluorescence generated contains a signal at the cross-correlation frequency, and its harmonics. The spatial distribution of the signal depends on the intensities of both lasers, and is the strongest at the focal point where both light sources have high photon flux. As a result, microscopic imaging with this technique results in strong axial depth discrimination and comparable spatial resolution as confocal microscopy. We will present data characterizing the point-spread function of pump-probe fluorescence microscopy by imaging fluorescent latex spheres. We ... [truncated at 150 words]
Ragan TM, So PTC, Berland KM, Yu W, Nienhaus GU, Gratton E.
Detection of single molecules using two-photon excitation.
APS March Meeting, St. Louis, Missouri, March 17-22, 1996.
We have developed a technique which enables us to detect single molecules in solution and on surfaces using two-photon excitation. A major experimental difficulty in observing single molecules (single chromophore) fluorescence is a low signal to background ratio. In one-photon excitation, fluorescence excitation (Raman and Rayleigh) overlaps with the fluorescence emission spectrum. Time gating techniques are often needed to reduce the scattered light. Two photon excitation is particularly suitable for single molecule detection. First, the excitation and emission wavelengths are widely separated, and filters can be chosen to greatly attenuate the detection of scattered light while maintaining high fluorescence emission collection efficiency. Second, the two-photon excitation volume can be as low as 0.1 femtoliters, which corresponds to an average of 0.06 molecules in a 1 nM sample. We show results for the detection of single molecules and single-chromophore labeled proteins in solution. Monte Carlo simulations of particle diffusion (including photobleaching ... [truncated at 150 words]
Yu W, So PTC, French TE, Mantulin WW, Nienhaus GU, Gratton E.
Kinetic study of single molecule by two-photon fluorescence microscopy.
APS March Meeting, St. Louis, Missouri, March 17-22, 1996.
Imaging of a single molecule provides us a new tool to study functions of proteins and other biomolecules. Two-photon excitation probes a sub-femtoliter volume. In addition to the advantage of high background rejection by two-photon excitation, which is crucial for efficient single molecule detection, this small probing volume permits us to work at nanomolar sample concentrations but still detecting a single molecule. Furthermore, in the far-field microscopy, the native conformational states and/or substates of a protein molecule is less biased than in the near field. We have used a single photon counting scanning microscope in combination with two-photon excitation to image a single molecule immobilized on a microscope quartz slide. By allowing a freely diffusing fluorescent substrate to randomly bind a single protein molecule, successive events of fluorescence emission at a unique spatial location contain kinetic information about the protein's function. By using this method, we have studied the kinetics ... [truncated at 150 words]
French TE, So PTC, Gratton E, Carrero J, Voss EW Jr.
Fluorescent antigen processing by macrophages.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), W-Pos279.We present a study of macrophage mediated antigen processing using laser scanning two-photon fluorescence lifetime microscopy. With this method, antigen processing can be continuously monitored non-destructively via the measured fluorescence intensity and lifetime. The primary antigen used was FITCnBSA (FITC, fluorescein-5-isothiocyanate; BSA, bovine serum albumin). Fluorescein moieties were covalently attached to BSA at sufficient concentration for efficient self quenching (typically 22 FITC per BSA molecule). Proteolysis of the antigen is indicated by an increase in the fluorescence intensity and lifetime. Absolute fluorescence intensity measurements in vivo are problematic because the local probe concentration also affects the observed intensity. When ratiometric measurement is not possible (as in this case), fluorescence lifetime is the only reliable way to measure the probe's state. Images of macrophages observed after incubation times of up to 36 hours indicate that the FITC-BSA is indeed proteolyzed. The average intervacuolar lifetime did increase with time. However after 24 ... [truncated at 150 words]
Dong CY, So PTC, Gratton E.
Pump-probe fluorescence microscopy: a new method for time-resolved imaging with high spatial resolution.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), W-Pos280.We present a unique time-resolved, fluorescence microscope using the pump-probe technique. In this microscope, two laser sources with different repetition frequencies are focused onto the fluorescent sample. The wavelengths of the lasers are chosen such that one laser excites the sample, and another laser induces stimulated emission from the molecules in the excited states. Due to the difference in the lasers' repetition frequencies, the fluorescence generated contains a signal at the cross-correlation frequency, and its harmonics. The spatial distribution of the signal depends on the intensities of both lasers, and is the strongest at the focal point where both light sources have high photon flux. As a result, microscopic imaging with this technique results in strong axial depth discrimination and comparable spatial resolution as confocal microscopy. We will present data characterizing the point-spread functions of pump-probe fluorescence microscopy by imaging fluorescent latex spheres. We will also present similar data showing ... [truncated at 150 words]
Ragan TM, Berland KM, So PTC, Yu W, Gratton E.
Imaging a single fluorophore using two-photon excitation.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), W-Pos292.We have developed a detection system for recording the time arrival of single photons with high throughput. We have incorporated this detection system into the two-photon excitation microscope. We have obtained single fluorophore detection sensitivity. The low back round inherent in two photon excitation and single-photon counting enable us to achieve very high sensitivity and large (15 bit) dynamic range. The sensitivity and dynamic range of this system has been characterized in model systems, The linearity of the instrument is investigated in terms of fluorescence signal intensity and pixel residence time. High sensitivity imaging of cells and macromolecular assemblies will be presented. Finally, the statistics of single chromophore photobleaching in a biological system has been studied. [Supported by NIH grant RR03155.
Berland KM, So PTC, Ragan TM, Yu W, Gratton E.
Two-photon excitation for low background fluorescence microscopy: detection of single molecules and single chromophores in solution.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), W-Pos295.The capability to observe single fluorescent molecule in solution opens up many exciting experimental possibilities, including the development of ultra-sensitive bioassays, and studies of single molecular chemical reactions or ligand binding. A major experimental difficulty in observing single molecule (single chromophore) fluorescence is a low signal to background ratio. In one-photon excitation, scattered fluorescence excitation (Raman and Rayleigh) overlaps with the fluorescence emission spectrum. Time gating techniques are often needed to reduce scattered light. Two-photon excitation is particularly suitable for single molecular detection. First, the excitation and emission wavelengths are widely separated, and filters can be chosen to greatly attenuate the detection of scattered light while maintaining high fluorescence emission collection efficiency. Second, the two-photon excitation volume can be as low as 0.1 femtoliters, which corresponds to an average of 0.06 molecules in a I nM sample. Single chromophore detection can be performed at nanomolar range and which maintains high ... [truncated at 150 words]
Fantini S, Franceschini MA, Cerussi AE, Maier JS, Walker SA, Barbieri BB, Chance B, Gratton E.
The effect of water in the quantitation of hemoglobin concentration in a tissue-like phantom by near-infrared spectroscopy.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), W-Pos343.The quantitative measurement of tissue oxygenation is of paramount importance to prevent dangerous hypoxic or hyperoxic conditions in ill patients. A non-invasive, accurate, real-time, in situ monitor of tissue oxygenation is one of the goals of near-infrared tissue spectroscopy. The quantitative optical oximeter developed so far in our laboratory which operates at 715 and 825 nm assume that the absorption of near-infrared light in tissue is solely due to oxy- and deoxy-hemoglobin. We have investigated the effect of a third chromophore (water) in the quantitative determination of hemoglobin concentration and saturation. Our study consists of in vitro measurements on an aqueous solution containing Liposyn bovine blood, and yeast, pH buffered at 7.2. The optical coefficients of the solution match those of biological tissue in the near-infrared (Ua-0.03-0.08 cm-, Us'-6 cm-') and the hemoglobin concentration (23 RM) is also of the same order of magnitude than that in tissue. We were ... [truncated at 150 words]
So PTC, Ragan TM, Yu W, Gratton E.
Two-photon 3-D single particle tracking: a scanning microscopy approach.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), W-Pos98.Single particle tracking is a powerful approach to study membrane protein diffusion, membrane compartmentalization and protein-cytoskeleton interaction. Tracking the dynamics of a single particle can resolve diffusive, directed or restricted motions. This type of information cannot be obtained with conventional techniques where the statistically average behavior of particles is monitored. These studies typically use wide field illumination and monitor the motion of either colliodial gold or fluorescent particles using area detectors. This type of technique suffers from two major limitations. First, only two dimensional motions can be followed. Many interesting biological processes, such as endocytosis kinetics, intracellular transports and diffusion in cytoplasm, can not be studied. The development of 3-D particle tracking using cylindrical optics by Verkman's group (Biophys. J. 67, 1994) has somewhat alleviated this problem. However, the axial tracking of this method is still confined to a range of 5 to 10 gm. Second, the time-resolution of the ... [truncated at 150 words]
Yu W, So PTC, French TE, Mantulin WW, Gratton E.
Kinetic imaging of a single molecule.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), Tu-Pos197.Imaging of a single molecule is a new technique to study biological functions and dynamics of a single protein or other biomolecules. Advances in fluorescence microscopy coupled with a high efficiency photon collection systems allow detection of individual fluorescent molecule by a diffraction limited laser beam. We have used a single photon counting scanning microscope in combination with two-photon excitation to image a single molecule immobilized on a microscope cover glass. The scanning rate at each pixel can attain 50 KHz. This allows us to detect protein kinetic events down to tens of microseconds. This system can be more versatile and flexible than using a cooled CCD camera (Funatsu et al., 1995 Nature Vol. 374) which only operates at the video-rate. Two-photon excitation probes a 100 attolite sampling volume. In addition to the advantage of high background rejection by two-photon excitation, which is crucial for efficient single molecule detection, this ... [truncated at 150 words]
Gratton E, di Iorio E, Giusti AM, Parasassi T.
Diffusion in membranes studied by fluorescence oxygen quenching.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), Su-Pos459.The diffusion of molecular oxygen in membrane has been measured by fluorescence oxygen quenching, using probes of different lifetimes. Probes with longer lifetime should probe a large environment. Molecular oxygen is highly soluble in the hydrophobic interior of the bilayer and its diffusion is strongly dependent on the phase states of lipids. In general, in the liquid-crystalline phase oxygen diffusion is much faster. The apparent Stem-Volmer constant in the two phospholipid phases depends both on the lifetime of the fluorescent probe and on its size, i.e., on the perturbation introduced by the probe in the bilayer. For 2-dimethylamino-6-lauroylnaphthalene (LAURDAN) the apparent Stern-Volmer constant differs by a factor of about 50 between the two phases, while for pyrene-butyric acid we can only observe a difference of a factor of about 2. Moreover, using long lifetime probes we observed that the fluorescence decay becomes multi-exponential as the oxygen concentration is increased. A ... [truncated at 150 words]
Parasassi T, Krasnowska EK, di Iorio E, Gratton E.
PRODAN and Laurdan partition between the lipid phases and water.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), Su-Pos458.The spectroscopic features of PRODAN and LAURDAN (6-propionyl- and 6-lauroyI-2- dimethylaminonaphthalene, respectively) in phospholipid vesicles of different phase states have been compared. PRODAN shows a higher partitioning in the liquid-crystalline with respect to the gel phase. Instead LAURDAN does not display a preferential partitioning between the phases and it is virtually non-fluorescent in the aqueous environments. PRODAN in water can be easily identified by the red emission band, particularly evident in gel phase vesicles. To separate the probe response arising from the lipid environment, a new method based on the measurement at three wavelengths has been developed for the determination of the generalized polarization (GP). The method effectively subtracts the contribution of PRODAN fluorescence arising from water. The three-wavelengths-GP can also be used to calculate PRODAN partition coefficient between lipids and the aqueous phase and between the gel and the liquid-crystalline phase of phospholipids. The presence of cholesterol in the ... [truncated at 150 words]
Levi M, Giusti AM, Cruz CJ, Wilson PV, Nguyen S, Gratton E, Sapora O, Parasassi T.
Cell membrane reversible "aging" detected by laurdan gp is associated with an increase in cholesterol content.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), Su-Pos367.Laurdan (2-dimethylamino-6-lauroylnaphthalene) GP (generalized polarization) has been measured in two human cell lines, promyelocytic HL60 cells and proerythroblastoid K562 cells, routinely subcultured twice per week in RPM1 1640 with 10% fetal calf serum. The GP measurements were performed on the asynchronously growing populations 24 and 96 hours after the medium renewal. The GP value increased linearly with time after the medium renewal. The increase in GP was reversible with the next medium renewal, i.e., the initial lower GP value was recovered after 24 hours from the passage in fresh medium. At 96 hours, there was a 30% increase in the GP in HL60 cells and a 20% increase in the GP in K562 cells. We tested the hypothesis that a major reason for the increase in GP is the aging induced change in membrane lipid composition. The results showed that from the first to the fifth day of asynchronous growth ... [truncated at 150 words]
Chen Y, Berland KM, So PTC, Mantulin WW, Gratton E.
Study of protein aggregation in solution using scanning two photon fluorescence high order correlation spectroscopy.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), W-Pos294.Two photon scanning fluctuation correlation spectroscopy is a powerful technique for measuring diffusion coefficients and particle number concentrations in dilute solutions of biomolecules (K. Berland, P. T. C. So and Enrico Gratton, 1995, Biophysical Journal, 68, 694-701). By comparing measured number concentrations with nominal concentrations (known weight concentrations), one can detect the presence of molecular aggregates. We have applied this technique to study protein association /dissociation equilibrium at dilute concentrations (sub micromolar). We demonstrate accurate detection of changes in oligomeric state as protein concentration, pH, or other relevant experimental parameters are varied. These measurements are shown for the proteins Malate Dehydrogenase (MDH), and rabbit muscle Phosphofructokinase (PFK). Also presented are kinetic measurements of dissociation following dilution using PFK. PFK has a complicated distribution of aggregate sizes. We have examined the oligomerization process by calculating high order correlation function. The intensity fluctuation is Poisson distributed up to I RM in less ... [truncated at 150 words]
Maier JS, Cerussi AE, Fantini S, Franceschini MA, Walker SA, Gratton E.
Experimental verification of a quantitative model to describe fluorescence in homogeneous tissues.
40th Annual Meeting of the Biophysical Society, New Baltimore, Maryland, 1996.
Biophys J. 1996; 70(2 Pt 2), W-Pos293.Fluorescence spectroscopy is a potentially valuable tool for the investigation of tissues because of its high specificity. In vivo fluorescence spectroscopy has several potential advantages over standard near-infrared tissue spectroscopy in non-invasive medical applications. The multiple scattering of light in tissues (with effective photon mean free paths less than 1 mm) complicates quantitative analysis of tissue fluorescence. Our work unites fluorescence spectroscopy with the diffusive theory for light transport in highly scattering media such as tissues. We developed a model which uses the photon density of the excitation light to derive the spatially extended source at the emission wavelengths. This model relates the measured emission photon density to the optical coefficients of the tissue, and the undistorted fluorescence spectrum, probe concentration, quantum yield, and lifetime of the probe. We demonstrate the effectiveness of this model in describing frequency-domain data collected on a tissue-like sample of Rhodamine B and ink dissolved ... [truncated at 150 words]
Reininger R, Hansen RWC, Gratton E, Mantulin WW, Weber G, Royer CA.
Studies of fluorescence dynamics in biological systems using the pulsed structure of the SRC.
APS X-ray Centennial Symposium and 7th Users Meeting for the APS, Argonne, IL, 16-20 Oct 1995.
A beamline for time-resolved fluorescence spectroscopy of biological systems is under construction at the Synchrotron Radiation Center. The fluorometer, operating in the frequency domain, will take advantage of the time structure of the synchrotron-radiation light pulses to determine fluorescence lifetimes. Using autocorrelation techniques, the instrument can achieve an ultimate time resolution on the order of picoseconds. Preliminary experiments have shown that reducing the intensity of one of the fifteen electron bunches in the storage ring allows measurement of harmonic frequencies equivalent to the single bunch mode. This significantly extends the range of lifetimes that can be measured. The wavelength range (down to 200 nm), the range of measurable lifetimes, and the stability and reproducibility of the storage ring pulses should make this beamline a versatile tool for the investigation of the complex fluorescence decay of biological systems.
Gratton E, So PTC, Berland KM, French TE, Dong CY, Yu W.
Lifetime-resolved two-photon fluorescence microscopy: a new tool for spectroscopy and imaging.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
Since the introduction by Webb's group of the two-photon excitation technique in fluorescence microscopy, several utilizations of this novel method have been proposed. We have developed a scanning microscope which uses two-photon excitation and fluorescence detection to provide lifetime information on a pixel basis. In our system, the frequency-domain method is used to measure the phase and modulation of the signal at every pixel with a residence time of about 40ms. This microscope provides the well-established advantages of two-photon excitation without sacrificing light intensity for the measurement of lifetime-resolved images. Several applications in biology will be presented. Using the laser scanning hardware and two separate pulsed lasers focused at the same point, it is possible to achieve excitation and stimulated emission on a pixel basis. Due to the interaction of the two lasers at the focal point, this stimulated emission microscope has similar sectioning capability as the ... [truncated at 150 words]
-
Adenylate Kinase: a spectroscopic characterization of conformational dynamics in Tryptophan mutants.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
Adenylate kinase catalyzes the transfer of a phosphoryl group ATP+AMP ´ 2ADP with a Mg2+ cofactor. The adenylate kinase from Escherichia coli (AKe) is a monomeric, single chain protein of 28,590D. Its amino acid sequence and ligand bound crystal structure are known. The enzyme has mixed amounts of secondary structure (50% a-helix; 20% b-sheet; 30% other structures) and distinct domains that undergo significant conformational motion with substrate binding. Wild type AKe, which is devoid of tryptophan, has been isolated, cloned, and purified to homogeneity. We have constructed a library of several single tryptophan (Trp) mutants. The Trp mutants are distributed around the enzyme, including both surface and interior locations in various domains, some of which interact with the substrate binding sites. The specific mutations discussed in this study are: Ser41Trp; Ala73Trp; Phe86Trp; Tyr133Trp; and Phe137Trp. We have used optical spectroscopic techniques to investigate the conformational dynamics of ... [truncated at 150 words]
Voss EW Jr, Cherukuri A, Coelho-Sampaio T, Carrero J, French TE, So PTC, Gratton E.
Fluorescent antigenic probes designed to elucidate intracellular steps and mechanisms involved in the macrophage-mediated antigen processing and presentation pathway.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
Densely substituted fluorescein conjugated bovine serum albumin (BSA) served as a soluble exogenous antigen and fluorescent probe for continuous monitoring of macrophage-mediated endocytosis, intracellular vacuolar encapsulation, and processing. The relatively non-fluorescent probe was designed to reflect intracellular antigen processing events (thiol reduction, protein unfolding, and proteolytic degradation) through changes in the fluorescence properties from the original quenched state. Since fluorescein is also sensitive to pH changes the probe was suited to monitor the acidic vacuoles that comprise the endocytic pathway. Kinetics of endocytosis were determined quantitatively through flow cytometry and two-photon fluorescence microscopy with time-resolved and intensity imaging. The kinetics of cellular uptake of the antigen showed a relatively rapid uptake (0-120 sec) followed by significant fluorescence quenching in the time frame of 120-240 sec. Flow cytometric and fluorescence lifetime measurements suggested that the fluorescence quenching may reflect a macromolecular binding intravacuolar event and was ... [truncated at 150 words]
Barcellona ML, Gratton E.
Nucleic acids torsional dynamics detection by anisotropy decay of DNA/DAPI complexes.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
The flexibility of calf thymus DNA and several polydeoxynucleotides was measured using the anisotropy decay of DAPI bound to DNA, a classical minor groove probe. DNA torsional dynamics were analyzed using Schurr's model in the infinite polymer length approximation. Time-resolved fluorescence depolarization was measured using a frequency-doubled mode-locked dye laser and frequency domain acquisition methods. At very high P/D ratios, the anisotropy decay is dominated by DNA torsional dynamics. The recovered values of the torsional elastic constant were in good agreement with literature values obtained using other DNA probes. The exact knowledge of the angle between the probe emission dipole transition moment and the helix axis is critical for the determination of the polymer elastic constant. At intermediate P/D ratios, energy transfer between dye molecules strongly contributes to the anisotropy decay. We have developed a statistical model that, when correct geometrical factors are ... [truncated at 150 words]
Gilmore AM, Debrunner PG, Hazlett TL, Govindjee.
Global fluorescence lifetime distribution analyses of non-photochemical quenching of photosystem II.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
Under saturating light intensities, plants activate a protection pathway to bleed off surplus excitation energy. This non-photochemical quenching process (NPQ) dissipates damaging excitation energy in the form of heat and rescues the plant's photosynthetic apparatus from photodamage. It has been suggested that the association of zeaxanthin (Zx) and antheraxanthin (Ax) with the antenna chlorophyll serves as the NPQ mechanism. The extent of NPQ can be monitored through the fluorescence intensities and lifetimes of chlorophyll a. The known heterogeneity of thylakoid photosystem II (PSII) complexes justifies the use of a lifetime distributional model as an appropriate analysis tool. Fluorescence lifetime data were collected and analyses were performed on isolated thylakoids under quenched (Fm') and unquenched (Fm) conditions. A single fluorescence lifetime distribution centered at 2.3 ns was resolved for thylakoids in the unquenched condition while the appearance of a second, 0.4 ns, lifetime distribution was observed under quenched conditions. The fractional ... [truncated at 150 words]
König K, So PTC, Mantulin WW, Gratton E, Berns MW, Tromberg BJ.
Two-photon excited autofluorescence in CIIO cells and motile spermatozoa.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
We report the first two-photon-excited autofluorescence measurements of motile cells in a single-beam gradient force optical trap (“optical tweezers”). Autofluorescence in the blue spectral region of optically-trapped human spermatozoa was excited with “continuous wave” NIR radiation of a tunable Ti:Sapphire laser (899-01, Coherent). In addition, two-photon NIR excited autofluorescence imaging and t-mapping was performed with laser scanning microradiation of a tunable femtosecond, Ti:Sapphire laser (Mira 900, Coherent). Cellular response on autofluorescence to two-photon absorption of short wavelength NIR radiation was compared with exposure to UVA light (one-photon absorption).
Sapora O, Giusti AM, Gratton E, Maggi A, Parasassi T, Marherita R.
Production and repair of membrane oxidative damage in mammalian cells after ionizing radiation.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
Oxidative damage produced by ionizing radiation on membrane lipids of mammalian cells has been studied by the measurement of 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence decay in two human cell lines, K562 and HL60. For both cell lines the width of the DPH continuous lifetime distribution decreases linearly as a function of the logarithm of the ionizing radiation dose, up to 10 Gy. The lowest dose at which the damage can be observed is 0.1 Gy. Repair experiments show that the damage produced by a single acute dose of 10 Gy is not repaired in the 24 hours following the cell exposure. Cells were also exposed daily to a dose of 0.1 Gy for eleven days, and the results show an additive and not repaired membrane damage. The fractionated exposure did not affect cells viability. Finally, the amount of the observed damage is related to the time ... [truncated at 150 words]
Krasnowska EK, Iorio E, Gratton E, Parasassi T.
Comparison of LAURDAN and PRODAN fluorescence in phospholipid phases.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
The fluorescence signals from PRODAN and LAURDAN (6-propionyl- and 6-lauroyl-2-dimethylamino-naphthalene, respectively) in phospholipid vesicles composed of the pure gel and liquid-crystalline phases and of coexisting domains of the two phases has been compared. PRODAN partitions between the membrane surface and the membrane interior depending on the phase state of phospholipids and shows appreciable fluorescence also in aqueous environment. In gel phase bilayers, PRODAN partitioning in water increases. In vesicles composed of coexisting phases, PRODAN also shows a preferential partitioning in the liquid-crystalline phase. Instead, LAURDAN remains in the membrane interior and does not display preferential partitioning between the phases. Hence, using PRODAN we cannot discriminate between the pure liquid-crystalline phase and coexisting phase domains as we do with LAURDAN. Especially, the wavelength dependence of PRODAN generalized polarization (GP) value in these two cases is similar. Interestingly, a different correlation between the variation of the emission GP and of the excitation ... [truncated at 150 words]
Iorio E, Giusti AM, Parasassi T, Gratton E.
Determination of membrane defects using oxygen quenching of membrane probes.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
Fluorescence oxygen quenching has been used as a technique to measure diffusion of small molecules in proteins and membranes. Molecular oxygen has a relatively high solubility in the non-polar membrane interior. Oxygen diffusion in model membranes is strongly dependent on the phase state of phospholipids. In the gel phase, quenching is generally strongly reduced, even for relatively long lived probes such as pyrene. Instead, the quenching rate increases dramatically in the liquid crystalline state. In the gel phase, the presence of a small number of membrane defects can enhance the rate of oxygen quenching. We have performed oxygen quenching experiments in DLPC and DPPC vesicles using fluorescent probes of different lifetimes, with and without cholesterol. We have collected steady-state and lifetime data. With long lifetime probes, an interesting observation is that the fluorescence decay becomes non-exponential when oxygen is added. This ... [truncated at 150 words]
Levi M, Giusti AM, Wilson P, Nguyen S, Gratton E, Sapora O, Parasassi T.
Cell membrane reversible “aging” detected by Laurdan GP is associated with an increase in cholesterol content.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
Laurdan (2-dimethylamino-6-lauroylnaphthalene) GP (generalized polarization) has been measured in two human cell lines, promyelocytic HL60 cells and proerythroblastoid K562 cells, routinely subcultured twice per week in RPM1 1640 with 10% fetal calf serum. The GP measurements were performed on the asynchronously growing populations 24 and 96 hours after the medium renewal. The GP value increased linearly with time after the medium renewal. The increase in GP was reversible with the next medium renewal, i.e., the initial lower GP value was recovered after 24 hours from the passage in fresh medium. At 96 hours, there was a 30% increase in the GP in HL60 cells and a 20% increase in the GP in K562 cells. We tested the hypothesis that a major reason for the increase in GP is the aging induced change in membrane lipid composition. We analyzed HL60 and K562 cell cholesterol content by GC and phospholipid ... [truncated at 150 words]
Bilderback T, Fulmer TJ, Mantulin WW, Glaser M.
Substrate binding causes movement in the ATP binding domain of Escherichia coli adenylate kinase.
3rd International Weber Symposium. Maui, Hawaii. July 30-August 2, 1995.
Crystallographic evidence suggests that there is a large hinged domain motion associated with substrate binding in adenylate kinase. To test this hypothesis, we initiated resonance energy transfer measurements of substrate binding. Adenylate kinase from Escherichia coli consists of three domains: the main body of the enzyme with a-helical and b-sheet secondary structure and domains that close over the AMP and ATP binding sites. Four single tryptophan mutants were constructed to map distances. Two tryptophan mutants were positioned at residues 133 (Y133W) and 137 (F137W), which are in the domain that closes over the ATP binding site. Mutant F86W, which is located near the AMP binding site, and mutant S41W, which is in the AMP binding domain, complete the mapping library. Energy transfer was measured between each of these tryptophans and 5-[[2-(iodoacetyl)-amino] ethyl] amino] naphthalene-1-sulfonic acid (IAEDANS) covalently bound to the single cysteine residue at position ... [truncated at 150 words]
Cerussi AE, Yu W, Fantini S, Franceschini MA, Gratton E.
Effects of curved boundaries on the measured linear transport coefficient of tissue-like phantoms in the near infrared.
39th Annual Meeting of the Biophysical Society, San Francisco, California, 1995.
Biophys J. 1995; 68(2 Pt 2), Th-Pos352.We are studying the effects of curved boundaries on photon density waves (PDW) in strongly scattering tissue-like phantoms. PDW are generated in highly scattering media by intensity modulated light sources, which are employed in the frequency domain experimental approach. These PDW are spherical waves in an infinite, homogeneous medium where scattering dominates over absorption. The measured frequency domain parameters, such as relative phase shift and intensity, can be translated into linear absorption (μa) and reduced scattering (μs') coefficients in both infinite and semi-infinite media. A commonly used assumption is that the angular photon density is nearly isotropic; it is composed of an isotropic term plus a smaller angular contribution. This diffusion approximation of the Boltzmann transport equation, however, is not valid in the presence of finite boundaries. Near a boundary, the space is not isotropic and the diffusion approximation is no longer valid. Although there has been success in recovering ... [truncated at 150 words]
Maier JS, Walker SA, Fantini S, Franceschini MA, Gratton E.
Tissue reduced scattering as a near-infrared spectroscopy tool: correlation between in-vivo blood glucose and reduced scattering.
39th Annual Meeting of the Biophysical Society, San Francisco, California, 1995.
Biophys J. 1995; 68(2 Pt 2), Th-Pos353.Most of the emphasis in near-infrared tissue spectroscopy is placed on the use of absorption characteristics of tissues and how they relate to underlying physiology or pathology. In order to accurately determine the absorption coefficient of bulk tissue at a near-infared wavelength one must separate the effect of tissue scattering from absorption, thereby determining the reduced scattering coefficient of the tissue. Development of physical models which connect changes in reduced scattering coefficient with changes in physiology will allow use of reduced scattering in answering physiological questions. One such model is the relationship between index of refraction mismatch and scattering. The effectiveness of an inhomogeneity as a scatterer is related to the difference between its refractive index and the refractive index of the surrounding medium. As the indices approach one another, the scattering caused by the inhomogeneity decreases. We consider changes in refractive index of the intracellular and extracellular fluid caused ... [truncated at 150 words]
Walker SA, Franceschini MA, Fandni S, Maier JS, Grauton E.
Spatial maps of optical coefficients of tissue like phantoms in turbid media using near infrared light.
39th Annual Meeting of the Biophysical Society, San Francisco, California, 1995.
Biophys J. 1995; 68(2 Pt 2), Th-Pos351.Optical tomography in tissue is a possible alternative to current methods for locating tumors in the human body (i.e. Magnetic Resonance Imaging. X-ray techniques). It has the advantages of safety, potability, and relatively low cost near infrared light is used because of its deep penetration into human tissue (typically several centimeters). In this wavelength region,human tissue is highly scattering, causing light to propagate in a diffusive manner. In turbid media, a high frequency (about 100 MHz) intensity modulated light source produces a "photon density wave". By measuring the phase and amplitude of this wave, one can recover the scattering and absorption coefficients (ms' and ma respectively) of a macroscopically homogeneous, highly scattering medium. In imaging applications, when macroscopic inhomogeneities are present, we use a precalibrated measurement protocol to recover the optical coefficients. This protocol consists of a single distance, single modulation frequency measurement in an microscopically homogeneous medium of known ... [truncated at 150 words]
Paraassi C, Gius AM, Jameson DM, Gratton E.
Cholesterol induces ordered microdomains in membranes.
39th Annual Meeting of the Biophysical Society, San Francisco, California, 1995.
Biophys J. 1995; 68(2 Pt 2), M-Pos406.Formation of ordered molecular microdomains in phopholipid vesicles at different cholesterol concentrations were studied using the Generalized Polarization function (GP) of LAURDAN fluorescence. By constructing a surface plot of the emission (or excitation) GP as a function of cholesterol mol% with respect to total phospholipid content, particular concentrations were identified, producing discontinuities of the GP surface. The changes in the fluorescent properties of LAURDAN have been interpreted as due to ordered lipid structure which inhibit water penetration in the membrane and affect LAURDAN GP. Those structures only occur in a very narrow range of cholesterol concentration. To further characterize these ordered molecular structures, oxygen quenching experiments were also performed. The diffusion of oxygen in gel-phase membranes is facilitated by the presence of packing defects introduced by the addition of cholesterol. However, oxygen quenching measurements showed that, at cholesterol concentrations at which ordered molecular structures are formed, oxygen diffusion is ... [truncated at 150 words]
Dong CY, So PTC, Gratton E.
Multiphoton pump-probe fluorescence spectroscopy by stimulated emission following excitation.
39th Annual Meeting of the Biophysical Society, San Francisco, California, 1995.
Biophys J. 1995; 68(2 Pt 2), Tu-Pos344.We are developing the technique of pump-probe fluorescence spectroscopy by stimulated emission following excitation. This method involves spatially overlapping two pulse laser sources at the sample. One beam (pump) is used to excite the molecules under study and a second beam (probe) is used to induce stimulated emission from the molecules still in the excited state. The two pulse trains are offset in frequency by an amount small compared to the laser repetition frequency. The presence of a probe beam at a different frequency means that the fluorescence from excited state molecules contains a component at the difference frequency and corresponding harmonics. The lower frequency crosscorrelation signal gives us picosecond information about the response of the chromophores at high laser frequency harmonics. For spectroscopic applications, the main advantage of this sample heterodyning technique is that we can study ultra-fast phenomena at higher frequencies using the same optical detector. In this ... [truncated at 150 words]
Yu W, So PTC, French TE, Gratton E.
Lipid domains in biological membranes studied by using a twophoton microscope.
39th Annual Meeting of the Biophysical Society, San Francisco, California, 1995.
Biophys J. 1995; 68(2 Pt 2), Tu-Pos342.We have investigated lipid domains in biological membranes using LAURDAN (2-dimethylamino-6-lauroyl-naphthalene) generalized polarization obtained from a scanning two-photon fluorescence microscope. The lipophilic fluorescence probe LAURDAN is sensitive to the polarity of its environment and its fluorescence spectra change with variations in membrane lipid composition as well as in the membrane phase state. The generalized polarization function(GP) is useful in exploring the existence of lipid microdomains in biological membranes. In conventional confocal microscopy, fluorescence imaging of a single cell stained with LAURDAN has been unsuccessful, primarily due to the probe's susceptibility to photobleaching. By taking advantages of two photon scanning fluorescence microscopy photo bleaching is localized and reduced to a minimum. We have obtained single cell three dimensional images of both fluorescence intensity and generalized polarization by the two photon scanning approach. From these images, regions characterized by different fluorescence intensities for the blue and red edge of the LAURDAN emission ... [truncated at 150 words]
Sutin JDB, So PTC, Gratton E.
Studies of confomational substates in glasses with fluorescence distribution of lifetimes.
39th Annual Meeting of the Biophysical Society, San Francisco, California, 1995.
Biophys J. 1995; 68(2 Pt 2), Tu-Pos315.Previous work has demonstrated that a single distribution of lifetimes model of fluorescence decay cannot be resolved from a multiple discrete exponentials model (Alcala, et al.; Biophys J 56: 587). In proteins, a distribution of lifetimes can be physically justified by the conformational substates model, where the width of the distribution arises from a heterogeneous conformational energy landscape. The temperature dependence of this model allows the distribution and discrete models to be distinguished. The conformational substates model also suggests proteins share some essential characteristics with glassy systems since both have heterogeneous landscapes (Silva, et al.; Biophys J: to be published). In this study, we are using N-acetyltryptophanamide (NATA) in glass forming glycerol:buffer solutions to investigate the dependence of the fluorescence decay on conformational substates. The measured width of the lifetime distribution increased with decreasing temperature but reached a plateau at a certain "critical" temperature, in agreement with the conformational substate ... [truncated at 150 words]
Berland KM, So PTC, Mantulin WW, Gratton E.
Molecular aggregation studies using scanning two photon fluctuation correlation spectroscopy.
39th Annual Meeting of the Biophysical Society, San Francisco, California, 1995.
Biophys J. 1995; 68(2 Pt 2), Tu-Pos284.There are many problems in biology and biochemistry in which it is inte
