Sohrabi A, Lefebvre AEYT, Harrison MJ, Condro MC, Sanazzaro TM, Safarians G, Solomon I, Bastola S, Kordbacheh S, Toh N, Kornblum HI, Digman MA, Seidlits SK.
Microenvironmental stiffness induces metabolic reprogramming in glioblastoma.
Cell Rep. 2023; 42(10), 113175.The mechanical properties of solid tumors influence tumor cell phenotype and the ability to invade surrounding tissues. Using bioengineered scaffolds to provide a matrix microenvironment for patient-derived glioblastoma (GBM) spheroids, this study demonstrates that a soft, brain-like matrix induces GBM cells to shift to a glycolysis-weighted metabolic state, which supports invasive behavior. We first show that orthotopic murine GBM tumors are stiffer than peritumoral brain tissues, but tumor stiffness is heterogeneous where tumor edges are softer than the tumor core. We then developed 3D scaffolds with μ-compressive moduli resembling either stiffer tumor core or softer peritumoral brain tissue. We demonstrate that the softer matrix microenvironment induces a shift in GBM cell metabolism toward glycolysis, which manifests in lower proliferation rate and increased migration activities. Finally, we show that these mechanical cues are transduced from the matrix via CD44 and integrin receptors to induce metabolic and phenotypic changes in cancer cells.
Fazel M, Jazani S, Scipioni L, Vallmitjana A, Zhu S, Gratton E, Digman MA, Pressé S.
Building fluorescence lifetime maps photon-by-photon by leveraging spatial correlations.
ACS Photonics. 2023; 10(10): 3558-3569. PMCID: PMC10890823Fluorescence lifetime imaging microscopy (FLIM) has become a standard tool in the quantitative characterization of subcellular environments. However, quantitative FLIM analyses face several challenges. First, spatial correlations between pixels are often ignored as signal from individual pixels is analyzed independently thereby limiting spatial resolution. Second, existing methods deduce photon ratios instead of absolute lifetime maps. Next, the number of fluorophore species contributing to the signal is unknown, while excited state lifetimes with <1 ns difference are difficult to discriminate. Finally, existing analyses require high photon budgets and often cannot rigorously propagate experimental uncertainty into values over lifetime maps and number of species involved. To overcome all of these challenges simultaneously and self-consistently at once, we propose the first doubly nonparametric framework. That is, we learn the number of species (using Beta-Bernoulli process priors) and absolute maps of these fluorophore species (using Gaussian process priors) by leveraging information from pulses not ... [truncated at 150 words]
Kettmayer C, Gratton E, Estrada LC.
Comparison of MSD analysis from single particle tracking with MSD from images. Getting the best of both worlds.
Methods Appl Fluoresc. 2023; 12(1), 015001.Fluorescence microscopy can provide valuable information about cell interior dynamics. Particularly, mean squared displacement (MSD) analysis is widely used to characterize proteins and sub-cellular structures' mobility providing the laws of molecular diffusion. The MSD curve is traditionally extracted from individual trajectories recorded by single-particle tracking-based techniques. More recently, image correlation methods like iMSD have been shown capable of providing averaged dynamic information directly from images, without the need for isolation and localization of individual particles. iMSD is a powerful technique that has been successfully applied to many different biological problems, over a wide spatial and temporal scales. The aim of this work is to review and compare these two well-established methodologies and their performance in different situations, to give an insight on how to make the most out of their unique characteristics. We show the analysis of the same datasets by the two methods. Regardless of the experimental differences in ... [truncated at 150 words]
Philipp N, Gratton E, Estrada L.
Measuring protein-membrane interaction through radial fluorescence correlation in 2 dimension.
Methods Appl Fluoresc. 2023; Online ahead of print.The cell membrane has a fundamental role in the cell life cycle but there's
still much to be learned about its heterogeneous structure, regulation, and protein interaction.
Additionally, the protein-membrane interaction is often overlooked when studying specific protein
dynamics. In this work, we present a new tool for a better understanding of protein dynamics
and membrane function using live cells and fast non-invasive techniques without the need for
individual particle tracking. To this end, we used the 2D-pair correlation function (2D-pCF) to
study protein interactions across cellular membranes. We performed numerical simulations and
confocal experiments using a GAP-mEGFP fusion construct known to interact with the plasmatic
membrane. Our results demonstrate that based on a quantitative correlation analysis as the 2D
pair correlation of the signal intensities, is possible to characterize protein-membrane interactions
in live systems and real-time. Combining experimental and numerical results this work presents
a new powerful approach to the study of the dynamic protein-membrane interaction.
Tharp K, Park S, Timblin G, Richards A, Twells N, Riley N, Peltan E, Shon J, Stevenson E, Tsui K, Palomba F, Lefebvre A, Soens R, Ayad, N, Hoeve-Scott JT, Healy K, Digman M, Dillin A, Bertozzi C, Swaney D, Mahal L, Cantor J, Paszek M, Weaver VM, Twells N.
The microenvironment dictates glycocalyx construction and immune surveillance.
Res Sq. 2023; Preprint. PMCID: PMC10462183Efforts to identify anti-cancer therapeutics and understand tumor-immune interactions are built with in vitro models that do not match the microenvironmental characteristics of human tissues. Using in vitro models which mimic the physical properties of healthy or cancerous tissues and a physiologically relevant culture medium, we demonstrate that the chemical and physical properties of the microenvironment regulate the composition and topology of the glycocalyx. Remarkably, we find that cancer and age-related changes in the physical properties of the microenvironment are sufficient to adjust immune surveillance via the topology of the glycocalyx, a previously unknown phenomenon observable only with a physiologically relevant culture medium.
Agustinus AS, Al-Rawi D, Dameracharla B, Raviram R, Jones BSCL, Stransky S, Scipioni L, Luebeck J, di Bona M, Norkunaite D, Myers RM, Duran M, Choi S, Weigelt B, Yomtoubian S, McPherson A, Toufektchan E, Keuper K, Mischel PS, Mittal V, Shah SP, Maciejowski J, Storchova Z, Gratton E, Ly P, Landau D, Bakhoum MF, Koche RP, Sidoli S, Bafna V, David Y, Bakhoum SF.
Epigenetic dysregulation from chromosomal transit in micronuclei.
Nature. 2023; 619(7968): 176-183. PMCID: PMC10322720Chromosomal instability (CIN) and epigenetic alterations are characteristics of advanced and metastatic cancers1-4, but whether they are mechanistically linked is unknown. Here we show that missegregation of mitotic chromosomes, their sequestration in micronuclei5,6 and subsequent rupture of the micronuclear envelope7 profoundly disrupt normal histone post-translational modifications (PTMs), a phenomenon conserved across humans and mice, as well as in cancer and non-transformed cells. Some of the changes in histone PTMs occur because of the rupture of the micronuclear envelope, whereas others are inherited from mitotic abnormalities before the micronucleus is formed. Using orthogonal approaches, we demonstrate that micronuclei exhibit extensive differences in chromatin accessibility, with a strong positional bias between promoters and distal or intergenic regions, in line with observed redistributions of histone PTMs. Inducing CIN causes widespread epigenetic dysregulation, and chromosomes that transit in micronuclei experience heritable abnormalities in their accessibility long after they have been reincorporated into the primary ... [truncated at 150 words]
Fazel M, Vallmitjana A, Scipioni L, Gratton E, Digman MA, Pressé S.
Fluorescence lifetime: Beating the IRF and interpulse window.
Biophys J. 2023; 122(4): 672-683. PMCID: PMC9989884Fluorescence lifetime imaging captures the spatial distribution of chemical species across cellular environments employing pulsed illumination confocal setups. However, quantitative interpretation of lifetime data continues to face critical challenges. For instance, fluorescent species with known in vitro excited-state lifetimes may split into multiple species with unique lifetimes when introduced into complex living environments. What is more, mixtures of species, which may be both endogenous and introduced into the sample, may exhibit 1) very similar lifetimes as well as 2) wide ranges of lifetimes including lifetimes shorter than the instrumental response function or whose duration may be long enough to be comparable to the interpulse window. By contrast, existing methods of analysis are optimized for well-separated and intermediate lifetimes. Here, we broaden the applicability of fluorescence lifetime analysis by simultaneously treating unknown mixtures of arbitrary lifetimes-outside the intermediate, Goldilocks, zone-for data drawn from a single confocal spot leveraging the tools of ... [truncated at 150 words]
Fang T, Huang YK, Wei J, Mena JEM, Lakey PSJ, Kleinman MT, Digman MA, Shiraiwa M.
Superoxide release by macrophages through NADPH oxidase activation dominating chemistry by isoprene secondary organic aerosols and quinones to cause oxidative damage on membranes.
Environ Sci Technol. 2022; 56(23): 17029-17038. PMCID: PMC9730850Oxidative stress mediated by reactive oxygen species (ROS) is a key process for adverse aerosol health effects. Secondary organic aerosols (SOA) account for a major fraction of fine particulate matter, and their inhalation and deposition into the respiratory tract causes the formation of ROS by chemical and cellular processes, but their relative contributions are hardly quantified and their link to oxidative stress remains uncertain. Here, we quantified cellular and chemical superoxide generation by 9,10-phenanthrenequinone (PQN) and isoprene SOA using a chemiluminescence assay combined with electron paramagnetic resonance spectroscopy as well as kinetic modeling. We also applied cellular imaging techniques to study the cellular mechanism of superoxide release and oxidative damage on cell membranes. We show that PQN and isoprene SOA activate NADPH oxidase in macrophages to release massive amounts of superoxide, overwhelming the superoxide formation by aqueous chemical reactions in the epithelial lining fluid. The activation dose for PQN is ... [truncated at 150 words]
Halbrook CJ, Thurston G, Boyer S, Anaraki C, Jiménez JA, McCarthy A, Steele NG, Kerk SA, Hong HS, Lin L, Law FV, Felton C, Scipioni L, Sajjakulnukit P, Andren A, Beutel AK, Singh R, Nelson BS, van Bergh FD, Krall AS, Mullen PJ, Zhang L, Batra S, Morton JP, Stanger BZ, Christofk HR, Digman MA, Beard DA, Viale A, Zhang J, Crawford HC, di Magliano MP, Jorgensen C, Lyssiotis CA.
Differential integrated stress response and asparagine production drive symbiosis and therapy resistance of pancreatic adenocarcinoma cells.
Nat Cancer. 2022; 3(11): 1386-1403. PMCID: PMC9701142The pancreatic tumor microenvironment drives deregulated nutrient availability. Accordingly, pancreatic cancer cells require metabolic adaptations to survive and proliferate. Pancreatic cancer subtypes have been characterized by transcriptional and functional differences, with subtypes reported to exist within the same tumor. However, it remains unclear if this diversity extends to metabolic programming. Here, using metabolomic profiling and functional interrogation of metabolic dependencies, we identify two distinct metabolic subclasses among neoplastic populations within individual human and mouse tumors. Furthermore, these populations are poised for metabolic cross-talk, and in examining this, we find an unexpected role for asparagine supporting proliferation during limited respiration. Constitutive GCN2 activation permits ATF4 signaling in one subtype, driving excess asparagine production. Asparagine release provides resistance during impaired respiration, enabling symbiosis. Functionally, availability of exogenous asparagine during limited respiration indirectly supports maintenance of aspartate pools, a rate-limiting biosynthetic precursor. Conversely, depletion of extracellular asparagine with PEG-asparaginase sensitizes tumors to mitochondrial ... [truncated at 150 words]
Vallmitjana A, Lepanto P, Irigoin F, Malacrida L.
Phasor-based multi-harmonic unmixing for in-vivo hyperspectral imaging.
Methods Appl Fluoresc. 2022; 11(1), 014001.Hyperspectral imaging (HSI) is a paramount technique in biomedical science, however, unmixing and quantification of each spectral component is a challenging task. Traditional unmixing relies on algorithms that need spectroscopic parameters from the fluorescent species in the sample. The phasor-based multi-harmonic unmixing method requires only the empirical measurement of the pure species to compute the pixel-wise photon fraction of every spectral component. Using simulations, we demonstrate the feasibility of the approach for up to 5 components and explore the use of adding a 6th unknown component representing autofluorescence. The simulations show that the method can be successfully used in typical confocal imaging experiments (with pixel photon counts between 101 and 103). As a proof of concept, we tested the method in living cells, using 5 common commercial dyes for organelle labeling and we easily and accurately separate them. Finally, we challenged the method by introducing a solvatochromic probe, 6-Dodecanoyl-N,N-dimethyl-2-naphthylamine (LAURDAN), ... [truncated at 150 words]
Wang XX, Xie C, Libby AE, Ranjit S, Levi J, Myakala K, Bhasin K, Jones BA, Orlicky DJ, Takahashi S, Dvornikov A, Kleiner DE, Hewitt SM, Adorini L, Kopp JB, Krausz KW, Rosenberg A, McManaman JL, Robertson CE, Ir D, Frank DN, Luo Y, Gonzalez FJ, Gratton E, Levi M.
The role of FXR and TGR5 in reversing and preventing progression of Western diet-induced hepatic steatosis, inflammation, and fibrosis in mice.
J Biol Chem. 2022; 298(11), 102530. PMCID: PMC9638804Nonalcoholic steatohepatitis (NASH) is the most common chronic liver disease in the US, partly due to the increasing incidence of metabolic syndrome, obesity, and type 2 diabetes. The roles of bile acids and their receptors, such as the nuclear receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, on the development of NASH are not fully clear. C57BL/6J male mice fed a Western diet (WD) develop characteristics of NASH, allowing determination of the effects of FXR and TGR5 agonists on this disease. Here we show that the FXR-TGR5 dual agonist INT-767 prevents progression of WD-induced hepatic steatosis, inflammation, and fibrosis, as determined by histological and biochemical assays and novel label-free microscopy imaging techniques, including third harmonic generation, second harmonic generation, and fluorescence lifetime imaging microscopy. Furthermore, we show INT-767 decreases liver fatty acid synthesis and fatty acid and cholesterol uptake, as well as liver inflammation. INT-767 markedly changed ... [truncated at 150 words]
Rahim MK, Zhao J, Patel HV, Lagouros HA, Kota R, Fernandez I, Gratton E, Haun JB.
Phasor analysis of fluorescence lifetime enables quantitative multiplexed molecular imaging of three probes.
Anal Chem. 2022; 94(41): 14185-14194.The excited-state lifetime is an intrinsic property of fluorescent molecules that can be leveraged for multiplexed imaging. An advantage of fluorescence lifetime-based multiplexing is that signals from multiple probes can be gathered simultaneously, whereas traditional spectral fluorescence imaging typically requires multiple images at different excitation and emission wavelengths. Additionally, lifetime and spectra could both be utilized to expand the multiplexing capacity of fluorescence. However, resolving exogenous molecular probes based exclusively on the fluorescence lifetime has been limited by technical challenges in analyzing lifetime data. The phasor approach to lifetime analysis offers a simple, graphical solution that has increasingly been used to assess endogenous cellular autofluorescence to quantify metabolic factors. In this study, we employed the phasor analysis of FLIM to quantitatively resolve three exogenous, antibody-targeted fluorescent probes with similar spectral properties based on lifetime information alone. First, we demonstrated that three biomarkers that were spatially restricted to the cell membrane, ... [truncated at 150 words]
Guaglianone G, Torrado B, Lin YF, Watkins MC, Wysocki VH, Gratton E, Nowick JS.
Elucidating the oligomerization and cellular interactions of a trimer derived from Aβ through fluorescence and mass spectrometric studies.
ACS Chem Neurosci. 2022; 13(16): 2473-2482. PMCID: PMC9389591Aβ oligomers play a central role in the neurodegeneration observed with Alzheimer's disease. Our laboratory has developed covalently stabilized trimers derived from residues 17-36 of Aβ as model systems for studying Aβ oligomers. In the current study, we apply the emerging techniques of fluorescence lifetime imaging microscopy (FLIM) and native mass spectrometry (native MS) to better understand the assembly and interactions of the oligomer model system 2AT-L in aqueous solutions and with cells. 2AT-L and fluorescently labeled 2AT-L analogues assemble in the membrane-like environment of SDS-PAGE, showing diffuse bands of oligomers in equilibrium. Native ion mobility-mass spectrometry (native IM-MS) of 2AT-L allows for the identification of discrete oligomers in solution and shows similar patterns of oligomer formation between 2AT-L and fluorescently labeled analogues. Fluorescence microscopy with SH-SY5Y cells reveals that fluorescently labeled 2AT-L analogues colocalize within lysosomes. FLIM studies with phasor analysis further elucidate the assembly of 2AT-L within cells ... [truncated at 150 words]
Chun SK, Fortin BM, Fellows RC, Habowski AN, Verlande A, Song WA, Mahieu AL, Lefebvre AEYT, Sterrenberg JN, Velez LM, Digman MA, Edwards RA, Pannunzio NR, Seldin MM, Waterman ML, Masri S.
Disruption of the circadian clock drives Apc loss of heterozygosity to accelerate colorectal cancer.
Sci Adv. 2022; 8(32), eabo2389. PMCID: PMC9365282An alarming rise in young onset colorectal cancer (CRC) has been reported; however, the underlying molecular mechanism remains undefined. Suspected risk factors of young onset CRC include environmental aspects, such as lifestyle and dietary factors, which are known to affect the circadian clock. We find that both genetic disruption and environmental disruption of the circadian clock accelerate Apc-driven CRC pathogenesis in vivo. Using an intestinal organoid model, we demonstrate that clock disruption promotes transformation by driving Apc loss of heterozygosity, which hyperactivates Wnt signaling. This up-regulates c-Myc, a known Wnt target, which drives heightened glycolytic metabolism. Using patient-derived organoids, we show that circadian rhythms are lost in human tumors. Last, we identify that variance between core clock and Wnt pathway genes significantly predicts the survival of patients with CRC. Overall, our findings demonstrate a previously unidentified mechanistic link between clock disruption and CRC, which has important implications for young onset ... [truncated at 150 words]
Sallaberry I, Luszczak A, Philipp N, Navarro GSC, Gabriel MV, Gratton E, Gamarnik AV, Estrada LC.
Author Correction: In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells.
Sci Rep. 2022; 12(1): 11343. PMCID: PMC9256625No abstract available
Yao Z, Brennan CK, Scipioni L, Chen H, Ng KK, Tedeschi G, Parag-Sharma K, Amelio AL, Gratton E, Digman MA, Prescher JA.
Multiplexed bioluminescence microscopy via phasor analysis.
Nat Methods. 2022; 19(7): 893-898.Bioluminescence imaging with luciferase-luciferin pairs is a well-established technique for visualizing biological processes across tissues and whole organisms. Applications at the microscale, by contrast, have been hindered by a lack of detection platforms and easily resolved probes. We addressed this limitation by combining bioluminescence with phasor analysis, a method commonly used to distinguish spectrally similar fluorophores. We built a camera-based microscope equipped with special optical filters to directly assign phasor locations to unique luciferase-luciferin pairs. Six bioluminescent reporters were easily resolved in live cells, and the readouts were quantitative and instantaneous. Multiplexed imaging was also performed over extended time periods. Bioluminescent phasor further provided direct measures of resonance energy transfer in single cells, setting the stage for dynamic measures of cellular and molecular features. The merger of bioluminescence with phasor analysis fills a long-standing void in imaging capabilities, and will bolster future efforts to visualize biological events in real time ... [truncated at 150 words]
Tentori P, Signore G, Camposeo A, Carretta A, Ferri G, Pingue P, Luin S, Pozzi D, Gratton E, Beltram F, Caracciolo G, Cardarelli F.
Fluorescence lifetime microscopy unveils the supramolecular organization of liposomal Doxorubicin.
Nanoscale. 2022; 14(25): 8901-8905.The supramolecular organization of Doxorubicin (DOX) within the standard Doxoves® liposomal formulation (DOX®) is investigated using visible light and phasor approach to fluorescence lifetime imaging (phasor-FLIM). First, the phasor-FLIM signature of DOX® is resolved into the contribution of three co-existing fluorescent species, each with its characteristic mono-exponential lifetime, namely: crystallized DOX (DOXc, 0.2 ns), free DOX (DOXf, 1.0 ns), and DOX bound to the liposomal membrane (DOXb, 4.5 ns). Then, the exact molar fractions of the three species are determined by combining phasor-FLIM with quantitative absorption/fluorescence spectroscopy on DOXc, DOXf, and DOXb pure standards. The final picture on DOX® comprises most of the drug in the crystallized form (∼98%), with the remaining fractions divided between free (∼1.4%) and membrane-bound drug (∼0.7%). Finally, phasor-FLIM in the presence of a DOX dynamic quencher allows us to suggest that DOXf is both encapsulated and non-encapsulated, and that DOXb is present on both liposome-membrane ... [truncated at 150 words]
Solano A, Lou J, Scipioni L, Gratton E, Hinde E.
Radial pair correlation of molecular brightness fluctuations maps protein diffusion as a function of oligomeric state within live cell nuclear architecture.
Biophys J. 2022; 121(11): 2152-2167.Nuclear proteins can modulate their DNA binding activity and the exploration volume available during DNA target search by self-associating into higher order oligomers. Directly tracking this process in the nucleoplasm of a living cell is, however, a complex task. Thus, here we present a microscopy method based on radial pair correlation of molecular brightness fluctuations (radial pCOMB) that can extract the mobility of a fluorescently tagged nuclear protein as a function of its oligomeric state and spatiotemporally map the anisotropy of this parameter with respect to nuclear architecture. By simply performing a rapid frame scan acquisition, radial pCOMB has the capacity to detect within each pixel, protein oligomer formation and the size dependent obstruction nuclear architecture imparts on this complex's transport across sub-micron distances. From application of radial pCOMB to an oligomeric transcription factor and DNA repair protein, we demonstrate that homo-oligomer formation differentially regulates chromatin accessibility and interaction with ... [truncated at 150 words]
Morris MA, Vallmitjana A, Grein F, Schneider T, Arts M, Jones CR, Nguyen BT, Hashemian MH, Malek M, Gratton E, Nowick JS.
Visualizing the mode of action and supramolecular assembly of teixobactin analogues in Bacillus subtilis.
Chem Sci. 2022; 13(26): 7747-7754. PMCID: PMC9258396Teixobactin has been the source of intensive study and interest as a promising antibiotic, because of its excellent activity against drug-resistant Gram-positive pathogens and its novel but not yet fully understood mechanism of action that precludes drug resistance. Recent studies have demonstrated that the mode of action of teixobactin is more complicated than initially thought, with supramolecular assembly of the antibiotic appearing to play a critical role in the binding process. Further studies of the interactions of teixobactin with bacteria and its molecular targets offer the promise of providing deeper insights into its novel mechanism of action and guiding the design of additional drug candidates and analogues. The current study reports the preparation and study of teixobactin analogues bearing a variety of fluorophores. Structured illumination microscopy of the fluorescent teixobactin analogues with B. subtilis enables super-resolution visualization of the interaction of teixobactin with bacterial cell walls and permits the observation ... [truncated at 150 words]
Fazel M, Jazani S, Scipioni L, Vallmitjana A, Gratton E, Digman MA, Pressé S.
High resolution fluorescence lifetime maps from minimal photon counts.
ACS Photonics. 2022; 9(3): 1015-1025. PMCID: PMC9278809Fluorescence lifetime imaging microscopy (FLIM) may reveal subcellular spatial lifetime maps of key molecular species. Yet, such a quantitative picture of life necessarily demands high photon budgets at every pixel under the current analysis paradigm, thereby increasing acquisition time and photodamage to the sample. Motivated by recent developments in computational statistics, we provide a direct means to update our knowledge of the lifetime maps of species of different lifetimes from direct photon arrivals, while accounting for experimental features such as arbitrary forms of the instrument response function (IRF) and exploiting information from empty laser pulses not resulting in photon detection. Our ability to construct lifetime maps holds for arbitrary lifetimes, from short lifetimes (comparable to the IRF) to lifetimes exceeding interpulse times. As our method is highly data efficient, for the same amount of data normally used to determine lifetimes and photon ratios, working within the Bayesian paradigm, we report ... [truncated at 150 words]
Vorontsova I, Vallmitjana A, Torrado B, Schilling TF, Hall JE, Gratton E, Malacrida L.
In vivo macromolecular crowding is differentially modulated by aquaporin 0 in zebrafish lens: Insights from a nanoenvironment sensor and spectral imaging.
Sci Adv. 2022; 8(7). PMCID: PMC8849302Macromolecular crowding is crucial for cellular homeostasis. In vivo studies of macromolecular crowding and water dynamics are needed to understand their roles in cellular physiology and fate determination. Macromolecular crowding in the lens is essential for normal optics, and an understanding of its regulation will help prevent cataract and presbyopia. Here, we combine the use of the nanoenvironmental sensor [6-acetyl-2-dimethylaminonaphthalene (ACDAN)] to visualize lens macromolecular crowding with in vivo studies of aquaporin 0 zebrafish mutants that disrupt its regulation. Spectral phasor analysis of ACDAN fluorescence reveals water dipolar relaxation and demonstrates that mutations in two zebrafish aquaporin 0s, Aqp0a and Aqp0b, alter water state and macromolecular crowding in living lenses. Our results provide in vivo evidence that Aqp0a promotes fluid influx in the deeper lens cortex, whereas Aqp0b facilitates fluid efflux. This evidence reveals previously unidentified spatial regulation of macromolecular crowding and spatially distinct roles for Aqp0 in the lens.
Torrado B, Malacrida L, Ranjit S.
Linear combination properties of the phasor space in fluorescence imaging.
Sensors. 2022; 22(3), 999. PMCID: PMC8840623The phasor approach to fluorescence lifetime imaging, and more recently hyperspectral fluorescence imaging, has increased the use of these techniques, and improved the ease and intuitiveness of the data analysis. The fit-free nature of the phasor plots increases the speed of the analysis and reduces the dimensionality, optimization of data handling and storage. The reciprocity principle between the real and imaginary space—where the phasor and the pixel that the phasor originated from are linked and can be converted from one another—has helped the expansion of this method. The phasor coordinates calculated from a pixel, where multiple fluorescent species are present, depends on the phasor positions of those components. The relative positions are governed by the linear combination properties of the phasor space. According to this principle, the phasor position of a pixel with multiple components lies inside the polygon whose vertices are occupied by the phasor positions of these individual ... [truncated at 150 words]
Vu T, Vallmitjana A, Gu J, La K, Xu Q, Flores J, Zimak J, Shiu J, Hosohama L, Wu J, Douglas C, Waterman ML, Ganesan A, Hedde PN, Gratton E, Zhao W.
Spatial transcriptomics using combinatorial fluorescence spectral and lifetime encoding, imaging and analysis.
Nat Commun. 2022; 13(1), 169. PMCID: PMC8748653Multiplexed mRNA profiling in the spatial context provides new information enabling basic research and clinical applications. Unfortunately, existing spatial transcriptomics methods are limited due to either low multiplexing or complexity. Here, we introduce a spatialomics technology, termed Multi Omic Single-scan Assay with Integrated Combinatorial Analysis (MOSAICA), that integrates in situ labeling of mRNA and protein markers in cells or tissues with combinatorial fluorescence spectral and lifetime encoded probes, spectral and time-resolved fluorescence imaging, and machine learning-based decoding. We demonstrate MOSAICA's multiplexing scalability in detecting 10-plex targets in fixed colorectal cancer cells using combinatorial labeling of five fluorophores with facile error-detection and removal of autofluorescence. MOSAICA's analysis is strongly correlated with sequencing data (Pearson's r = 0.96) and was further benchmarked using RNAscopeTM and LGC StellarisTM. We further apply MOSAICA for multiplexed analysis of clinical melanoma Formalin-Fixed Paraffin-Embedded (FFPE) tissues. We finally demonstrate simultaneous co-detection of protein and mRNA in cancer ... [truncated at 150 words]
Sallaberry I, Luszczak A, Philipp N, Navarro GSC, Gabriel MV, Gratton E, Gamarnik AV, Estrada LC.
In vivo pair correlation microscopy reveals dengue virus capsid protein nucleocytoplasmic bidirectional movement in mammalian infected cells.
Sci Rep. 2021; 11(1), 24415. PMCID: PMC8709865Flaviviruses are major human disease-causing pathogens, including dengue virus (DENV), Zika virus, yellow fever virus and others. DENV infects hundreds of millions of people per year around the world, causing a tremendous social and economic burden. DENV capsid (C) protein plays an essential role during genome encapsidation and viral particle formation. It has been previously shown that DENV C enters the nucleus in infected cells. However, whether DENV C protein exhibits nuclear export remains unclear. By spatially cross-correlating different regions of the cell, we investigated DENV C movement across the nuclear envelope during the infection cycle. We observed that transport takes place in both directions and with similar translocation times (in the ms time scale) suggesting a bidirectional movement of both C protein import and export.Furthermore, from the pair cross-correlation functions in cytoplasmic or nuclear regions we found two populations of C molecules in each compartment with fast and slow ... [truncated at 150 words]
Ma D, Hernandez GA, Lefebvre AEYT, Alshetaiwi H, Blake K, Dave KR, Rauf M, Williams JW, Davis RT, Evans KT, Longworth A, Masoud MYG, Lee R, Edwards RA, Digman MA, Kessenbrock K, Lawson DA.
Patient-derived xenograft culture-transplant system for investigation of human breast cancer metastasis.
Commun Biol. 2021; 4(1): 1268. PMCID: PMC8571269Metastasis is a fatal disease where research progress has been hindered by a lack of authentic experimental models. Here, we develop a 3D tumor sphere culture-transplant system that facilitates the growth and engineering of patient-derived xenograft (PDX) tumor cells for functional metastasis assays in vivo. Orthotopic transplantation and RNA sequencing (RNA-seq) analyses show that PDX tumor spheres maintain tumorigenic potential, and the molecular marker and global transcriptome signatures of native tumor cells. Tumor spheres display robust capacity for lentiviral engineering and dissemination in spontaneous and experimental metastasis assays in vivo. Inhibition of pathways previously reported to attenuate metastasis also inhibit metastasis after sphere culture, validating our approach for authentic investigations of metastasis. Finally, we demonstrate a new role for the metabolic enzyme NME1 in promoting breast cancer metastasis, providing proof-of-principle that our culture-transplant system can be used for authentic propagation and engineering of patient tumor cells for functional studies of ... [truncated at 150 words]
Nicolai E, Pieri M, Gratton E, Motolese G, Bernardini S.
Bacterial infection diagnosis and antibiotic prescription in 3 h as an answer to antibiotic resistance: the case of urinary tract infections.
Antibiotics (Basel). 2021; 10(10): 1168. PMCID: PMC8532666Current methods for the diagnosis of urinary tract infections with antimicrobial susceptibility testing take 2-3 days and require a clinical laboratory. The lack of a rapid, point-of-care antibiotic susceptibility test (AST) has contributed to the misuse of antibiotics when treating urinary tract infections (UTIs) and consequently to the rise of multi-drug-resistant organisms. The current clinical approach has led to reduced treatment options and increased costs of diagnosis and therapy. To address this issue, novel diagnostics are needed for the timely determination of antimicrobial susceptibility. We present a rapid, point-of-care, phenotypic AST device that can report the antibiotic susceptibility/resistance of a uropathogen to a panel of antibiotics in as few as 3 h by utilizing fluorescent-labelling chemistry and a highly sensitive particle-counting instrument. We analysed 744 urine samples from the outpatients and inpatients of two Italian hospitals. The 130 UTI-positive patient urine samples we found were measured using a panel of ... [truncated at 150 words]
Shi B, Li W, Song Y, Wang Z, Ju R, Ulman A, Hu J, Palomba F, Zhao Y, Le JP, Jarrard W, Dimoff D, Digman MA, Gratton E, Zang C, Jiang H.
UTX condensation underlies its tumour-suppressive activity.
Nature. 2021; 597(7878): 726-731. PMCID: PMC9008583UTX (also known as KDM6A) encodes a histone H3K27 demethylase and is an important tumour suppressor that is frequently mutated in human cancers1. However, as the demethylase activity of UTX is often dispensable for mediating tumour suppression and developmental regulation2-8, the underlying molecular activity of UTX remains unknown. Here we show that phase separation of UTX underlies its chromatin-regulatory activity in tumour suppression. A core intrinsically disordered region (cIDR) of UTX forms phase-separated liquid condensates, and cIDR loss caused by the most frequent cancer mutation of UTX is mainly responsible for abolishing tumour suppression. Deletion, mutagenesis and replacement assays of the intrinsically disordered region demonstrate a critical role of UTX condensation in tumour suppression and embryonic stem cell differentiation. As shown by reconstitution in vitro and engineered systems in cells, UTX recruits the histone methyltransferase MLL4 (also known as KMT2D) to the same condensates and enriches the H3K4 methylation activity ... [truncated at 150 words]
Lefebvre AEYT, Ma D, Kessenbrock K, Lawson DA, Digman MA.
Automated segmentation and tracking of mitochondria in live-cell time-lapse images.
Nat Methods. 2021; 18(9): 1091-1102.Mitochondria display complex morphology and movements, which complicates their segmentation and tracking in time-lapse images. Here, we introduce Mitometer, an algorithm for fast, unbiased, and automated segmentation and tracking of mitochondria in live-cell two-dimensional and three-dimensional time-lapse images. Mitometer requires only the pixel size and the time between frames to identify mitochondrial motion and morphology, including fusion and fission events. The segmentation algorithm isolates individual mitochondria via a shape- and size-preserving background removal process. The tracking algorithm links mitochondria via differences in morphological features and displacement, followed by a gap-closing scheme. Using Mitometer, we show that mitochondria of triple-negative breast cancer cells are faster, more directional, and more elongated than those in their receptor-positive counterparts. Furthermore, we show that mitochondrial motility and morphology in breast cancer, but not in normal breast epithelia, correlate with metabolic activity. Mitometer is an unbiased and user-friendly tool that will help resolve fundamental questions regarding ... [truncated at 150 words]
Hedde PN, Cinco R, Malacrida L, Kamaid A, Gratton E.
Phasor-based hyperspectral snapshot microscopy allows fast imaging of live, three-dimensional tissues for biomedical applications.
Commun Biol. 2021; 4(1): 721. PMCID: PMC8195998Hyperspectral imaging is highly sought after in many fields including mineralogy and geology, environment and agriculture, astronomy and, importantly, biomedical imaging and biological fluorescence. We developed ultrafast phasor-based hyperspectral snapshot microscopy based on sine/cosine interference filters for biomedical imaging not feasible with conventional hyperspectral detection methods. Current approaches rely on slow spatial or spectral scanning limiting their application in living biological tissues, while faster snapshot methods such as image mapping spectrometry and multispectral interferometry are limited in spatial and/or spectral resolution, are computationally demanding, and imaging devices are very expensive to manufacture. Leveraging light sheet microscopy, phasor-based hyperspectral snapshot microscopy improved imaging speed 10-100 fold which, combined with minimal light exposure and high detection efficiency, enabled hyperspectral metabolic imaging of live, three-dimensional mouse tissues not feasible with other methods. As a fit-free method that does not require any a priori information often unavailable in complex and evolving biological systems, the ... [truncated at 150 words]
Torrado B, Dvornikov A, Gratton E.
Method of transmission filters to measure emission spectra in strongly scattering media.
Biomed Opt Express. 2021; 12(7): 3760-3774. PMCID: PMC8367243We describe a method based on a pair of transmission filters placed in the emission path of a microscope to resolve the emission wavelength of every point in an image. The method can be applied to any type of imaging device that provides the light in the wavelength transmission range of the filters. Unique characteristics of the filter approach are that the light does not need to be collimated and the wavelength response does not depend on the scattering of the sample or tissue. The pair of filters are used to produce the spectral phasor of the transmitted light, which is sufficient to perform spectral deconvolution over a broad wavelength range. The method is sensitive enough to distinguish free and protein-bound NADH and can be used in metabolic studies.
Tedeschi G, Scipioni L, Papanikolaou M, Abbott GW, Digman MA.
Fluorescence Fluctuation Spectroscopy enables quantification of potassium channel subunit dynamics and stoichiometry.
Sci Rep. 2021; 11(1), 10719. PMCID: PMC8140153Voltage-gated potassium (Kv) channels are a family of membrane proteins that facilitate K+ ion diffusion across the plasma membrane, regulating both resting and action potentials. Kv channels comprise four pore-forming α subunits, each with a voltage sensing domain, and they are regulated by interaction with β subunits such as those belonging to the KCNE family. Here we conducted a comprehensive biophysical characterization of stoichiometry and protein diffusion across the plasma membrane of the epithelial KCNQ1-KCNE2 complex, combining total internal reflection fluorescence (TIRF) microscopy and a series of complementary Fluorescence Fluctuation Spectroscopy (FFS) techniques. Using this approach, we found that KCNQ1-KCNE2 has a predominant 4:4 stoichiometry, while non-bound KCNE2 subunits are mostly present as dimers in the plasma membrane. At the same time, we identified unique spatio-temporal diffusion modalities and nano-environment organization for each channel subunit. These findings improve our understanding of KCNQ1-KCNE2 channel function and suggest strategies for elucidating the ... [truncated at 150 words]
Vallmitjana A, Torrado B, Gratton E.
Phasor-based image segmentation: machine learning clustering techniques.
Biomed Opt Express. 2021; 12(6): 410-3422. PMCID: PMC8221971The phasor approach is a well-established method for data visualization and image analysis in spectral and lifetime fluorescence microscopy. Nevertheless, it is typically applied in a user-dependent manner by manually selecting regions of interest on the phasor space to find distinct regions in the fluorescence images. In this paper we present our work on using machine learning clustering techniques to establish an unsupervised and automatic method that can be used for identifying populations of fluorescent species in spectral and lifetime imaging. We demonstrate our method using both synthetic data, created by sampling photon arrival times and plotting the distributions on the phasor plot, and real live cells samples, by staining cellular organelles with a selection of commercial probes.
Malacrida L, Ranjit S, Jameson DM, Gratton E.
The phasor plot: a universal circle to advance fluorescence lifetime analysis and interpretation.
Annu Rev Biophys. 2021; 50: 575-593.The phasor approach to fluorescence lifetime imaging has become a common method to analyze complicated fluorescence signals from biological samples. The appeal of the phasor representation of complex fluorescence decays in biological systems is that a visual representation of the decay of entire cells or tissues can be used to easily interpret fundamental biological states related to metabolism and oxidative stress. Phenotyping based on autofluorescence provides new avenues for disease characterization and diagnostics. The phasor approach is a transformation of complex fluorescence decays that does not use fits to model decays and therefore has the same information content as the original data. The phasor plot is unique for a given system, is highly reproducible, and provides a robust method to evaluate the existence of molecular interactions such as Förster resonance energy transfer or the response of ion indicators. Recent advances permitquantification of multiple components from phasor plots in fluorescence lifetime ... [truncated at 150 words]
Scipioni L, Rossetta A, Tedeschi G, Gratton E.
Phasor S-FLIM: a new paradigm for fast and robust spectral fluorescence lifetime imaging.
Nat Methods. 2021; 18(5): 542-550.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 acquisition and processing. By integrating technological and computational advances, we developed a robust and unbiased spectral FLIM (S-FLIM) system. Our method, Phasor S-FLIM, combines true parallel multichannel digital frequency domain electronics with a multidimensional phasor approach to extract detailed and precise information about the photophysics of fluorescent specimens at optical resolution. To show the flexibility of the Phasor S-FLIM technology and its applications to the biological and biomedical field, we address four common, yet challenging, problems: the blind unmixing of spectral and lifetime signatures from multiple unknown species, the unbiased bleedthrough- and background-free Förster resonance energy transfer analysis of biosensors, the photophysical characterization of environment-sensitive probes in living cells and parallel four-color FLIM imaging in tumor spheroids.
Gunther G, Malacrida L, Jameson DM, Gratton E, Sánchez SA.
LAURDAN since Weber: The quest for visualizing membrane heterogeneity.
Acc Chem Res. 2021; 54(4): 976-987. PMCID: PMC8552415Conspectus Any chemist studying the interaction of molecules with lipid assemblies will eventually be confronted by the topic of membrane bilayer heterogeneity and may ultimately encounter the heterogeneity of natural membranes. In artificial bilayers, heterogeneity is defined by phase segregation that can be in the nano- and micrometer range. In biological bilayers, heterogeneity is considered in the context of small (10-200 nm) sterol and sphingolipid-enriched heterogeneous and highly dynamic domains. Several techniques can be used to assess membrane heterogeneity in living systems. Our approach is to use a fluorescent reporter molecule immersed in the bilayer, which, by changes in its spectroscopic properties, senses physical-chemistry aspects of the membrane. This dye in combination with microscopy and fluctuation techniques can give information about membrane heterogeneity at different temporal and spatial levels: going from average fluidity to number and diffusion coefficient of nanodomains. LAURDAN (6-dodecanoyl-2-(dimethylamino) naphthalene), is a fluorescent probe designed and synthesized ... [truncated at 150 words]
Ranjit S, Lanzanò L, Libby AE, Gratton E, Levi M.
Advances in fluorescence microscopy techniques to study kidney function.
Nat Rev Nephrol. 2021; 17(2): 128-144.Fluorescence microscopy, in particular immunofluorescence microscopy, has been used extensively for the assessment of kidney function and pathology for both research and diagnostic purposes. The development of confocal microscopy in the 1950s enabled imaging of live cells and intravital imaging of the kidney; however, confocal microscopy is limited by its maximal spatial resolution and depth. More recent advances in fluorescence microscopy techniques have enabled increasingly detailed assessment of kidney structure and provided extraordinary insights into kidney function. For example, nanoscale precise imaging by rapid beam oscillation (nSPIRO) is a super-resolution microscopy technique that was originally developed for functional imaging of kidney microvilli and enables detection of dynamic physiological events in the kidney. A variety of techniques such as fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and Förster resonance energy transfer (FRET) enable assessment of interaction between proteins. The emergence of other super-resolution techniques, including super-resolution stimulated emission depletion ... [truncated at 150 words]
Sameni S, Zhang R, Digman MA.
The phasor FLIM method reveals a link between a change in energy metabolism and mHtt protein spread in healthy mammalian cells when co-cultured with Huntington diseased cells.
Methods Appl Fluoresc. 2021; 9(1), 015005.Huntington Disease (HD) is a late-onset autosomal neurodegenerative disease characterized by the aggregations of mutant Huntingtin proteins (mHTT). A glutamine stretch (PolyQ) at the N-terminal of the Huntingtin protein is generated by the abnormal expansion of CAG trinucleotide repeats in exon 1 of the HTT gene. While the resulting polyQ aggregates are the predominate feature of HD , the intercellular spread of the expanded protein and the effect upon this transfer inside healthy cells have not yet fully understood. Here, we have employed the phasor Fluorescence Lifetime Imaging Microscopy (FLIM) method to measure NADH fluorescence lifetime change after the internalization of the PolyQ protein. Based on our analysis, we have found a significant decrease in the fraction of bound NADH in both cytoplasmic and nucleus regions when cells are co-cultured or when healthy cells uptake the supernatant containing polyQ proteins and aggregates. Overall, our FLIM study combined with confocal fluorescence ... [truncated at 150 words]
Leemans SW, Dvornikov AS, Gallagher T, Gratton E.
AO DIVER: Development of a three-dimensional adaptive optics system to advance the depth limits of multiphoton imaging.
APL Photonics. 2020; 5(12), 120801.Multiphoton microscopy (MPM) can non-invasively measure the dynamic biochemical properties deep in scattering biological samples and has the potential to accelerate clinical research with advances in deep tissue imaging. However, in most samples, the imaging depth of MPM is limited to fractions of a millimeter due to blurring caused by refractive index mismatching throughout tissue and background fluorescence, overshadowing the signal in conventional MPM. To overcome these challenges, we developed a novel 3D adaptive optics (AO) system that uses an interpolated network of endogenous guide stars to focus laser light more efficiently into highly scattering samples. The synergistic combination of our AO system with DIVER detection technology enables millimeter-scale imaging with diffraction-limited resolution with optimization times between 15 s and 65 s. We characterized the algorithm and wavefront interpolation performance in a flat 2D sample and in 3D using fluorescent beads embedded in gels of various optical heterogeneity. We also ... [truncated at 150 words]
Greco CM, Cervantes M, Fustin JM, Ito K, Ceglia N, Samad M, Shi J, Koronowski KB, Forne I, Ranjit S, Gaucher J, Kinouchi K, Kojima R, Gratton E, Li W, Baldi P, Imhof A, Okamura H, Sassone-Corsi P.
S-adenosyl-l-homocysteine hydrolase links methionine metabolism to the circadian clock and chromatin remodeling.
Sci Adv. 2020; 6(51), eabc5629. PMCID: PMC7744083Circadian gene expression driven by transcription activators CLOCK and BMAL1 is intimately associated with dynamic chromatin remodeling. However, how cellular metabolism directs circadian chromatin remodeling is virtually unexplored. We report that the S-adenosylhomocysteine (SAH) hydrolyzing enzyme adenosylhomocysteinase (AHCY) cyclically associates to CLOCK-BMAL1 at chromatin sites and promotes circadian transcriptional activity. SAH is a potent feedback inhibitor of S-adenosylmethionine (SAM)-dependent methyltransferases, and timely hydrolysis of SAH by AHCY is critical to sustain methylation reactions. We show that AHCY is essential for cyclic H3K4 trimethylation, genome-wide recruitment of BMAL1 to chromatin, and subsequent circadian transcription. Depletion or targeted pharmacological inhibition of AHCY in mammalian cells markedly decreases the amplitude of circadian gene expression. In mice, pharmacological inhibition of AHCY in the hypothalamus alters circadian locomotor activity and rhythmic transcription within the suprachiasmatic nucleus. These results reveal a previously unappreciated connection between cellular metabolism, chromatin dynamics, and circadian regulation.
Vallmitjana A, Torrado B, Dvornikov A, Ranjit S, Gratton E.
Blind resolution of lifetime components in individual pixels of fluorescence lifetime images using the phasor approach.
J Phys Chem B. 2020; 124(45): 10126-10137.The phasor approach is used in fluorescence lifetime imaging microscopy for several purposes, notably to calculate the metabolic index of single cells and tissues. An important feature of the phasor approach is that it is a fit-free method allowing immediate and easy to interpret analysis of images. In a recent paper, we showed that three or four intensity fractions of exponential components can be resolved in each pixel of an image by the phasor approach using simple algebra, provided the component phasors are known. This method only makes use of the rule of linear combination of phasors rather than fits. Without prior knowledge of the components and their single exponential decay times, resolution of components and fractions is much more challenging. Blind decomposition has been carried out only for cuvette experiments wherein the statistics in terms of the number of photons collected is very good. In this paper, we show ... [truncated at 150 words]
Makaremi S, Rose M, Ranjit S, Digman MA, Bowdish DME, Moran-Mirabal JM.
Lateral diffusion of CD14 and TLR2 in macrophage plasma membrane assessed by raster image correlation spectroscopy and single particle tracking.
Sci Rep. 2020; 10(1): 19375. PMCID: PMC7652837The diffusion of membrane receptors is central to many biological processes, such as signal transduction, molecule translocation, and ion transport, among others; consequently, several advanced fluorescence microscopy techniques have been developed to measure membrane receptor mobility within live cells. The membrane-anchored receptor cluster of differentiation 14 (CD14) and the transmembrane toll-like receptor 2 (TLR2) are important receptors in the plasma membrane of macrophages that activate the intracellular signaling cascade in response to pathogenic stimuli. The aim of the present work was to compare the diffusion coefficients of CD14 and TLR2 on the apical and basal membranes of macrophages using two fluorescence-based methods: raster image correlation spectroscopy (RICS) and single particle tracking (SPT). In the basal membrane, the diffusion coefficients obtained from SPT and RICS were found to be comparable and revealed significantly faster diffusion of CD14 compared with TLR2. In addition, RICS showed that the diffusion of both receptors was ... [truncated at 150 words]
Ranjit S, Henriksen K, Dvornikov A, Delsante M, Rosenberg A, Levi M, Gratton E.
Phasor approach to autofluorescence lifetime imaging FLIM can be a qantitative biomarker of chronic renal Parenchymal injury.
Kidney Int. 2020; 98(5): 1341-1346. PMCID: PMC7606347Diabetic kidney disease continues to be the leading cause of chronic kidney disease, often advancing to end stage kidney disease. In addition to the well characterized glomerular alterations including mesangial expansion, podocyte injury, and glomerulosclerosis, tubulointerstitial fibrosis is also an important component of diabetic kidney injury. Similarly, tubulointerstitial fibrosis is a critical component of any chronic kidney injury. Therefore, sensitive and quantitative identification of tubulointerstitial fibrosis is critical for the assessment of long-term prognosis of kidney disease. Here, we employed phasor approach to fluorescence lifetime imaging, commonly known as FLIM, to understand tissue heterogeneity and calculate changes in the tissue autofluorescence lifetime signatures due to diabetic kidney disease. FLIM imaging was performed on cryostat sections of snap-frozen biopsy material of patients with diabetic nephropathy. There was an overall increase in phase lifetime (τphase) with increased disease severity. Multicomponent phasor analysis shows the distinctive differences between the different disease states. Thus, ... [truncated at 150 words]
Chen YC, Sood C, Marin M, Aaron J, Gratton E, Salaita K, Melikyan GB.
Super-resolution fluorescence imaging reveals that Serine incorporator protein 5 inhibits human immunodeficiency virus fusion by disrupting envelope glycoprotein clusters.
ACS Nano. 2020; 14(9): 10929-10943. PMCID: PMC8274448Serine incorporator protein 5 (SERINC5) is the host anti-retroviral factor that reduces HIV-1 infectivity by incorporating into virions and inhibiting the envelope glycoprotein (Env) mediated virus fusion with target cells. We and others have shown that SERINC5 incorporation into virions alters the Env structure and sensitizes the virus to broadly neutralizing antibodies targeting cryptic Env epitopes. We have also found that SERINC5 accelerates the loss of Env function over time compared to control viruses. However, the exact mechanism by which SERINC5 inhibits HIV-1 fusion is not understood. Here, we utilized 2D and 3D super-resolution microscopy to examine the effect of SERINC5 on the distribution of Env glycoproteins on single HIV-1 particles. We find that, in agreement with a previous report, Env glycoproteins form clusters on the surface of mature virions. Importantly, incorporation of SERINC5, but not SERINC2, which lacks antiviral activity, disrupted Env clusters without affecting the overall Env content. ... [truncated at 150 words]
Hedde PN, Abram TJ, Jain A, Nakajima R, de Assis RR, Pearce T, Jasinskas A, Toosky MN, Khan S, Felgner PL, Gratton E, Zhao W.
A modular microarray imaging system for highly specific COVID-19 antibody testing.
Lab Chip. 2020; 20(18): 3302-3309. PMCID: PMC7263497To detect the presence of antibodies in blood against SARS-CoV-2 in a highly sensitive and specific manner, here we describe a robust, inexpensive ($200), 3D-printable portable imaging platform (TinyArray imager) that can be deployed immediately in areas with minimal infrastructure to read coronavirus antigen microarrays (CoVAMs) that contain a panel of antigens from SARS-CoV-2, SARS-1, MERS, and other respiratory viruses. Application includes basic laboratories and makeshift field clinics where a few drops of blood from a finger prick could be rapidly tested in parallel for the presence of antibodies to SARS-CoV-2 with a test turnaround time of only 2-4 h. To evaluate our imaging device, we probed and imaged coronavirus microarrays with COVID-19-positive and negative sera and achieved a performance on par with a commercial microarray reader 100× more expensive than our imaging device. This work will enable large scale serosurveillance, which can play an important role in the months ... [truncated at 150 words]
Harris CR, Millman KJ, van der Walt SJ, Gommers R, Virtanen P, Cournapeau D, Wieser E, Taylor J, Berg S, Smith NJ, Kern R, Picus M, Hoyer S, van Kerkwijk MH, Brett M, Haldane A, Río JFd, Wiebe M, Peterson P, Gérard-Marchant P, Sheppard K, Reddy T, Weckesser W, Abbasi H, Gohlke C, Oliphant TE.
Array programming with NumPy.
Nature. 2020; 585(7825): 357-362. PMCID: PMC7759461Array programming provides a powerful, compact and expressive syntax for accessing, manipulating and operating on data in vectors, matrices and higher-dimensional arrays. NumPy is the primary array programming library for the Python language. It has an essential role in research analysis pipelines in fields as diverse as physics, chemistry, astronomy, geoscience, biology, psychology, materials science, engineering, finance and economics. For example, in astronomy, NumPy was an important part of the software stack used in the discovery of gravitational waves1 and in the first imaging of a black hole2. Here we review how a few fundamental array concepts lead to a simple and powerful programming paradigm for organizing, exploring and analysing scientific data. NumPy is the foundation upon which the scientific Python ecosystem is constructed. It is so pervasive that several projects, targeting audiences with specialized needs, have developed their own NumPy-like interfaces and array objects. Owing to its central position ... [truncated at 150 words]
Palczewska G, Boguslawski J, Stremplewsk P, Kornaszewski L, Zhang J, Dong Z, Liang XX, Gratton E, Vogel A, Wojtkowski M, Palczewski K.
Noninvasive two-photon optical biopsy of retinal fluorophores.
Proc Natl Acad Sci USA. 2020; 117(36): 22532-22543. PMCID: PMC7486747High-resolution imaging techniques capable of detecting identifiable endogenous fluorophores in the eye along with genetic testing will dramatically improve diagnostic capabilities in the ophthalmology clinic and accelerate the development of new treatments for blinding diseases. Two-photon excitation (TPE)-based imaging overcomes the filtering of ultraviolet light by the lens of the human eye and thus can be utilized to discover defects in vitamin A metabolism during the regeneration of the visual pigments required for the detection of light. Combining TPE with fluorescence lifetime imaging (FLIM) and spectral analyses offers the potential of detecting diseases of the retina at earlier stages before irreversible structural damage has occurred. The main barriers to realizing the benefits of TPE for imaging the human retina arise from concerns about the high light exposure typically needed for informative TPE imaging and the requirement to correlate the ensuing data with different states of health and disease. To overcome ... [truncated at 150 words]
Li W, Hu J, Sh B, Palomba F, Digman MA, Gratton E, Jiang H.
Biophysical properties of AKAP95 protein condensates regulate splicing and tumorigenesis.
Nat Cell Biol. 2020; 22(8): 960-972. PMCID: PMC7425812It remains unknown if biophysical or material properties of biomolecular condensates regulate cancer. Here we show that AKAP95, a nuclear protein that regulates transcription and RNA splicing, plays an important role in tumorigenesis by supporting cancer cell growth and suppressing oncogene-induced senescence. AKAP95 forms phase-separated and liquid-like condensates in vitro and in nucleus. Mutations of key residues to different amino acids perturb AKAP95 condensation in opposite directions. Importantly, the activity of AKAP95 in splice regulation is abolished by disruption of condensation, significantly impaired by hardening of condensates, and regained by substituting its condensation-mediating region with other condensation-mediating regions from irrelevant proteins. Moreover, the abilities of AKAP95 in regulating gene expression and supporting tumorigenesis require AKAP95 to form condensates with proper liquidity and dynamicity. These results link phase separation to tumorigenesis and uncover an important role of appropriate biophysical properties of protein condensates in gene regulation and cancer.
Malacrida L, Hedde PN, Torrado B, Gratton E.
Barriers to diffusion in cells: visualization of membraneless particles in the nucleus.
Biophysicist (Rockv). 2020; 1(2): 9. PMCID: PMC9000293Transient barriers are fundamental to cell supramolecular organization and assembly. Discontinuities between spaces can be generated by a physical barrier but also by thermodynamic barriers achieved by phase separation of molecules. However, because of the transient nature and the lack of a visible barrier, the existence of phase separation is difficult to demonstrate experimentally. We describe an approach based on the 2-dimensional pair correlation function (2D-pCF) analysis of the spatial connectivity in a cell. The educational aim of the article is to present both a model suitable for explaining diffusion barrier measurements to a broad range of courses and examples of biological situations. If there are no barriers to diffusion, particles could diffuse equally in all directions. In this situation the pair correlation function introduced in this article is independent of the direction and is uniform in all directions. However, in the presence of obstacles, the shape of the 2D-pCF ... [truncated at 150 words]
Hedde PN, Bouzin M, Abram TJ, Chen X, Toosky MN, Vu T, Li Y, Zhao W, Gratton E.
Rapid isolation of rare targets from large fluid volumes.
Sci Rep. 2020; 10(1), 12458. PMCID: PMC7385493apidly isolating rare targets from larger, clinically relevant fluid volumes remains an unresolved problem in biomedicine and diagnosis. Here, we describe how 3D particle sorting can enrich targets at ultralow concentrations over 100-fold within minutes not possible with conventional approaches. Current clinical devices based on biochemical extraction and microfluidic solutions typically require high concentrations and/or can only process sub-milliliter volumes in time. In a proof-of-concept application, we isolated bacteria from whole blood as demanded for rapid sepsis diagnosis where minimal numbers of bacteria need to be found in a 1–10 mL blood sample. After sample encapsulation in droplets and target enrichment with the 3D particle sorter within a few minutes, downstream analyses were able to identify bacteria and test for antibiotic susceptibility, information which is critical for successful treatment of bloodstream infections.
Cervantes M, Forné I, Ranjit S, Gratton E, Imhof A, Sassone-Corsi P.
BMAL1 associates with NOP58 in the nucleolus and contributes to pre-rRNA processing.
iScience. 2020; 23(6), 101151. PMCID: PMC7256328The transcription factor BMAL1 is a core element of the circadian clock that contributes to cyclic control of genes transcribed by RNA polymerase II. By using biochemical cellular fractionation and immunofluorescence analyses we reveal a previously uncharacterized nucleolar localization for BMAL1. We used an unbiased approach to determine the BMAL1 interactome by mass spectrometry and identified NOP58 as a prominent nucleolar interactor. NOP58, a core component of the box C/D small nucleolar ribonucleoprotein complex, associates with Snord118 to control specific pre-ribosomal RNA (pre-rRNA) processing steps. These results suggest a non-canonical role of BMAL1 in ribosomal RNA regulation. Indeed, we show that BMAL1 controls NOP58-associated Snord118 nucleolar levels and cleavage of unique pre-rRNA intermediates. Our findings identify an unsuspected function of BMAL1 in the nucleolus that appears distinct from its canonical role in the circadian clock system.
Vallmitjana A, Dvornikov A, Torrado B, Jameson DM, Ranjit S, Gratton E.
Resolution of 4 components in the same pixel in FLIM images using the phasor approach.
Methods Appl Fluoresc. 2020; 8(3), 035001.In several cellular systems, the phasor FLIM approach has shown the existence of more than 2 components in the same pixel, a typical example being free and bound NADH. In order to properly quantify the concentrations and the spatial distributions of fluorescence components associated with different molecular species we developed a general method to resolve 3 and 4 components in the same pixel using the phasor approach. The method is based on the law of linear combination of components valid after transformation of the decay curves to phasors for each pixel in the image. In principle, the linear combination rule is valid for an arbitrary number of components. For 3 components we use only the phasor position for the first harmonic, which has a small error, while for 4 components we need the phasor location at higher harmonics that have intrinsically more noise. As a result of the noise in ... [truncated at 150 words]
Takahashi S, Luo Y, Ranjit S, Xie C, Libby AE, Orlicky DJ, Dvornikov A, Wang XX, Myakala K, Jones BA, Bhasin K, Wang D, McManaman JL, Krausz KW, Gratton E, Ir D, Robertson CE, Frank DN, Gonzalez FJ, Levi M.
Bile acid sequestration reverses liver injury and prevents progression of NASH in western diet-fed mice.
J Biol Chem. 2020; 295(14): 4733-4747. PMCID: PMC7135973Non-alcoholic fatty liver disease (NAFLD) is a rapidly rising problem in the 21st century and is a leading cause of chronic liver disease that can lead to end-stage liver diseases, including cirrhosis and hepatocellular cancer. Despite this rising epidemic, no pharmacological treatment has yet been established to treat this disease. The rapidly increasing prevalence of NAFLD and its aggressive form, nonalcoholic steatohepatitis (NASH), requires novel therapeutic approaches to prevent disease progression. Alterations in microbiome dynamics and dysbiosis play an important role in liver disease, and these may represent targetable pathways to treat liver disorders. Improving microbiome properties or restoring normal bile acid metabolism may prevent or slow the progression of liver diseases such as NASH. Importantly, aberrant systemic circulation of bile acids can greatly disrupt metabolic homeostasis. Bile acid sequestrants (BAS) are orally administered polymers that bind bile acids in the intestine forming nonabsorbable complexes. BAS interrupts intestinal ... [truncated at 150 words]
Planes N, Vanderheyden PPML, Gratton E, Caballero-George C.
Image mean square displacement to study the lateral mobility of Angiotensin II type 1 and Endothelin 1 type A receptors on living cells.
Microsc Res Tech. 2020; 83(4): 381-392.The lateral mobility of membrane receptors provides insights into the molecular interactions of protein binding and the complex dynamic plasma membrane. The image mean square displacement (iMSD) analysis is a method used to extract qualitative and quantitative information of the protein diffusion law and infers how diffusion dynamic processes may change when the cellular environment is modified. The aim of the study was to describe the membrane diffusing properties of two G-protein-coupled receptors namely Angiotensin II type 1 (AT1 ) and Endothelin 1 type A (ETA ) receptors and their corresponding receptor-ligand complexes in living cells using total internal reflection fluorescent microscopy and iMSD analysis. This study showed that both AT1 and ETA receptors displayed a mix of three modes of diffusion: free, confined, and partially confined. The confined mode was the predominant at the plasma membrane of living cells and was not affected by ligand binding. However, the local ... [truncated at 150 words]
Pascua SM, McGahey GE, Ma N, Wang JJ, Digman MA.
Caffeine and Cisplatin effectively targets the metabolism of a triple-negative breast cancer cell line assessed via Phasor-FLIM.
Int J Mol Sci. 2020; 21(7), E2443. PMCID: PMC7177700Triple-negative tumor cells, a malignant subtype of breast cancer, lack a biologically targeted therapy. Given its DNA repair inhibiting properties, caffeine has been shown to enhance the effectiveness of specific tumor chemotherapies. In this work, we have investigated the effects of caffeine, cisplatin, and a combination of the two as potential treatments in energy metabolism for three cell lines, triple-negative breast cancer (MDA-MB-231), estrogen-receptor lacking breast cancer (MCF7) and breast epithelial cells (MCF10A) using a sensitive label-free approach, phasor-fluorescence lifetime imaging microscopy (phasor-FLIM). We found that solely using caffeine to treat MDA-MB-231 shifts their metabolism towards respiratory-chain phosphorylation with a lower ratio of free to bound NADH, and a similar trend is seen in MCF7. However, MDA-MB-231 cells shifted to a higher ratio of free to bound NADH when cisplatin was added. The combination of cisplatin and caffeine together reduced the survival rate for MDA-MD231 and shifted their energy metabolism ... [truncated at 150 words]
Haensel D, Jin S, Sun P, Cinco R, Dragan M, Nguyen Q, Cang Z, Gong Y, Vu R, MacLean AL, Kessenbrock K, Gratton E, Nie Q, Dai X.
Defining epidermal basal cell states during skin homeostasis and wound healing using single-cell transcriptomics.
Cell Rep. 2020; 30(11): 3932-3947.e6. PMCID: PMC7218802Our knowledge of transcriptional heterogeneities in epithelial stem and progenitor cell compartments is limited. Epidermal basal cells sustain cutaneous tissue maintenance and drive wound healing. Previous studies have probed basal cell heterogeneity in stem and progenitor potential, but a comprehensive dissection of basal cell dynamics during differentiation is lacking. Using single-cell RNA sequencing coupled with RNAScope and fluorescence lifetime imaging, we identify three non-proliferative and one proliferative basal cell state in homeostatic skin that differ in metabolic preference and become spatially partitioned during wound re-epithelialization. Pseudotemporal trajectory and RNA velocity analyses predict a quasi-linear differentiation hierarchy where basal cells progress from Col17a1Hi/Trp63Hi state to early-response state, proliferate at the juncture of these two states, or become growth arrested before differentiating into spinous cells. Wound healing induces plasticity manifested by dynamic basal-spinous interconversions at multiple basal transcriptional states. Our study provides a systematic view of epidermal cellular dynamics, supporting a revised ... [truncated at 150 words]
Liang Z, Lou J, Scipioni L, Gratton E, Hinde E.
Quantifying nuclear wide chromatin compaction by phasor analysis of histone Förster resonance energy transfer (FRET) in frequency domain fluorescence lifetime imaging microscopy (FLIM) data.
Data Brief. 2020; 30, 105401. PMCID: PMC7152662The nanometer spacing between nucleosomes throughout global chromatin organisation modulates local DNA template access, and through continuous dynamic rearrangements, regulates genome function [1]. However, given that nucleosome packaging occurs on a spatial scale well below the diffraction limit, real time observation of chromatin structure in live cells by optical microscopy has proved technically difficult, despite recent advances in live cell super resolution imaging [2]. One alternative solution to quantify chromatin structure in a living cell at the level of nucleosome proximity is to measure and spatially map Förster resonance energy transfer (FRET) between fluorescently labelled histones - the core protein of a nucleosome [3]. 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 in the presence of cellular autofluorescence [4]. Here we share FLIM data recording histone ... [truncated at 150 words]
Perinbam K, Chacko JV, Kannan A, Digman MA, Siryaporn A.
A shift in central metabolism accompanies virulence activation in Pseudomonas aeruginosa.
mBio. 2020; 11(2), e02730-18. PMCID: PMC7064766The availability of energy has significant impact on cell physiology. However, the role of cellular metabolism in bacterial pathogenesis is not understood. We investigated the dynamics of central metabolism during virulence induction by surface sensing and quorum sensing in early-stage biofilms of the multidrug-resistant bacterium Pseudomonas aeruginosa We established a metabolic profile for P. aeruginosa using fluorescence lifetime imaging microscopy (FLIM), which reports the activity of NADH in live cells. We identified a critical growth transition period during which virulence is activated. We performed FLIM measurements and direct measurements of NADH and NAD+ concentrations during this period. Here, planktonic (low-virulence) and surface-attached (virulence-activated) populations diverged into distinct metabolic states, with the surface-attached population exhibiting FLIM lifetimes that were associated with lower levels of enzyme-bound NADH and decreasing total NAD(H) production. We inhibited virulence by perturbing central metabolism using citrate and pyruvate, which further decreased the enzyme-bound NADH fraction and total ... [truncated at 150 words]
Davis RT, Blake K, Ma D, Gabra MBI, Hernandez GA, Phung AT, Yang Y, Maurer D, Lefebvre AEYT, Alshetaiwi H, Xiao Z, Liu J, Locasale JW, Digman MA, Mjolsness E, Kong M, Werb Z, Lawson DA.
Transcriptional diversity and bioenergetic shift in human breast cancer metastasis revealed by single-cell RNA sequencing.
Nat Cell Biol. 2020; 22(3): 310-320.Although metastasis remains the cause of most cancer-related mortality, mechanisms governing seeding in distal tissues are poorly understood. Here, we establish a robust method for the identification of global transcriptomic changes in rare metastatic cells during seeding using single-cell RNA sequencing and patient-derived-xenograft models of breast cancer. We find that both primary tumours and micrometastases display transcriptional heterogeneity but micrometastases harbour a distinct transcriptome program conserved across patient-derived-xenograft models that is highly predictive of poor survival of patients. Pathway analysis revealed mitochondrial oxidative phosphorylation as the top pathway upregulated in micrometastases, in contrast to higher levels of glycolytic enzymes in primary tumour cells, which we corroborated by flow cytometric and metabolomic analyses. Pharmacological inhibition of oxidative phosphorylation dramatically attenuated metastatic seeding in the lungs, which demonstrates the functional importance of oxidative phosphorylation in metastasis and highlights its potential as a therapeutic target to prevent metastatic spread in patients with breast ... [truncated at 150 words]
Titlow J, Robertson F, Järvelin A, Ish-Horowicz D, Smith C, Gratton E, Davis I.
Syncrip/hnRNP Q is required for activity-induced Msp300/Nesprin-1 expression and new synapse formation.
J Cell Biol. 2020; 219(3), e201903135. PMCID: PMC7055005Memory and learning involve activity-driven expression of proteins and cytoskeletal reorganization at new synapses, requiring posttranscriptional regulation of localized mRNA a long distance from corresponding nuclei. A key factor expressed early in synapse formation is Msp300/Nesprin-1, which organizes actin filaments around the new synapse. How Msp300 expression is regulated during synaptic plasticity is poorly understood. Here, we show that activity-dependent accumulation of Msp300 in the postsynaptic compartment of the Drosophila larval neuromuscular junction is regulated by the conserved RNA binding protein Syncrip/hnRNP Q. Syncrip (Syp) binds to msp300 transcripts and is essential for plasticity. Single-molecule imaging shows that msp300 is associated with Syp in vivo and forms ribosome-rich granules that contain the translation factor eIF4E. Elevated neural activity alters the dynamics of Syp and the number of msp300:Syp:eIF4E RNP granules at the synapse, suggesting that these particles facilitate translation. These results introduce Syp as an important early acting activity-dependent regulator ... [truncated at 150 words]
Tetreau G, Banneville AS, Andreeva EA, Brewster AS, Hunter MS, Sierra RG, Teulon JM, Young ID, Burke N, Grünewald TA, Beaudouin J, Snigireva I, Fernandez-Luna MT, Burt A, Park HW, Signor L, Bafna JA, Sadir R, Fenel D, Boeri-Erba E, Bacia M, Zala N, Laporte F, Després L, Weik M, Boutet S, Rosenthal M, Coquelle N, Burghammer M, Cascio D, Sawaya MR, Winterhalter M, Gratton E, Gutsche I, Federici B, Pellequer JL, Sauter NK, Colletier JP.
Serial femtosecond crystallography on in vivo-grown crystals drives elucidation of mosquitocidal Cyt1Aa bioactivation cascade.
Nat Commun. 2020; 11(1): 1153. PMCID: PMC7052140Cyt1Aa is the one of four crystalline protoxins produced by mosquitocidal bacterium Bacillus thuringiensis israelensis (Bti) that has been shown to delay the evolution of insect resistance in the field. Limiting our understanding of Bti efficacy and the path to improved toxicity and spectrum has been ignorance of how Cyt1Aa crystallizes in vivo and of its mechanism of toxicity. Here, we use serial femtosecond crystallography to determine the Cyt1Aa protoxin structure from sub-micron-sized crystals produced in Bti. Structures determined under various pH/redox conditions illuminate the role played by previously uncharacterized disulfide-bridge and domain-swapped interfaces from crystal formation in Bti to dissolution in the larval mosquito midgut. Biochemical, toxicological and biophysical methods enable the deconvolution of key steps in the Cyt1Aa bioactivation cascade. We additionally show that the size, shape, production yield, pH sensitivity and toxicity of Cyt1Aa crystals grown in Bti can be controlled by single atom substitution.
Abram TJ, Cherukury H, Ou CY, Vu T, Toledano M, Li Y, Grunwald JT, Toosky MN, Tifrea DF, Slepenkin A, Chong J, Kong L, Pozo DVD, La KT, Labanieh L, Zimak J, Shen B, Huang SS, Gratton E, Peterson EM, Zhao W.
Rapid bacterial detection and antibiotic susceptibility testing in whole blood using one-step, high throughput blood digital PCR.
Lab Chip. 2020; 20(3): 477-489. PMCID: PMC7250044Sepsis due to antimicrobial resistant pathogens is a major health problem worldwide. The inability to rapidly detect and thus treat bacteria with appropriate agents in the early stages of infections leads to excess morbidity, mortality, and healthcare costs. Here we report a rapid diagnostic platform that integrates a novel one-step blood droplet digital PCR assay and a high throughput 3D particle counter system with potential to perform bacterial identification and antibiotic susceptibility profiling directly from whole blood specimens, without requiring culture and sample processing steps. Using CTX-M-9 family ESBLs as a model system, we demonstrated that our technology can simultaneously achieve unprecedented high sensitivity (10 CFU per ml) and rapid sample-to-answer assay time (one hour). In head-to-head studies, by contrast, real time PCR and BioRad ddPCR only exhibited a limit of detection of 1000 CFU per ml and 50-100 CFU per ml, respectively. In a blinded test inoculating clinical isolates ... [truncated at 150 words]
Sameni S, Zhang R, Digman MA.
Number and molecular brightness analysis reveals Htt25Q protein aggregation upon the uptake of Htt97Q aggregates.
Biochem Biophys Res Commun. 2020; 522(1): 133-137.Number and molecular Brightness (N&B) analysis is a powerful method used to monitor protein aggregation in living cells. Here, we used the N&B method to characterize the unexpanded HTT protein oligomerization after the internalization of the mutant HTT (mHTT) which contains a CAG repeat extensions encoding for long polyglutamine (polyQ) proteins resulting in misfolding and aggregation. HEK cells expressing Htt25Q-mCherry proteins were infected with Htt97Q-EGFP aggregates, by cell to cell uptake, in cultured conditions resulting in an increasing population of dimers and tetramers compared to our controls. This study shows for the first time the impact of protein aggregation in the unexpanded Htt25Q-mCherry expressing cells that occurs from cell to cell transfer of the expanded Htt97Q-EGFP. These results signify the sporadic behavior of the polyQ inclusion that gives insight into the mechanism of protein dynamics as a consequence of secreted mHTT aggregates.
Pedrote MM, Motta MF, Ferretti GDS, Norberto DR, Spohr TCLS, Lima FRS, Gratton E, Silva JL, de Oliveira GAP.
Oncogenic gain of function in Glioblastoma is linked to mutant p53 Amyloid oligomers.
iScience. 2020; 23(2), 100820. PMCID: PMC6976948Tumor-associated p53 mutations endow cells with malignant phenotypes, including chemoresistance. Amyloid-like oligomers of mutant p53 transform this tumor suppressor into an oncogene. However, the composition and distribution of mutant p53 oligomers are unknown and the mechanism involved in the conversion is sparse. Here, we report accumulation of a p53 mutant within amyloid-like p53 oligomers in glioblastoma-derived cells presenting a chemoresistant gain-of-function phenotype. Statistical analysis from fluorescence fluctuation spectroscopy, pressure-induced measurements, and thioflavin T kinetics demonstrates the distribution of oligomers larger than the active tetrameric form of p53 in the nuclei of living cells and the destabilization of native-drifted p53 species that become amyloid. Collectively, these results provide insights into the role of amyloid-like mutant p53 oligomers in the chemoresistance phenotype of malignant and invasive brain tumors and shed light on therapeutic options to avert cancer.
Castro-Castillo V, Gajardo J, Sandoval-Altamirano C, Gratton E, Sanchez S, Malacrida L, Gunther G.
CAPRYDAA, an anthracene dye analog to LAURDAN: a comparative study using cuvette and microscopy.
J Mater Chem B. 2020; 8(1). PMCID: PMC7029800We synthesized an anthracene derivative with solvatochromic properties to be used as a molecular probe for membrane dynamics and supramolecular organization. A nine carbon atom acyl chain and a dimethylamino substitution were introduced at positions 2 and 6 of the anthracene ring, respectively. This derivative, 2-nonanoyl-6-(dimethylamino)anthracene (termed CAPRYDAA), is a molecular probe designed to mimic the well-known membrane probe LAURDAN's location and response in the lipid membranes. Due to the larger distance between the electron donor and acceptor groups, its absorption and emission bands are red-shifted according to the polarity of the media. The photophysical behavior of CAPRYDAA was measured in homogeneous media, synthetic bilayer and cells, both in a cuvette and in a fluorescence microscope, using one and two-photon excitation. Our results show a comparable physicochemical behavior of CAPRYDAA with LAURDAN, but with the advantage of using visible light (488 nm) as an excitation source. CAPRYDAA was also excitable ... [truncated at 150 words]
Hedde PN, Staaf E, Singh SB, Johansson S, Gratton E.
Pair correlation analysis maps the dynamic two-dimensional organization of natural killer cell receptors at the synapse.
ACS Nano. 2019; 13(2): 14274-14282. PMCID: PMC8427743In living systems, the contact between cells is the basis of recognition, differentiation, and orchestration of an immune response. Obstacles and barriers to biomolecular motion, especially for receptors at cellular synapses, critically control these functions by creating an anisotropic environment. Whereas conventional fluorescence fluctuation methods, such as fluorescence correlation spectroscopy or fluorescence recovery after photobleaching, can only measure the isotropic diffusion of molecules, the two-dimensional pair correlation function (2D-pCF) approach probes the anisotropic paths at different spatial locations within an image, allowing the creation of high-resolution maps that can visualize and quantify how molecules move in a living cell. In this work, we show how the 2D-pCF method maps the environment in cellular synapses as perceived by natural killer (NK) cell receptors. In cultured human HLA null 721.221 cells, 2D-pCF reveals the motion of inhibitory receptor HLA-Cw4-YFP coexpressed with KIR3DL1 to be highly directional around specific loci, while these restrictions ... [truncated at 150 words]
Sitiwin E, Madigan MC, Gratton E, Cherepanoff S, Conway RM, Whan R, Macmillan A.
Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques.
Sci Rep. 2019; 9(1), 18585. PMCID: PMC6901595Choroidal melanocytes (HCMs) are melanin-producing cells in the vascular uvea of the human eye (iris, ciliary body and choroid). These cranial neural crest-derived cells migrate to populate a mesodermal microenvironment, and display cellular functions and extracellular interactions that are biologically distinct to skin melanocytes. HCMs (and melanins) are important in normal human eye physiology with roles including photoprotection, regulation of oxidative damage and immune responses. To extend knowledge of cytoplasmic melanins and melanosomes in label-free HCMs, a non-invasive 'fit-free' approach, combining 2-photon excitation fluorescence lifetimes and emission spectral imaging with phasor plot segmentation was applied. Intracellular melanin-mapped FLIM phasors showed a linear distribution indicating that HCM melanins are a ratio of two fluorophores, eumelanin and pheomelanin. A quantitative histogram of HCM melanins was generated by identifying the image pixel fraction contributed by phasor clusters mapped to varying eumelanin/pheomelanin ratio. Eumelanin-enriched dark HCM regions mapped to phasors with shorter lifetimes and ... [truncated at 150 words]
Oneto M, Scipioni L, Sarmento MJ, Cainero I, Pelicci S, Furia L, Pelicci PG, Dellino GI, Bianchini P, Faretta M, Gratton E, Diaspro A, Lanzanò L.
Nanoscale distribution of nuclear sites by super-resolved image cross-correlation spectroscopy.
Biophys J. 2019; 117(11): 2054-2065. PMCID: PMC6895719Deciphering the spatiotemporal coordination between nuclear functions is important to understand its role in the maintenance of human genome. In this context, super-resolution microscopy has gained considerable interest because it can be used to probe the spatial organization of functional sites in intact single-cell nuclei in the 20-250 nm range. Among the methods that quantify colocalization from multicolor images, image cross-correlation spectroscopy (ICCS) offers several advantages, namely it does not require a presegmentation of the image into objects and can be used to detect dynamic interactions. However, the combination of ICCS with super-resolution microscopy has not been explored yet. Here, we combine dual-color stimulated emission depletion (STED) nanoscopy with ICCS (STED-ICCS) to quantify the nanoscale distribution of functional nuclear sites. We show that super-resolved ICCS provides not only a value of the colocalized fraction but also the characteristic distances associated to correlated nuclear sites. As a validation, we quantify the ... [truncated at 150 words]
Ranjit S, Datta R, Dvornikov AS, Gratton E.
Multicomponent analysis of phasor plot in a single pixel to calculate changes of metabolic trajectory in biological systems.
J Phys Chem A. 2019; 123(45): 9865-9873.Phasor FLIM in cells undergoing oxidative stress and in mice liver sections have shown presence of a third autofluorescent component indicative of lipid droplets along with free and enzyme bound NADH with similar emission. This third component affects position and shape of the phasor distribution pushing it away from the metabolic trajectory. Phasor rule of addition is still valid and was exploited here to create a multicomponent analysis where the phasor distribution can be reassigned to the metabolic trajectory and changes in metabolism can be detected independently of the intensity of this third component. Calculation of multiple components from FLIM imaging data of biological systems is a difficult process, especially if different fluorescent species are present at a particular pixel. This paper describes the methodology that can be used to separate these multiple components when they are present in the phasor signature acquired in a single pixel of an image.
Ranjit S, Malacrida L, Stakic M, Gratton E.
Determination of the metabolic index using the fluorescence lifetime of free and bound NADH in the phasor approach.
J Biophotonics. 2019; 12(11), e201900156. PMCID: PMC6842045The fluorescence lifetime of nicotinamide adenine dinucleotide (NADH) is commonly used in conjunction with the phasor approach as a molecular biomarker to provide information on cellular metabolism of autofluorescence imaging of cells and tissue. However, in the phasor approach, the bound and free lifetime defining the phasor metabolic trajectory is a subject of debate. The fluorescence lifetime of NADH increases when bound to an enzyme, in contrast to the short multiexponential lifetime displayed by NADH in solution. The extent of fluorescence lifetime increase depends on the enzyme to which NADH is bound. With proper preparation of lactate dehydrogenase (LDH) using oxalic acid (OA) as an allosteric factor, bound NADH to LDH has a lifetime of 3.4 ns and is positioned on the universal semicircle of the phasor plot, inferring a monoexponential lifetime for this species. Surprisingly, measurements in the cellular environments with different metabolic states show a linear trajectory between ... [truncated at 150 words]
Gabriel M, Anzalone A, Gratton E, Estrada LC.
A tracking-based nanoimaging method for fast detection of surfaces' inhomogeneities using gold nanoparticles.
Microsc Res Tech. 2019; 82(11): 1835-1842.The localization of surfaces inhomogeneities is central to many areas of technology, chemistry and biology, ranging from surface defects in industry to the identification and screening of early bio-defects inside cells. The development of methods that enable direct, sensitive, and rapid detection of those inhomogeneities is both relevant and timely. To address this challenge, we developed a far-field nanoimaging method to detect the presence of surface's nanodefects that modify the signal emitted by gold nanoparticles (AuNPs) under laser irradiation. Our technique is based on the formation of hot spots due to the confinement of light in the proximity of the AuNP, whose positions depend on the polarization direction of the incident beam. An inhomogeneity is detected as an increase in the intensity collected from the hot spots when a laser beam is orbiting the nanoparticle and the incident polarization direction of the laser beam is changed periodically.
Murata MM, Kong X, Moncada E, Chen Y, Imamura H, Wang P, Berns MW, Yokomori K, Digman MA.
NAD+ consumption by PARP1 in response to DNA damage triggers metabolic shift critical for damaged cell survival.
Mol Biol Cell. 2019; 30(20): 2584-2597. PMCID: PMC6740200DNA damage signaling is critical for the maintenance of genome integrity and cell fate decision. Poly(ADP-ribose) polymerase 1 (PARP1) is a DNA damage sensor rapidly activated in a damage dose- and complexity-dependent manner playing a critical role in the initial chromatin organization and DNA repair pathway choice at damage sites. However, our understanding of a cell-wide consequence of its activation in damaged cells is still limited. Using the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and fluorescence-based biosensors in combination with laser microirradiation, we found a rapid cell-wide increase of the bound NADH fraction in response to nuclear DNA damage, which is triggered by PARP-dependent NAD+ depletion. This change is linked to the metabolic balance shift to oxidative phosphorylation (oxphos) over glycolysis. Inhibition of oxphos, but not glycolysis, resulted in parthanatos due to rapid PARP-dependent ATP deprivation, indicating that oxphos becomes critical for damaged cell survival. The results reveal ... [truncated at 150 words]
Ma N, de Mochel NR, Pham PD, Yoo TY, Cho KWY, Digman MA.
Label-free assessment of pre-implantation embryo quality by the Fluorescence Lifetime Imaging Microscopy (FLIM)-phasor approach.
Sci Rep. 2019; 9(1), 13206. PMCID: PMC6744410Development of quantitative, safe and rapid techniques for assessing embryo quality provides significant advances in Assisted Reproductive Technologies (ART). Instead of assessing the embryo quality by the standard morphologic evaluation, we apply the phasor-FLIM (Fluorescence Lifetime Imaging Microscopy) method to capture endogenous fluorescent biomarkers of pre-implantation embryos as a non-morphological caliber for embryo quality. Here, we identify, under hypoxic and non-hypoxic conditions, the unique spectroscopic trajectories at different stages of mouse pre-implantation development, which is referred to as the developmental, or "D-trajectory", that consists of fluorescence lifetime from different stages of mouse pre-implantation embryos. The D-trajectory correlates with intrinsic fluorescent species from a distinctive energy metabolism and oxidized lipids, as seen with Third Harmonic Generation (THG) that changes over time. In addition, we have defined a non-morphological Embryo Viability Index (EVI) to distinguish pre-implantation embryo quality using the Distance Analysis (DA), a machine learning algorithm to process the fluorescence lifetime ... [truncated at 150 words]
Trivedi P, Palomba F, Niedzialkowska E, Digman MA, Gratton E, Stukenberg PT.
The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex.
Nat Cell Biol. 2019; 21(9): 1127-1137. PMCID: PMC7341897The inner centromere is a region on every mitotic chromosome that enables specific biochemical reactions that underlie properties, such as the maintenance of cohesion, the regulation of kinetochores and the assembly of specialized chromatin, that can resist microtubule pulling forces. The chromosomal passenger complex (CPC) is abundantly localized to the inner centromeres and it is unclear whether it is involved in non-kinase activities that contribute to the generation of these unique chromatin properties. We find that the borealin subunit of the CPC drives phase separation of the CPC in vitro at concentrations that are below those found on the inner centromere. We also provide strong evidence that the CPC exists in a phase-separated state at the inner centromere. CPC phase separation is required for its inner-centromere localization and function during mitosis. We suggest that the CPC combines phase separation, kinase and histone code-reading activities to enable the formation of a ... [truncated at 150 words]
Dong Y, Sameni S, Digman MA, Brewer GJ.
Reversibility of age-related oxidized free NADH redox states in Alzheimer's disease neurons by imposed external Cys/CySS redox shifts.
Sci Rep. 2019; 9(1), 11274. PMCID: PMC6677822Redox systems including extracellular cysteine/cystine (Cys/CySS), intracellular glutathione/oxidized glutathione (GSH/GSSG) and nicotinamide adenine dinucleotide reduced/oxidized forms (NADH/NAD+) are critical for maintaining redox homeostasis. Aging as a major risk factor for Alzheimer's disease (AD) is associated with oxidative shifts, decreases in anti-oxidant protection and dysfunction of mitochondria. Here, we examined the flexibility of mitochondrial-specific free NADH in live neurons from non-transgenic (NTg) or triple transgenic AD-like mice (3xTg-AD) of different ages under an imposed extracellular Cys/CySS oxidative or reductive condition. We used phasor fluorescence lifetime imaging microscopy (FLIM) to distinguish free and bound NADH in mitochondria, nuclei and cytoplasm. Under an external oxidative stress, a lower capacity for maintaining mitochondrial free NADH levels was found in old compared to young neurons and a further decline with genetic load. Remarkably, an imposed Cys/CySS reductive state rejuvenated the mitochondrial free NADH levels of old NTg neurons by 71% and old 3xTg-AD neurons by ... [truncated at 150 words]
Levi M, Gratton E, Forster IC, Hernando N, Wagner CA, Biber J, Sorribas V, Murer H.
Mechanisms of phosphate transport.
Nat Rev Nephrol. 2019; 15(8): 482-500.Over the past 25 years, successive cloning of SLC34A1, SLC34A2 and SLC34A3, which encode the sodium-dependent inorganic phosphate (Pi) cotransport proteins 2a-2c, has facilitated the identification of molecular mechanisms that underlie the regulation of renal and intestinal Pi transport. Pi and various hormones, including parathyroid hormone and phosphatonins, such as fibroblast growth factor 23, regulate the activity of these Pi transporters through transcriptional, translational and post-translational mechanisms involving interactions with PDZ domain-containing proteins, lipid microdomains and acute trafficking of the transporters via endocytosis and exocytosis. In humans and rodents, mutations in any of the three transporters lead to dysregulation of epithelial Pi transport with effects on serum Pi levels and can cause cardiovascular and musculoskeletal damage, illustrating the importance of these transporters in the maintenance of local and systemic Pi homeostasis. Functional and structural studies have provided insights into the mechanism by which these proteins transport Pi, whereas in vivo ... [truncated at 150 words]
Dvornikov A, Malacrida L, Gratton E.
The DIVER microscope for imaging in scattering media.
Methods Protoc. 2019; 2(2): 53. PMCID: PMC6632175We describe an advanced DIVER (Deep Imaging Via Emission Recovery) detection system for two-photon fluorescence microscopy that allows imaging in multiple scattering media, including biological tissues, up to a depth of a few mm with micron resolution. This detection system is more sensitive to low level light signals than conventional epi-detection used in two-photon fluorescence microscopes. The DIVER detector efficiently collects scattered emission photons from a wide area of turbid samples at almost any entrance angle in a 2π spherical angle. Using an epi-detection scheme only photons coming from a relatively small area of a sample and at narrow acceptance angle can be detected. The transmission geometry of the DIVER imaging system makes it exceptionally suitable for Second and Third Harmonic Generation (SHG, THG) signal detection. It also has in-depth fluorescence lifetime imaging (FLIM) capability. Using special optical filters with sin-cos spectral response, hyperspectral analysis of images acquired in-depth in ... [truncated at 150 words]
Sanborn CD, Chacko JV, Digman MA, Ardo S.
Interfacial and nanoconfinement effects decrease the excited-state acidity of polymer-bound photoacids.
Chem. 2019; 5(6): 1648-1670.Photo-initiated ion transport on the nanoscale is relevant to various fields
including energy conversion, neuron triggering, and biomimetic processes. To
study this phenomenon, we synthesized and evaluated photoacid-modified
polymers, which generate hydrated protons in response to visible-light excita-
tion. Pyrenol photoacid dye molecules were covalently bonded within conical
nanopores that had been track etched in poly(ethylene terephthalate). The
data suggest that 90% of the nanopore surfaces were modified with photoacids and that photoacids were, on average, bound through three sulfonamide groups. In comparison to photoacids dissolved in an aqueous solution, photoacids bound specifically to the nanoporous tip region were, on average, weaker acids in their ground and excited states, which we presume is due to differences in surface potential or solvation environment in the confined nanopores. These results suggest that ion transport initiated by nanoconfined photoacids will require careful molecular engineering to enable efficient light-to-ionic energy conversion.
Polyzos AA, Lee DY, Datta R, Hauser M, Budworth H, Holt A, Mihalik S, Goldschmidt P, Frankel K, Trego K, Bennett MJ, Vockley J, Xu K, Gratton E, McMurray CT.
Metabolic reprogramming in astrocytes distinguishes region-specific neuronal susceptibility in Huntington mice.
Cell Metab. 2019; 29(6): 1258-1273. PMCID: PMC6583797The basis for region-specific neuronal toxicity in Huntington disease is unknown. Here, we show that region-specific neuronal vulnerability is a substrate-driven response in astrocytes. Glucose is low in HdhQ(150/150) animals, and astrocytes in each brain region adapt by metabolically reprogramming their mitochondria to use endogenous, non-glycolytic metabolites as an alternative fuel. Each region is characterized by distinct metabolic pools, and astrocytes adapt accordingly. The vulnerable striatum is enriched in fatty acids, and mitochondria reprogram by oxidizing them as an energy source but at the cost of escalating reactive oxygen species (ROS)-induced damage. The cerebellum is replete with amino acids, which are precursors for glucose regeneration through the pentose phosphate shunt or gluconeogenesis pathways. ROS is not elevated, and this region sustains little damage. While mhtt expression imposes disease stress throughout the brain, sensitivity or resistance arises from an adaptive stress response, which is inherently region specific. Metabolic reprogramming may have ... [truncated at 150 words]
Rodriguez-Agudo D, Malacrida L, Kakiyama G, Sparrer T, Fortes C, Maceyka M, Sublerh MA, Windle JJ, Gratton E, Pandak WM, Gil G.
StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis.
J Lipid Res. 2019; 60(6): 1087-1098. PMCID: PMC6547630How plasma membrane (PM) cholesterol is controlled is poorly understood. Ablation of the gene encoding the ER stress steroidogenic acute regulatory-related lipid transfer domain 5 (StarD5) leads to a decrease in PM cholesterol content, a decrease in cholesterol efflux, and an increase in intracellular neutral lipid accumulation in macrophages, the major cell type that expresses StarD5. ER stress increases StarD5 expression in mouse hepatocytes, which results in an increase in accessible PM cholesterol in wild type but not in StarD5-/- hepatocytes. StarD5-/- mice store higher levels of cholesterol and triglycerides and leads to altered expression of cholesterol-regulated genes. In vitro, a recombinant GST-StarD5 protein transfers cholesterol between synthetic liposomes. StarD5 overexpression leads to a marked increase in PM cholesterol. Phasor analysis of LAURDAN FLIM data revealed an increase in PM fluidity in StarD5-/- macrophages. Taken together, these studies show that StarD5 is a stress responsive protein that regulates PM cholesterol ... [truncated at 150 words]
Chen H, Ma N, Kagawa K, Kawahito S, Digman M, Gratton E.
Widefield multifrequency fluorescence lifetime imaging using a two-tap complementary metal-oxide semiconductor camera with lateral electric field charge modulators.
J Biophotonics. 2019; 12(5), e201800223.Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) measures the fluorescence lifetime of entire images in a fast and efficient manner. We report a widefield FD-FLIM system based on a complementary metal-oxide semiconductor camera equipped with two-tap true correlated double sampling lock-in pixels and lateral electric field charge modulators. Owing to the fast intrinsic response and modulation of the camera, our system allows parallel multifrequency FLIM in one measurement via fast Fourier transform. We demonstrate that at a fundamental frequency of 20 MHz, 31-harmonics 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. These results indicate that our widefield multifrequency FD-FLIM system is a ... [truncated at 150 words]
Planes N, Digman MA, Vanderheyden PPML, Gratton E, Caballero-George C.
Number and brightness analysis to study spatio-temporal distribution of the angiotensin II AT1 and the endothelin-1 ETA receptors: Influence of ligand binding.
Biochim Biophys Acta Gen Subj. 2019; 1863(5): 917-924.The angiotensin II AT1 and the endothelin 1 ETA receptors play a crucial role in the pathogenesis of cardiovascular diseases like hypertension, heart failure, stroke, pulmonary hypertension, and cardiac hypertrophy. Both receptors are members of the rhodopsion-like superfamily of G protein-coupled receptors which can exist as monomers, dimers, and higher order aggregates. Recently, oligomerization of these two receptors have been described by several biophysical methods based mainly on luminescence and fluorescence energy transfer. Since this oligomerization can occur either spontaneously or it can be induced by ligand-binding, the aim of this work was to address whether the oligomerization of these receptors occurs upon ligand-binding. For this purpose the Number and Brightness analysis, a method that allows the identification, localization, and quantification of protein aggregates in the plasma membrane of a single cell, was used. An advantage of this method is that it is not limited to certain dyes specially required ... [truncated at 150 words]
Lou J, Scipioni L, Wright BK, Bartolec TK, Zhang J, Masamsetti VP, Gaus K, Gratton E, Cesare AJ, Hinde E.
Phasor histone FLIM-FRET microscopy quantifies spatiotemporal rearrangement of chromatin architecture during the DNA damage response.
Proc Natl Acad Sci USA. 2019; 116(15): 7323-7332. PMCID: PMC6462080To investigate how chromatin architecture is spatiotemporally organized at a double-strand break (DSB) repair locus, we established a biophysical method to quantify chromatin compaction at the nucleosome level during the DNA damage response (DDR). The method is based on phasor image-correlation spectroscopy of histone fluorescence lifetime imaging microscopy (FLIM)-Förster resonance energy transfer (FRET) microscopy data acquired in live cells coexpressing H2B-eGFP and H2B-mCherry. This multiplexed approach generates spatiotemporal maps of nuclear-wide chromatin compaction that, when coupled with laser microirradiation-induced DSBs, quantify the size, stability, and spacing between compact chromatin foci throughout the DDR. Using this technology, we identify that ataxia-telangiectasia mutated (ATM) and RNF8 regulate rapid chromatin decompaction at DSBs and formation of compact chromatin foci surrounding the repair locus. This chromatin architecture serves to demarcate the repair locus from the surrounding nuclear environment and modulate 53BP1 mobility.
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Visualizing the regulation of SLC34 proteins at the apical membrane.
Pflugers Arch. 2019; 471(4): 533-542. PMCID: PMC6436987The cloning of the renal NaPi-2a (SLC34A1) and NaPi-2c (SLC34A3) phosphate transporters has made it possible to characterize the molecular and biophysical regulation of renal proximal tubular reabsorption of inorganic phosphate (Pi). Dietary factors, such as Pi and K, and several hormones and phosphatonins, including parathyroid hormone (PTH), fibroblast growth factor 23 (FGF23), and glucocorticoids, regulate the transporters through various transcriptional, translational, and post-translational mechanisms that involve acute trafficking via endocytosis or exocytosis, interactions with PDZ domain proteins, lipid microdomains, and diffusion and clustering in the apical brush border membrane. The visualization of these trafficking events by means of novel microscopy techniques that includes fluorescence lifetime imaging microscopy (FLIM), Förster resonance energy transfer (FRET), fluctuation correlation spectroscopy (FCS), and modulation tracking (MT), is the primary focus of this review.
Ou CY, Vu T, Grunwald JT, Toledano M, Zimak J, Toosky M, Shen B, Zell JA, Gratton E, Abram TJ, Zhao W.
An ultrasensitive test for profiling circulating tumor DNA using integrated comprehensive droplet digital detection.
Lab Chip. 2019; 19(6): 993-1005. PMCID: PMC6559803Current cancer detection systems lack the required sensitivity to reliably detect minimal residual disease (MRD) and recurrence at the earliest stages when treatment would be most effective. To address this issue, we present a novel liquid biopsy approach that utilizes an integrated comprehensive droplet digital detection (IC3D) digital PCR system which combines microfluidic droplet partitioning, fluorescent multiplex PCR chemistry, and our rapid 3D, large-volume droplet counting technology. The IC3D ddPCR assay can detect cancer-specific, ultra-rare genomic targets due to large sample input and high degree of partitioning. We first demonstrate our droplet digital PCR assay can robustly detect common cancer mutants including KRAS G12D spiked in wild-type genomic background or isolated from patient samples with 100% specificity. We then demonstrate that the IC3D ddPCR system can detect oncogenic KRAS G12D mutant alleles against a background of wild-type genomes at a sensitivity of 0.00125-0.005% with a false positive rate of 0% ... [truncated at 150 words]
Begarani F, D'Autilia F, Signore G, Grosso AD, Cecchini M, Gratton E, Beltram F, Cardarelli F.
Capturing metabolism-dependent solvent dynamics in the lumen of a trafficking lysosome.
ACS Nano. 2019; 13(2): 1670-1682.The eukaryotic cell compartmentalizes into spatially confined, membrane-enclosed, intracellular structures ( e. g., organelles, endosomes, and vesicles). Here, peculiar physicochemical properties of the local environment occur and participate in the regulation of ongoing molecular processes. In spite of the huge amount of available environmental probes, experiments on subcellular structures are severely challenged by their three-dimensional (3D) movement. This bottleneck is tackled here 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 at high temporal resolution: microseconds along the orbit, milliseconds between orbits. The lysosome is selected as the intracellular target, together with 6-acetyl-2-dimethylaminonaphthalene (ACDAN) as probe of the physicochemical properties of the intralysosomal environment. Generalized polarization (GP) analysis of ACDAN emission is used to get a quantitative view on intralysosomal solvent dipolar relaxation. Thus, raster image correlation spectroscopy (RICS) analysis reveals that ... [truncated at 150 words]
Hedde PN, Abram T, Vu T, Zhao W, Gratton E.
Fluorescence lifetime detection with particle counting devices.
Biomed Opt Express. 2019; 10(3): 1223-1233. PMCID: PMC6420272Fluorescence-based single particle counting devices have become very powerful tools for human health-related applications such as the detection of blood-borne pathogens. Instead of passing the sample fluid through a thin tube or microfluidic chip, as it is commonly practiced in flow cytometers and sorter devices, single particle counters scan the fluid volume by rotation and translation of the sample container. Hence, single particle counters are not limited by the fluid flow friction and can scan a large volume in a short timeframe while maintaining high sensitivity. A single particle can be detected in a milliliter of the fluid sample within minutes, and diagnostics are being developed using this principle. Until now, signal detection with particle counters has been based on signal intensity and signal separation into multiple wavelength bands coupled with multiple detectors, which limits the number of species that can be resolved. In this paper, we applied fluorescence lifetime ... [truncated at 150 words]
Dong Y, Digman MA, Brewer GJ.
Age- and AD-related redox state of NADH in subcellular compartments by fluorescence lifetime imaging microscopy.
Geroscience. 2019; 41(1): 51-67. PMCID: PMC6423217Nicotinamide adenine dinucleotide (reduced form: NADH) serves as a vital redox-energy currency for reduction-oxidation homeostasis and fulfilling energetic demands. While NADH exists as free and bound forms, only free NADH is utilized for complex I to power oxidative phosphorylation, especially important in neurons. Here, we studied how much free NADH remains available for energy production in mitochondria of old living neurons. We hypothesize that free NADH in neurons from old mice is lower than the levels in young mice and even lower in neurons from the 3xTg-AD Alzheimer's disease (AD) mouse model. To assess free NADH, we used lifetime imaging of NADH autofluorescence with 2-photon excitation to be able to resolve the pool of NADH in mitochondria, cytoplasm, and nuclei. Primary neurons from old mice were characterized by a lower free/bound NADH ratio than young neurons from both non-transgenic (NTg) and more so in 3xTg-AD mice. Mitochondrial compartments maintained 26 ... [truncated at 150 words]
Ma N, Kamalakshakurup G, Aghaamoo M, Lee AP, Digman MA.
Label-Free Metabolic Classification of Single Cells in Droplets Using the Phasor Approach to Fluorescence Lifetime Imaging Microscopy.
Cytometry A. 2019; 95(1): 93-100. PMCID: PMC6613543Characterization of single cell metabolism is imperative for understanding subcellular functional and biochemical changes associated with healthy tissue development and the progression of numerous diseases. However, single-cell analysis often requires the use of fluorescent tags and cell lysis followed by genomic profiling to identify the cellular heterogeneity. Identifying individual cells in a noninvasive and label-free manner is crucial for the detection of energy metabolism which will discriminate cell types and most importantly critical for maintaining cell viability for further analysis. Here, we have developed a robust assay using the droplet microfluidic technology together with the phasor approach to fluorescence lifetime imaging microscopy to study cell heterogeneity within and among the leukemia cell lines (K-562 and Jurkat). We have extended these techniques to characterize metabolic differences between proliferating and quiescent cells-a critical step toward label-free single cancer cell dormancy research. The result suggests a droplet-based noninvasive and label-free method to distinguish ... [truncated at 150 words]
Scipioni L, Lanzanò L, Diaspro A, Gratton E.
Comprehensive correlation analysis for super-resolution dynamic fingerprinting of cellular compartments using the Zeiss Airyscan detector.
Nat Commun. 2018; 9(1), 5120. PMCID: PMC6269422The availability of the Airyscan detector in the Zeiss LSM 880 has made possible the development of a new concept in fluctuation correlation spectroscopy using super-resolution. The Airyscan unit acquires data simultaneously on 32 detectors arranged in a hexagonal array. This detector opens up the possibility to use fluctuation methods based on time correlation at single points or at a number of points simultaneously, as well as methods based on spatial correlation in the area covered by the detector. Given the frame rate of this detector, millions of frames can be acquired in seconds, providing a robust statistical basis for fluctuation data. We apply the comprehensive analysis to the molecular fluctuations of free GFP diffusing in live cells at different subcellular compartments to show that at the nanoscale different cell environments can be distinguished by the comprehensive fluctuation analysis.
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LAURDAN fluorescence and phasor plots reveal the effects of a H2O2 bolus in NIH-3T3 fibroblast membranes dynamics and hydration.
Free Radic Biol Med. 2018; 128: 144-156. PMCID: PMC6175669Fluorescence spectroscopy, coupled with microscopy, opens new frontiers for the study of dynamic processes with high spatio-temporal resolution. The application of phasor plots to FLIM and hyperspectral imaging demonstrate unprecedented capabilities to study complex photophysics at the subcellular level. Using these approaches we studied the effects of an H2O2 bolus on NIH-3T3 membranes dynamics monitored by LAURDAN fluorescence. Exposure of NIH-3T3 cells to a bolus of H2O2 modifies the cell membranes and, in particular, the plasma membrane in a complex manner. The LAURDAN results reveal that the peroxide treatment decreases membrane fluidity but surprisingly increases dipolar relaxation around the excited probe. Using the Multidimensional-phasor approach we elucidated the complex photophysics of LAURDAN incorporated into cell membrane after H2O2 exposure. The results indicate the occurrence of LAURDAN fast-diffusion from gel↔ld phases in membranes exposed to a H2O2 bolus. An ad hoc hypothesis is presented to interpret the results in the context ... [truncated at 150 words]
Mah EJ, Lefebvre AEYT, McGahey GE, Yee AF, Digman MA.
Collagen density modulates triple-negative breast cancer cell metabolism through adhesion-mediated contractility.
Sci Rep. 2018; 8(1), 17094. PMCID: PMC6244401Extracellular matrix (ECM) mechanical properties upregulate cancer invasion, cell contractility, and focal adhesion formation. Alteration in energy metabolism is a known characteristic of cancer cells (i.e., Warburg effect) and modulates cell invasion. There is little evidence to show if collagen density can alter cancer cell metabolism. We investigated changes in energy metabolism due to collagen density in five breast cell lines by measuring the fluorescence lifetime of NADH. We found that only triple-negative breast cancer cells, MDA-MB231 and MDA-MB468 cells, had an increased population of bound NADH, indicating an oxidative phosphorylation (OXPHOS) signature, as collagen density decreased. When inhibiting ROCK and cell contractility, MDA-MB231 cells on glass shifted from glycolysis (GLY) to OXPHOS, confirming the intricate relationship between mechanosensing and metabolism. MCF10A cells showed less significant changes in metabolism, shifting towards GLY as collagen density decreased. The MCF-7 and T-47D, less invasive breast cancer cells, compared to the MDA-MB231 and ... [truncated at 150 words]
Ranjit S, Malacrida L, Gratton E.
Differences between FLIM phasor analyses for data collected with the Becker and Hickl SPC830 card and with the FLIMbox card.
Microsc Res Tech. 2018; 81(9): 980-989. PMCID: PMC6240382The phasor approach to FLIM (Fluorescence Lifetime Imaging Microscopy) is becoming popular due to the powerful fit free analysis and the visualization of the decay at each point in images of cells and tissues. However, although several implementation of the method are offered by manufactures of FLIM accessories for microscopes, the details of the conversion of the decay to phasors at each point in an image requires some consideration. Here, we show that if the decay is not properly acquired, the apparently simple phasor transformation can provide incorrect phasor plots and the results may be misinterpreted. In particular, we show the disagreement in experimental data acquired on the same samples using the two cards (FLIMbox, frequency domain and Becker & Hickl BH 830, time domain) and the effect produced by using the BH 830 card with different settings. This difference in data acquisition translates to the assignment of phasor components ... [truncated at 150 words]
Ranjit S, Malacrida L, Jameson DM, Gratton E.
Fit-free analysis of fluorescence lifetime imaging data using the phasor approach.
Nat. Protoc. 2018; 13(9): 1979-2004.Fluorescence lifetime imaging microscopy (FLIM) is used in diverse disciplines, including biology, chemistry and biophysics, but its use has been limited by the complexity of the data analysis. The phasor approach to FLIM has the potential to markedly reduce this complexity and at the same time provide a powerful visualization of the data content. Phasor plots for fluorescence lifetime analysis were originally developed as a graphical representation of excited-state fluorescence lifetimes for in vitro systems. The method's simple mathematics and specific rules avoid errors and confusion common in the study of complex and heterogeneous fluorescence. In the case of FLIM, the phasor approach has become a powerful method for simple and fit-free analyses of the information contained in the many thousands of pixels constituting an image. At present, the phasor plot is used not only for FLIM, but also for hyperspectral imaging, wherein phasors provide an unprecedented understanding of heterogeneous ... [truncated at 150 words]
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Selective plane illumination microscopy with a light sheet of uniform thickness formed by an electrically tunable lens.
Microsc Res Tech. 2018; 81(9): 924-928. PMCID: PMC5479743Light sheet microscopy is a powerful technique for rapid, three-dimensional fluorescence imaging of large specimen such as drosophila and zebrafish embryos. Yet, beam divergence results in a loss of axial resolution at the periphery of the light sheet. Here, we demonstrate how an electrically tunable lens can be utilized to maintain the minimal, diffraction-limited thickness of the light sheet over a wide field of view (>600 µm) at high frame rates (40 fps). This mode of operation is necessary for the application of fluorescence fluctuation spectroscopy in images.
Hellenkamp B, Schmid S, Doroshenko O, Opanasyuk O, Kühnemuth R, Adariani SR, Ambrose B, Aznauryan M, Barth A, Birkedal V, Bowen ME, Chen H, Cordes T, Eilert T, Fijen C, Gebhardt C, Götz M, Gouridis G, Gratton E, Ha T, Hao P, Hanke CA, Hartmann A, Hendrix J, Hildebrandt LL, Hirschfeld V, Hohlbein J, Hua B, Hübner CG, Kallis E, Kapanidis AN, Kim JY, Krainer G, Lamb DC, Lee NK, Lemke EA, Levesque B, Levitus M, McCann JJ, Naredi-Rainer N, Nettels D, Ngo T, Qiu R, Robb NC, Röcker C, Sanabria H, Schlierf M, Schröder T, Schuler B, Seidel H, Streit L, Thurn J, Tinnefeld P, Tyagi S, Vandenberk N, Vera AM, Weninger KR, Wünsch B, Yanez-Orozco IS, Michaelis J, Seidel CAM, Craggs TD, Hugel T.
Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.
Nat Methods. 2018; 15(9): 669-676. PMCID: PMC6121742Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.
Pedrote MM, de Oliveira GAP, Felix AL, Mota MF, de Marques MA, Soares IN, Iqbal A, Norberto DR, Gomes AMO, Gratton E, Cino EA, Silva JL.
Aggregation-primed molten globule conformers of the p53 core domain provide potential tools for studying p53C aggregation in cancer.
J Biol Chem. 2018; 293(29): 11374-11387. PMCID: PMC6065177The functionality of the tumor suppressor p53 is altered in more than 50% of human cancers, and many individuals with cancer exhibit amyloid-like buildups of aggregated p53. An understanding of what triggers the pathogenic amyloid conversion of p53 is required for the further development of cancer therapies. Here, perturbation of the p53 core domain (p53C) with sub-denaturing concentrations of guanidine hydrochloride and high hydrostatic pressure revealed native-like molten globule (MG) states, a subset of which were highly prone to amyloidogenic aggregation. We found that MG conformers of p53C, likely representing population-weighted averages of multiple states, have different volumetric properties, as determined by pressure perturbation and size-exclusion chromatography. We also found that they bind the fluorescent dye 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid (bis-ANS) and have a native-like tertiary structure that occludes the single Trp residue in p53. Fluorescence experiments revealed conformational changes of the single Trp and Tyr residues before p53 unfolding and the ... [truncated at 150 words]
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Hyperspectral imaging in highly scattering media by the spectral phasor approach using two filters.
Biomed Opt Express. 2018; 9(8): 3503-3511. PMCID: PMC6191637Hyperspectral 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. 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 2 filters that accept uncollimated light over a large aperture are sufficient to calculate the spectral phasor rather than displaying the entire spectrum. Using the properties of the spectral phasors, we can resolve spectral components and perform the type of data analyses that are usually performed in hyperspectral image analysis.
Kim SM, Nguyen TT, Ravi A, Kubiniok P, Finicle BT, Jayashankar V, Malacrida L, Hou J, Robertson J, Gao D, Chernoff J, Digman MA, Potma EO, Tromberg BJ, Thibault P, Edinger AL.
PTEN deficiency and AMPK activation promote nutrient scavenging and anabolism in prostate cancer cells.
Cancer Discov. 2018; 8(7): 866-883. PMCID: PMC6030497We report that PTEN-deficient prostate cancer cells use macropinocytosis to survive and proliferate under nutrient stress. PTEN loss increased macropinocytosis only in the context of AMPK activation revealing a general requirement for AMPK in macropinocytosis and a novel mechanism by which AMPK promotes survival under stress. In prostate cancer cells, albumin uptake did not require macropinocytosis, but necrotic cell debris proved a specific macropinocytic cargo. Isotopic labeling confirmed that macropinocytosed necrotic cell proteins fueled new protein synthesis in prostate cancer cells. Supplementation with necrotic debris, but not albumin, also maintained lipid stores suggesting that macropinocytosis can supply nutrients other than amino acids. Non-transformed prostatic epithelial cells were not macropinocytic, but patient-derived prostate cancer organoids and xenografts and autochthonous prostate tumors all exhibited constitutive macropinocytosis, and blocking macropinocytosis limited prostate tumor growth. Macropinocytosis of extracellular material by prostate cancer cells is a previously unappreciated tumor-microenvironment interaction that could be targeted therapeutically.
Anzalone A, Chacko JV, Nishi RA, Dumont C, Smith D, Shea LD, Digman MA, Cummings BJ, Anderson AJ.
Feasibility study on mouse live imaging after spinal cord injury and poly(lactide-co-glycolide) bridge implantation.
J Biomed Opt. 2018; 23(6): 1-6.Spinal cord injury (SCI) causes permanent paralysis below the damaged area. SCI is linked to neuronal death, demyelination, and limited ability of neuronal fibers to regenerate. Regeneration capacity is limited by the presence of many inhibitory factors in the spinal cord environment. The use of poly(lactide-co-glycolide) (PLG) bridges has demonstrated the ability to sustain long-term regeneration after SCI in a cervical hemisection mouse model. Critically, imaging of regenerating fibers and the myelination status of these neuronal filaments is a severe limitation to progress in SCI research. We used a transgenic mouse model that selectively expresses fluorescent reporters (eGFP) in the neuronal fibers of the spinal cord. We implanted a PLG bridge at C5 vertebra after hemisection and evaluated in live animals' neuronal fibers at the bridge interface and within the bridge 8 weeks postimplant. These in vivo observations were correlated with in situ evaluation 12 weeks postimplantation. We sectioned the ... [truncated at 150 words]
Davey RJ, Digman MA, Gratton E, Moens PDJ.
Quantitative image mean squared displacement (iMSD) analysis of the dynamics of Profilin 1 at the membrane of live cells.
Methods. 2018; 140-141: 119-125. PMCID: PMC5995621Image mean square displacement analysis (iMSD) is a method allowing the mapping of diffusion dynamics of molecules in living cells. However, it can also be used to obtain quantitative information on the diffusion processes of fluorescently labelled molecules and how their diffusion dynamics change when the cell environment is modified. In this paper, we describe the use of iMSD to obtain quantitative data of the diffusion dynamics of a small cytoskeletal protein, profilin 1 (pfn1), at the membrane of live cells and how its diffusion is perturbed when the cells are treated with Cytochalasin D and/or the interactions of pfn1 are modified when its actin and polyphosphoinositide binding sites are mutated (pfn1-R88A). Using total internal reflection fluorescence microscopy images, we obtained data on isotropic and confined diffusion coefficients, the proportion of cell areas where isotropic diffusion is the major diffusion mode compared to the confined diffusion mode, the size of ... [truncated at 150 words]
Malacrida L, Rao E, Gratton E.
Comparison between iMSD and 2D-pCF analysis for molecular motion studies on in vivo cells: the case of the epidermal growth factor receptor.
Methods. 2018; 140-141: 74-84. PMCID: PMC5999564Image correlation analysis has evolved to become a valuable method of analysis of the diffusional motion of molecules in every points of a live cell. Here we compare the iMSD and the 2D-pCF approaches that provide complementary information. The iMSD method provides the law of diffusion and it requires spatial averaging over a small region of the cell. The 2D-pCF does not require spatial averaging and it gives information about obstacles for diffusion at pixel resolution. We show the analysis of the same set of data by the two methods to emphasize that both methods could be needed to have a comprehensive understanding of the molecular diffusional flow in a live cell.
Lee DH, Li X, Ma N, Digman MA, Lee AP.
Rapid and label-free identification of single leukemia cells from blood in a high-density microfluidic trapping array by fluorescence lifetime imaging microscopy.
Lab Chip. 2018; 18(9): 1349-1358.The rapid screening and isolation of single leukemia cells from blood has become critical for early leukemia detection and tumor heterogeneity interrogation. However, due to the size overlap between leukemia cells and the more abundant white blood cells (WBCs), the isolation and identification of leukemia cells individually from peripheral blood is extremely challenging and often requires immunolabeling or cytogenetic assays. Here we present a rapid and label-free single leukemia cell identification platform that combines: (1) high-throughput size-based separation of hemocytes via a single-cell trapping array, and (2) leukemia cell identification through phasor approach and fluorescence lifetime imaging microscopy (phasor-FLIM), to quantify changes between free/bound nicotinamide adenine dinucleotide (NADH) as an indirect measurement of metabolic alteration in living cells. The microfluidic trapping array designed with 1600 highly-packed addressable single-cell traps can simultaneously filter out red blood cells (RBCs) and trap WBCs/leukemia cells, and is compatible with low-magnification imaging and fast-speed fluorescence ... [truncated at 150 words]
Kobylkevich BM, Sarkar A, Carlberg BR, Huang L, Ranjit S, Graham DM, Messerli MA.
Reversing the direction of galvanotaxis with controlled increases in boundary layer viscosity.
Phys Biol. 2018; 15(3): 036005. PMCID: PMC5970543Weak external electric fields (EFs) polarize cellular structure and direct most migrating cells (galvanotaxis) toward the cathode, making it a useful tool during tissue engineering and for healing epidermal wounds. However, the biophysical mechanisms for sensing weak EFs remain elusive. We have reinvestigated the mechanism of cathode-directed water flow (electro-osmosis) in the boundary layer of cells, by reducing it with neutral, viscous polymers. We report that increasing viscosity with low molecular weight polymers decreases cathodal migration and promotes anodal migration in a concentration dependent manner. In contrast, increased viscosity with high molecular weight polymers does not affect directionality. We explain the contradictory results in terms of porosity and hydraulic permeability between the polymers rather than in terms of bulk viscosity. These results provide the first evidence for controlled reversal of galvanotaxis using viscous agents and position the field closer to identifying the putative electric field receptor, a fundamental, outside-in signaling ... [truncated at 150 words]
Staaf E, Hedde PN, Singh SB, Piguet J, Gratton E, Johansson S.
Educated natural killer cells show dynamic movement of the activating receptor NKp46 and confinement of the inhibitory receptor Ly49A.
Sci Signal. 2018; 11(517), eaai9200. PMCID: PMC5925740Educated natural killer (NK) cells have inhibitory receptors specific for self major histocompatibility complex (MHC) class I molecules and kill cancer cells more efficiently than do NK cells that do not have such receptors (hyporesponsive NK cells). The mechanism behind this functional empowerment through education has so far not been fully described. In addition, distinctive phenotypic markers of educated NK cells at the single-cell level are lacking. We developed a refined version of the image mean square displacement (iMSD) method (called iMSD carpet analysis) and used it in combination with single-particle tracking to characterize the dynamics of the activating receptor NKp46 and the inhibitory receptor Ly49A on resting educated versus hyporesponsive murine NK cells. Most of the NKp46 and Ly49A molecules were restricted to microdomains; however, individual NKp46 molecules resided in these domains for shorter periods and diffused faster on the surface of educated, compared to hyporesponsive, NK cells. In ... [truncated at 150 words]
Kong L, Murata MM, Digman MA.
Absence of REV3L promotes p53-regulated cancer cell metabolism in cisplatin-treated lung carcinoma cells.
Biochem Biophys Res Commun. 2018; 496(1): 199-204. PMCID: PMC6327949Lung cancer is one of the deadliest cancers in the world because of chemo-resistance to the commonly used cisplatin-based treatments. The use of low fidelity DNA polymerases in the translesional synthesis (TLS) DNA damage response pathway that repairs lesions caused by cisplatin also presents a mutational carcinogenic burden on cells that needs to be regulated by the tumor suppressor protein p53. However, there is much debate over the roles of the reversionless 3-like (REV3L) protein responsible for TLS and p53 in regulating cancer cell metabolism. In this study, the fluorescence lifetime of the metabolic coenzyme NADH reveals that the absence of REV3L can promote the p53-mediated upregulation of oxidative phosphorylation in cisplatin-treated H1299 lung carcinoma cells and increases cancer cell sensitivity to this platinum-based chemotherapy. These results demonstrate a previously unrecognized relationship between p53 and REV3L in cancer cell metabolism and may lead to improvements in chemotherapy treatment plans that ... [truncated at 150 words]
Mäntylä E, Chacko JV, Aho V, Parrish CR, Shahin V, Kann M, Digman MA, Gratton E, Vihinen-Ranta M.
Viral highway to nucleus exposed by image correlation analyses.
Sci Rep. 2018; 8(1): 1152. PMCID: PMC5773500Parvoviral genome translocation from the plasma membrane into the nucleus is a coordinated multistep process mediated by capsid proteins. We used fast confocal microscopy line scan imaging combined with image correlation methods including auto-, pair- and cross-correlation, and number and brightness analysis, to study the parvovirus entry pathway at the single-particle level in living cells. Our results show that the endosome-associated movement of virus particles fluctuates from fast to slow. Fast transit of single cytoplasmic capsids to the nuclear envelope is followed by slow movement of capsids and fast diffusion of capsid fragments in the nucleoplasm. The unique combination of image analyses allowed us to follow the fate of intracellular single virus particles and their interactions with importin β revealing previously unknown dynamics of the entry pathway.
Sameni S, Malacrida L, Tan Z, Digman MA.
Alteration in fluidity of cell plasma membrane in Huntington disease revealed by spectral phasor Analysis.
Sci Rep. 2018; 8(1): 734. PMCID: PMC5768877Huntington disease (HD) is a late-onset genetic neurodegenerative disorder caused by expansion of cytosine-adenine-guanine (CAG) trinucleotide in the exon 1 of the gene encoding the polyglutamine (polyQ). It has been shown that protein degradation and lipid metabolism is altered in HD. In many neurodegenerative disorders, impaired lipid homeostasis is one of the early events in the disease onset. Yet, little is known about how mutant huntingtin may affect phospholipids membrane fluidity. Here, we investigated how membrane fluidity in the living cells (differentiated PC12 and HEK293 cell lines) are affected using a hyperspectral imaging of widely used probes, LAURDAN. Using phasor approach, we characterized the fluorescence of LAURDAN that is sensitive to the polarity of the immediate environment. LAURDAN is affected by the physical order of phospholipids (lipid order) and reports the membrane fluidity. We also validated our results using a different fluorescent membrane probe, Nile Red (NR). The plasma membrane ... [truncated at 150 words]
Wang XX, Wang D, Luo Y, Myakala K, Dobrinskikh E, Rosenberg AZ, Levi J, Kopp JB, Field A, Hill A, Lucia S, Qiu L, Jiang T, Peng Y, Orlicky D, Garcia G, Herman-Edelstein M, D’Agati V, Henriksen K, Adorini L, Pruzanski M, Xie C, Krausz KW, Gonzalez FJ, Ranjit S, Dvornikov A, Gratton E, Levi M.
FXR/TGR5 dual agonist prevents progression of nephropathy in diabetes and obesity.
J Am Soc Nephrol. 2018; 29(1). PMCID: PMC5748904Bile acids are ligands for the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5. We have shown that FXR and TGR5 have renoprotective roles in diabetes- and obesity-related kidney disease. Here, we determined whether these effects are mediated through differential or synergistic signaling pathways. We administered the FXR/TGR5 dual agonist INT-767 to DBA/2J mice with streptozotocin-induced diabetes, db/db mice with type 2 diabetes, and C57BL/6J mice with high-fat diet-induced obesity. We also examined the individual effects of the selective FXR agonist obeticholic acid (OCA) and the TGR5 agonist INT-777 in diabetic mice. The FXR agonist OCA and the TGR5 agonist INT-777 modulated distinct renal signaling pathways involved in the pathogenesis and treatment of diabetic nephropathy. Treatment of diabetic DBA/2J and db/db mice with the dual FXR/TGR5 agonist INT-767 improved proteinuria and prevented podocyte injury, mesangial expansion, and tubulointerstitial fibrosis. INT-767 exerted coordinated effects on multiple ... [truncated at 150 words]
Malacrida L, Hedde PN, Ranjit S, Cardarelli F, Gratton E.
Visualization of barriers and obstacles to molecular diffusion in live cells by spatial pair-cross-correlation in two dimensions.
Biomed Opt Express. 2018; 9(1): 303-321. PMCID: PMC5772584Despite recent advances in optical super-resolution, we lack a method that can visualize the path followed by diffusing molecules in the cytoplasm or in the nucleus of cells. Fluorescence correlation spectroscopy (FCS) provides molecular dynamics at the single molecule level by averaging the behavior of many molecules over time at a single spot, thus achieving very good statistics but at only one point in the cell. Earlier image-based methods including raster-scan and spatiotemporal image correlation need spatial averaging over relatively large areas, thus compromising spatial resolution. Here, we use 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 the average path followed by molecules is created.
Gach JS, Bouzin M, Wong MP, Chromikova V, Gorlani A, Yu KT, Sharma B, Gratton E, Forthal DN.
Human Immunodeficiency Virus type-1 (HIV-1) evades antibody-dependent phagocytosis.
PLoS Pathog. 2017; 13(12), e1006793. PMCID: PMC5760106Fc gamma receptor (FcyR)-mediated antibody functions play a crucial role in preventing HIV infection. One such function, antibody-dependent phagocytosis (ADP), is thought to be involved in controlling other viral infections, but its role in HIV infection is unknown. We measured the ability of HIV-specific polyclonal and monoclonal antibodies (mAbs) to mediate the internalization of HIV-1 virions and HIV-1-decorated cells by phagocytes. To measure ADP of virions, we primarily used a green-fluorescent protein-expressing molecular clone of HIV-1JRFL, an R5, clinical isolate, in combination with polyclonal HIVIG or mAbs known to capture and/or neutralize HIV-1. THP-1 and U937 cells, as well as freshly isolated primary monocytes from healthy individuals, were used as phagocytic effector cells, and uptake of virions was measured by cytometry. We surprisingly found minimal or no ADP of virions with any of the antibodies. However, after coating virions with gp41 or with gp41-derived peptides, gp41- (but not gp120-) specific ... [truncated at 150 words]
Trinh AL, Chen H, Chen Y, Hu Y, Li Z, Siegel ER, Linskey ME, Wang PH, Digman MA, Zhou YH.
Tracking functional tumor cell subpopulations of malignant glioma by phasor fluorescence lifetime imaging microscopy of NADH.
Cancers (Basel). 2017; 9(12), E168. PMCID: PMC5742816Intra-tumoral heterogeneity is associated with therapeutic resistance of cancer and there exists a need to non-invasively identify functional tumor subpopulations responsible for tumor recurrence. 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. Using FLIM, we identified a significant shift towards longer NADH fluorescence lifetimes, suggesting an increase in the fraction of protein-bound NADH, in the invasive stem-like tumor-initiating cell (STIC) subpopulation relative to the tumor mass-forming cell (TMC) subpopulation of malignant gliomas. By applying our previously studied model to transition glioma from a majority of STIC to a majority of TMC in serum-adherent culture conditions following serial passages, we compared changes in NADH states, cellular respirations (oxidative phosphorylation and glycolysis), EGFR expression, and cell-growth speed over passages. We identified a significant positive ... [truncated at 150 words]
Bohórquez-Hernández A, Gratton E, Pacheco J, Asanov A, Vaca L.
Cholesterol modulates the cellular localization of Orai1 channels and its disposition among membrane domains.
Biochim Biophys Acta. 2017; 1862(12): 1481-1490. PMCID: PMC5902182Store Operated Calcium Entry (SOCE) is one of the most important mechanisms for calcium mobilization in to the cell. Two main proteins sustain SOCE: STIM1 that acts as the calcium sensor in the endoplasmic reticulum (ER) and Orai1 responsible for calcium influx upon depletion of ER. There are many studies indicating that SOCE is modulated by the cholesterol content of the plasma membrane (PM). However, a myriad of questions remain unanswered concerning the precise molecular mechanism by which cholesterol modulates SOCE.
In the present study we found that reducing PM cholesterol results in the internalization of Orai1 channels, which can be prevented by overexpressing caveolin 1 (Cav1). Furthermore, Cav1 and Orai1 associate upon SOCE activation as revealed by FRET and coimmunoprecipitation assays. The effects of reducing cholesterol were not limited to an increased rate of Orai1 internalization, but also, affects the lateral movement of Orai1, inducing movement in a linear pattern ... [truncated at 150 words]
Digiacomo L, Cardarelli F, Pozzi D, Palchetti S, Digman MA, Gratton E, Capriotti AL, Mahmoudi M, Caracciolo G.
An apolipoprotein-enriched biomolecular corona switches the cellular uptake mechanism and trafficking pathway of lipid nanoparticles.
Nanoscale. 2017; 9(44): 17254-17262. PMCID: PMC5700750Following exposure to biological milieus (e.g. after systemic administration), nanoparticles (NPs) get covered by an outer biomolecular corona (BC) that defines many of their biological outcomes, such as the elicited immune response, biodistribution, and targeting abilities. In spite of this, the role of BC in regulating the cellular uptake and the subcellular trafficking properties of NPs has remained elusive. Here, we tackle this issue by employing multicomponent (MC) lipid NPs, human plasma (HP) and HeLa cells as models for nanoformulations, biological fluids, and target cells, respectively. By conducting confocal fluorescence microscopy experiments and image correlation analyses, we quantitatively demonstrate that the BC promotes a neat switch of the cell entry mechanism and subsequent intracellular trafficking, from macropinocytosis to clathrin-dependent endocytosis. Nano-liquid chromatography tandem mass spectrometry identifies apolipoproteins as the most abundant components of the BC tested here. Interestingly, this class of proteins target the LDL receptors, which are overexpressed in ... [truncated at 150 words]
Malacrida L, Jameson DM, Gratton E.
A multidimensional phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes.
Sci Rep. 2017; 7(1): 9215. PMCID: PMC5569084Mammalian cell membranes have different phospholipid composition and cholesterol content, displaying a profile of fluidity that depends on their intracellular location. Among the dyes used in membrane studies, LAURDAN has the advantage to be sensitive to the lipid composition as well as to membrane fluidity. The LAURDAN spectrum is sensitive to the lipid composition and dipolar relaxation arising from water penetration, but disentangling lipid composition from membrane fluidity can be obtained if time resolved spectra could be measured at each cell location. Here we describe a method in which spectral and lifetime information obtained in different measurements at the same plane in a cell are used in the phasor plot providing a solution to analyze multiple lifetime or spectral data through a common visualization approach. We exploit a property of phasor plots based on the reciprocal role of the phasor plot and the image. In the phasor analysis each pixel ... [truncated at 150 words]
Hedde PN, Malacrida L, Ahrar S, Siryaporn A, Gratton E.
sideSPIM - selective plane illumination based on a conventional inverted microscope.
Biomed Opt Express. 2017; 8(9): 3918-3937. PMCID: PMC5611913Previously described selective plane illumination microscopy techniques typically offset ease of use and sample handling for maximum imaging performance or vice versa. Also, to reduce cost and complexity while maximizing flexibility, it is highly desirable to implement light sheet microscopy such that it can be added to a standard research microscope instead of setting up a dedicated system. We devised a new approach termed sideSPIM that provides uncompromised imaging performance and easy sample handling while, at the same time, offering new applications of plane illumination towards fluidics and high throughput 3D imaging of multiple specimen. Based on an inverted epifluorescence microscope, all of the previous functionality is maintained and modifications to the existing system are kept to a minimum. At the same time, our implementation is able to take full advantage of the speed of the employed sCMOS camera and piezo stage to record data at rates of up to ... [truncated at 150 words]
Liu L, Zhang SX, Liao W, Farhoodi HP, Wong CW, Chen CC, Ségaliny AI, Chacko JV, Nguyen LP, Lu M, Polovin G, Pone EJ, Downing TL, Lawson DA, Digman MA, Zhao W.
Mechanoresponsive stem cells to target cancer metastases through biophysical cues.
Sci Transl Med. 2017; 9(400), aan2966. PMCID: PMC5890431Despite decades of effort, little progress has been made to improve the treatment of cancer metastases. To leverage the central role of the mechanoenvironment in cancer metastasis, we present a mechanoresponsive cell system (MRCS) to selectively identify and treat cancer metastases by targeting the specific biophysical cues in the tumor niche in vivo. Our MRCS uses mechanosensitive promoter-driven mesenchymal stem cell (MSC)-based vectors, which selectively home to and target cancer metastases in response to specific mechanical cues to deliver therapeutics to effectively kill cancer cells, as demonstrated in a metastatic breast cancer mouse model. Our data suggest a strong correlation between collagen cross-linking and increased tissue stiffness at the metastatic sites, where our MRCS is specifically activated by the specific cancer-associated mechano-cues. MRCS has markedly reduced deleterious effects compared to MSCs constitutively expressing therapeutics. MRCS indicates that biophysical cues, specifically matrix stiffness, are appealing targets for cancer treatment due to ... [truncated at 150 words]
Stortz M, Presman DM, Bruno L, Annibale P, Dansey MV, Burton G, Gratton E, Pecci A, Levi V.
Mapping the dynamics of the Glucocorticoid Receptor within the nuclear landscape.
Sci Rep. 2017; 7(1), 6219. PMCID: PMC5524710The distribution of the transcription machinery among different sub-nuclear domains raises the question on how the architecture of the nucleus modulates the transcriptional response. Here, we used fluorescence fluctuation analyses to quantitatively explore the organization of the glucocorticoid receptor (GR) in the interphase nucleus of living cells. We found that this ligand-activated transcription factor diffuses within the nucleus and dynamically interacts with bodies enriched in the coregulator NCoA-2, DNA-dependent foci and chromatin targets. The distribution of the receptor among the nuclear compartments depends on NCoA-2 and the conformation of the receptor as assessed with synthetic ligands and GR mutants with impaired transcriptional abilities. Our results suggest that the partition of the receptor in different nuclear reservoirs ultimately regulates the concentration of receptor available for the interaction with specific targets, and thus has an impact on transcription regulation.
Herrington KA, Trinh AL, Dang C, O’Shaughnessy E, Hahn KM, Gratton E, Digman MA, Sütterlin C.
Spatial analysis of Cdc42 activity reveals a role for plasma membrane-associated Cdc42 in centrosome regulation.
Mol Biol Cell. 2017; 28(15): 2135-2145. PMCID: PMC5509425The ability of the small GTPase Cdc42 to regulate diverse cellular processes depends on tight spatial control of its activity. Cdc42 function is best understood at the plasma membrane (PM), where it regulates cytoskeletal organization and cell polarization. Active Cdc42 has also been detected at the Golgi, but its role and regulation at this organelle is only partially understood. Here we analyze the spatial distribution of Cdc42 activity by monitoring the dynamics of the Cdc42 FLARE biosensor using the phasor approach to FLIM-FRET. Phasor analysis revealed that Cdc42 is active at all Golgi cisternae, and that this activity is controlled by Tuba and ARHGAP10, two Golgi-associated Cdc42 regulators. To our surprise, FGD1, another Cdc42 GEF at the Golgi, was not required for Cdc42 regulation at the Golgi, although its depletion lowered Cdc42 activity at the PM. Similarly, changes in Golgi morphology did not affect Cdc42 activity at the Golgi, but ... [truncated at 150 words]
Bhattacharjee A, Datta R, Gratton E, Hochbaum AI.
Metabolic fingerprinting of bacteria by fluorescence lifetime imaging microscopy.
Sci Rep. 2017; 7(1), 3743. PMCID: PMC5473825Bacterial populations exhibit a range of metabolic states influenced by their environment, intra- and interspecies interactions. The identification of bacterial metabolic states and transitions between them in their native environment promises to elucidate community behavior and stochastic processes, such as antibiotic resistance acquisition. In this work, we employ two-photon fluorescence lifetime imaging microscopy (FLIM) to create a metabolic fingerprint of individual bacteria and populations. FLIM of autofluorescent reduced nicotinamide adenine dinucleotide (phosphate), NAD(P)H, has been previously exploited for label-free metabolic imaging of mammalian cells. However, NAD(P)H FLIM has not been established as a metabolic proxy in bacteria. Applying the phasor approach, we create FLIM-phasor maps of Escherichia coli, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, Bacillus subtilis, and Staphylococcus epidermidis at the single cell and population levels. The bacterial phasor is sensitive to environmental conditions such as antibiotic exposure and growth phase, suggesting that observed shifts in the phasor are representative ... [truncated at 150 words]
Ranjit S, Dvornikov A, Dobrinskikh E, Wang X, Luo Y, Levi M, Gratton E.
Measuring the effect of a Western diet on liver tissue architecture by FLIM autofluorescence and harmonic generation microscopy.
Biomed Opt Express. 2017; 8(7): 3143-3154. PMCID: PMC5508820The phasor approach to auto-fluorescence lifetime imaging was used to identify and characterize a long lifetime species (LLS) (~7.8 ns) in livers of mice fed with a Western diet. The size of the areas containing this LLS species depends on the type of diet and the size distribution shows Western diet has much larger LLS sizes. Combination of third harmonic generation images with FLIM identified the LLS species with fat droplets and the droplet size distribution was estimated. Second harmonic generation microscopy combined with phasor FLIM shows that there is an increase in fibrosis with a Western diet. A new decomposition in three components of the phasor plot shows that a Western diet is correlated with a higher fraction of free NADH, signifying more reducing condition and more glycolytic condition. Multiparametric analysis of phasor distribution shows that from the distribution of phasor points, a Western diet fed versus a low ... [truncated at 150 words]
Ceccarelli A, di Venere A, Nicolai E, de Luca A, Rosato N, Gratton E, Mei G, Caccuri AM.
New insight into the interaction of TRAF2 C-terminal domain with lipid raft microdomains.
Biochim Biophys Acta. 2017; 1862(9): 813-822. PMCID: PMC5562040In this study we provide the first evidence of the interaction of a truncated-TRAF2 with lipid raft microdomains. We have analyzed this interaction by measuring the diffusion coefficient of the protein in large and giant unilamellar vesicles (LUVs and GUVs, respectively) obtained both from synthetic lipid mixtures and from natural extracts. Steady-state fluorescence measurements performed with synthetic vesicles indicate that this truncated form of TRAF2 displays a tighter binding to raft-like LUVs with respect to the control (POPC-containing LUVs), and that this process depends on the protein oligomeric state. Generalized Polarization measurements and spectral phasor analysis revealed that truncated-TRAF2 affects the membrane fluidity, especially when vesicles are heated up at physiological temperature. The addition of nanomolar concentration of TRAF2 in GUVs also seems to exert a mechanical action, as demonstrated by the formation of intraluminal vesicles, a process in which ganglioside GM1 plays a crucial role.
Liang EI, Mah EJ, Yee AF, Digman MA.
Correlation of focal adhesion assembly and disassembly with cell migration on nanotopography.
Integr Biol (Camb). 2017; 9(2): 145-155. PMCID: PMC5399776Selective cell adhesion is desirable to control cell growth and migration on biomedical implants. Mesenchymal cell migration is regulated through focal adhesions (FAs) and can be modulated by their microenvironment including changes in surface topography. We use the Number and Molecular Brightness (N&B) imaging analysis to provide a unique perspective on FA assembly and disassembly. This imaging analysis provides a map of real-time fluctuations of protein monomers, dimers, and higher order aggregates of paxillin proteins in FA during assembly and disassembly. We show a dynamic view of how nanostructured surfaces (nanoline gratings or nanopillars) regulate single molecular dynamics. In particular, we report that the smallest nanopillars (100 nm spacing) gave rise to a low percentage population of disassembly adhesion cluster size of ~2 paxillin proteins/cluster whereas the larger nanopillars (380 nm spacing) gave rise to a much larger population of bigger disassembling cluster of ~3-5 paxillin proteins. Cells were more ... [truncated at 150 words]
de Vega GB, Ceballos JA, Anzalone A, Digman MA, Gratton E.
A laser-scanning confocal microscopy study of carrageenan in red algae from seaweed farms near the Caribbean entrance of the Panama Canal.
J Appl Phycol. 2017; 29(1): 495-508.Kappaphycus alvarezii (Doty) Doty ex P.C. Silva, a red macroalga, is a commercial source of carrageenan, a widely used polysaccharide compound important in the food and pharmaceutical industries, in nanotechnology, and in pharmacological applications. Carrageenan is found mainly in the cell wall and in the intercellular matrix. This is the first study to propose the characterization of carrageenans in vitro, using the auto-fluorescence properties of the alga treated with different polyamines: putrescine, spermidine, and spermine. This study suggests a four-phase cultivation sequence for seaweed farmers to enhance and assess the potential carrageenan yield of their crops. In phase 1, seedlings were treated with each of the polyamines. Explants were subsequently transferred through two additional culture phases before being planted on the sea farms in phase 4 and then harvested after 60 days for analysis. Images from transverse sections of 11 representative cultured K. alvarezii samples were obtained at 561 nm ... [truncated at 150 words]
Romero-López M, Trinh AL, Sobrino A, Hatch MMS, Keating MT, Fimbres C, Lewis DE, Gershon PD, Botvinick EL, Digman MA, Lowengrub JS, Hughes CCW.
Recapitulating the human tumor microenvironment: Colon tumor-derived extracellular matrix promotes angiogenesis and tumor cell growth.
Biomaterials. 2017; 116: 118-129. PMCID: PMC5226635Extracellular matrix (ECM) is an essential and dynamic component of all tissues and directly affects cellular behavior by providing both mechanical and biochemical signaling cues. Changes in ECM can alter tissue homeostasis, potentially leading to promotion of cellular transformation and the generation of tumors. Therefore, understanding ECM compositional changes during cancer progression is vital to the development of targeted treatments. Previous efforts to reproduce the native 3D cellular microenvironment have utilized protein gels and scaffolds that incompletely recapitulate the complexity of native tissues. Here, we address this problem by extracting and comparing ECM from normal human colon and colon tumor that had metastasized to liver. We found differences in protein composition and stiffness, and observed significant differences in vascular network formation and tumor growth in each of the reconstituted matrices, both in vitro and in vivo. We studied free/bound ratios of NADH in the tumor and endothelial cells using Fluorescence Lifetime Imaging ... [truncated at 150 words]
Porcaro F, Miao Y, Kota R, Haun JB, Polzonetti G, Battocchio C, Gratton E.
Fluctuation spectroscopy analysis of glucose capped gold nanoparticles.
Langmuir. 2016; 32(50): 13409-13417. PMCID: PMC5470844In this work, we report the synthesis and biophysical studies carried out on a new kind of biocompatible and very stable gold nanoparticle (GNP) stabilized with glucose through a PEG linker (AuNP-PEG-Glu). The synthetic path was optimized to obtain nanoparticles of controlled sizes. ζ-potential and dynamic light scattering measurements allowed assessment of the nanodimension, dispersity, surface charge, and stability of our GNPs. Confocal microscopy demonstrated qualitatively that glucose molecules are successfully bonded to GNP surfaces. For our study, we selected nanoparticles with diameter in a range that maximizes the internalization efficiency in cells (40 nm). A detailed investigation about the biophysical proprieties of AuNP-PEG-Glu was carried out by means of fluorescence correlation spectroscopy (FCS) and orbital tracking techniques. This work gives new insights about the uptake mechanism of gold nanoparticles capped with glucose molecules.
Chen CC, Liu L, Ma F, Wong CW, Guo XE, Chacko JV, Farhoodi HP, Zhang SX, Zimak J, Ségaliny A, Riazifar M, Pham V, Digman MA, Pone EJ, Zhao W.
Elucidation of exosome migration across the blood-brain barrier model in vitro.
Cell Mol Bioeng. 2016; 9(4): 509-529. PMCID: PMC5382965The delivery of therapeutics to the central nervous system remains a major challenge in part due to the presence of the blood–brain barrier (BBB). Recently, cell-derived vesicles, particularly exosomes, have emerged as an attractive vehicle for targeting drugs to the brain, but whether or how they cross the BBB remains unclear. Here, we investigated the interactions between exosomes and brain microvascular endothelial cells (BMECs) in vitro under conditions that mimic the healthy and inflamed BBB in vivo. Transwell assays revealed that luciferase-carrying exosomes can cross a BMEC monolayer under stroke-like, inflamed conditions (TNF-α activated) but not under normal conditions. Confocal microscopy showed that exosomes are internalized by BMECs through endocytosis, co-localize with endosomes, in effect primarily utilizing the transcellular route of crossing. Together, these results indicate that cell-derived exosomes can cross the BBB model under stroke-like conditions in vitro. This study encourages further development of engineered exosomes as drug delivery ... [truncated at 150 words]
Malacrida L, Astrada S, Briva A, Bollati-Fogolín M, Gratton E, Bagatolli LA.
Spectral Phasor analysis of LAURDAN fluorescence in live A549 lung cells to study the hydration and time evolution of intracellular lamellar body-like structures.
Biochim Biophys Acta. 2016; 1858(11): 2625-2635. PMCID: PMC5045802Using LAURDAN spectral imaging and spectral phasor analysis we concurrently studied the growth and hydration state of subcellular organelles (Lamellar Body-like, LB-like) from live A549 lung cancer cells at different post-confluence days. Our results reveal a time dependent two-step process governing the size and hydration of these intracellular LB-like structures. Specifically, a first step (days 1 to 7) is characterized by an increase in their size, followed by a second one (days 7 to 14) where the organelles display a decrease in their global hydration properties. Interestingly, our results also show that their hydration properties significantly differ from those observed in well-characterized artificial lamellar model membranes, challenging the notion that a pure lamellar membrane organization is present in these organelles at intracellular conditions. Finally, these LB-like structures show a significant increase in their hydration state upon secretion, suggesting a relevant role of entropy during this process.
Ranjit S, Dobrinskikh E, Montford J, Dvornikov A, Lehman A, Orlicky DJ, Nemenoff R, Gratton E, Levi M, Furgeson S.
Label-free fluorescence lifetime and second harmonic generation imaging microscopy improves quantification of experimental renal fibrosis.
Kidney Int. 2016; 90(5): 1123-1128. PMCID: PMC5473685All forms of progressive renal diseases develop a final pathway of tubulointerstitial fibrosis and glomerulosclerosis. Renal fibrosis is usually quantified using histological staining, a process that is time-consuming and pathologist dependent. Here we develop a fast and operator-independent method to measure fibrosis utilizing the murine unilateral ureteral obstruction model which manifests a time-dependent fibrotic increase in obstructed kidneys while the contralateral kidneys are used as controls. After ureteral obstruction, kidneys were analyzed at 7, 14, and 21 days. Fibrosis was quantified using fluorescence lifetime imaging (FLIM) and second harmonic generation (SHG) in a Deep Imaging via Enhanced photon Recovery deep tissue imaging microscope. This microscope was developed for deep tissue along with second and third harmonic generation imaging and has extraordinary sensitivity toward harmonic generation. SHG data suggest the presence of more fibrillar collagen in the obstructed kidneys. The combination of short-wavelength FLIM and SHG analysis results in a robust ... [truncated at 150 words]
Aguilar-Arnal L, Ranjit S, Stringari C, Orozco-Solis R, Gratton E.
Spatial dynamics of SIRT1 and the subnuclear distribution of NADH species.
Proc Natl Acad Sci USA. 2016; 113(45): 12715-12720. PMCID: PMC5111721Sirtuin 1 (SIRT1) is an NAD+-dependent deacetylase that functions as metabolic sensor of cellular energy and modulates biochemical pathways in the adaptation to changes in the environment. SIRT1 substrates include histones and proteins related to enhancement of mitochondrial function as well as antioxidant protection. Fluctuations in intracellular NAD+ levels regulate SIRT1 activity, but how SIRT1 enzymatic activity impacts on NAD+ levels and its intracellular distribution remains unclear. Here, we show 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 in single living cells, we reveal that NAD+ metabolism in the nucleus is linked to subnuclear dynamics of active SIRT1. These results reveal a ... [truncated at 150 words]
Sameni S, Syed A, Marsh JL, Digman MA.
The phasor-FLIM fingerprints reveal shifts from OXPHOS to enhanced glycolysis in Huntington Disease.
Sci Rep. 2016; 6: 34755. PMCID: PMC5054433Huntington disease (HD) is an autosomal neurodegenerative disorder caused by the expansion of Polyglutamine (polyQ) in exon 1 of the Huntingtin protein. Glutamine repeats below 36 are considered normal while repeats above 40 lead to HD. Impairment in energy metabolism is a common trend in Huntington pathogenesis; however, this effect is not fully understood. Here, we used the phasor approach and Fluorescence Lifetime Imaging Microscopy (FLIM) to measure changes between free and bound fractions of NADH as a indirect measure of metabolic alteration in living cells. Using Phasor-FLIM, pixel maps of metabolic alteration in HEK293 cell lines and in transgenic Drosophila expressing expanded and unexpanded polyQ HTT exon1 in the eye disc were developed. We found a significant shift towards increased free NADH, indicating an increased glycolytic state for cells and tissues expressing the expanded polyQ compared to unexpanded control. In the nucleus, a further lifetime shift occurs towards higher ... [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.
Biol Reprod. 2016; 95(6): 129. PMCID: PMC5315427Previous work characterizing ovarian bioenergetics has defined follicular metabolism by measuring metabolic byproducts 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 fluorescent lifetime imaging microscopy (Phasor FLIM) at 740 nm 2-photon excitation to examine the spatial distribution 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 pre-pubertal ovaries. We obtained subcellular resolution Phasor FLIM images of primordial through primary follicles and quantified the free/bound NADH ratio (relative NADH/NAD+) separately 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 increasing ... [truncated at 150 words]
Qiu Y, Lin CY, Hinkle P, Plett TS, Yang C, Chacko JV, Digman MA, Yeh LH, Hsu JP, Siwy ZS.
Highly charged particles cause a larger current blockage in micropores compared to neutral particles.
ACS Nano. 2016; 10(9): 8413-8422.Single pores in the resistive-pulse technique are used as an analytics tool to detect, size, and characterize physical as well as chemical properties of individual objects such as molecules and particles. Each object passing through a pore causes a transient change of the transmembrane current called a resistive pulse. In high salt concentrations when the pore diameter is significantly larger than the screening Debye length, it is assumed that the particle size and surface charge can be determined independently from the same experiment. In this article we challenge this assumption and show that highly charged hard spheres can cause a significant increase of the resistive-pulse amplitude compared to neutral particles of a similar diameter. As a result, resistive pulses overestimate the size of charged particles by even 20%. The observation is explained by the effect of concentration polarization created across particles in a pore, revealed by numerical modeling of ionic ... [truncated at 150 words]
Digiacomo L, Digman MA, Gratton E, Caracciolo G.
Development of an image Mean Square Displacement (iMSD)-based method as a novel approach to study the intracellular trafficking of nanoparticles.
Acta Biomater. 2016; 42: 189-198. PMCID: PMC5483853Fluorescence microscopy and spectroscopy techniques are commonly used to investigate complex and interacting biological systems (e.g. proteins and nanoparticles in living cells), since these techniques can explore intracellular dynamics with high time resolution at the nanoscale. Here we extended one of the Image Correlation Spectroscopy (ICS) methods, i.e. the image Mean Square Displacement, in order to study 2-dimensional diffusive and flow motion in confined systems, whose driving speed is uniformly distributed in a variable angular range. Although these conditions are not deeply investigated in the current literature, they can be commonly found in the intracellular trafficking of nanocarriers, which diffuse in the cytoplasm and/or may move along the cytoskeleton in different directions. The proposed approach could reveal the underlying system's symmetry using methods derived from fluorescence correlation concepts and could recover dynamic and geometric features which are commonly done by single particle analyses. Furthermore, it improves the characterization of low-speed ... [truncated at 150 words]
Serebryannyy LA, Parilla M, Annibale P, Cruz CM, Laster K, Gratton E, Kudryashov D, Kosak ST, Gottardi CJ, de Lanerolle P.
Persistent nuclear actin filaments inhibit transcription by RNA polymerase II.
J Cell Sci. 2016; 129(18): 3412-3425. PMCID: PMC5047679Actin is abundant in the nucleus and it is clear that nuclear actin has important functions. Yet, mystery surrounds the absence of classical actin filaments in the nucleus. To address this question, we investigated how polymerizing nuclear actin into persistent nuclear actin filaments affected transcription by RNA polymerase II. Nuclear filaments impaired nuclear actin dynamics by polymerizing and sequestering nuclear actin. Polymerizing actin into stable nuclear filaments disrupted the interaction of actin with RNA polymerase II and correlated with impaired RNA polymerase II localization, dynamics, gene recruitment, and reduced global transcription and cell proliferation. Polymerizing and crosslinking nuclear actin in vitro similarly disrupted the actin/RNA polymerase II interaction and inhibited transcription. These data rationalize the general absence of stable actin filaments in mammalian somatic nuclei. They also suggest a dynamic pool of nuclear actin is required for the proper localization and activity of RNA polymerase II.
Chen H, Gratton E, Digman MA.
Self-assisted optothermal trapping of gold nanorods under two-photon excitation.
Methods Appl Fluoresc. 2016; 4(3): 035003. PMCID: PMC5497913We report a 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 enhance the optothermal trapping of other particles. This trapping phenomenon is the net result of thermophoresis and a convective flow caused by localized heating. The heating is due to the conversion of absorbed photons into heat at GNR's longitudinal surface plasmon resonance (LSPR) wavelength. First, we investigated the optothermal trapping of GNRs at their LSPR wavelength on the glass surface with as low as 0.5 mW laser power. The trapping range was observed to be larger than a typical field of view, e.g. 210 µm × 210 µm here. Second, by adjusting the distance between the laser focus and the glass surface, ring patterns of GNRs on the glass surface were obtained. These patterns could be controlled by the laser ... [truncated at 150 words]
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Imaging in turbid media: a transmission detector gives 2-3 order of magnitude enhanced sensitivity compared to epi-detection schemes.
Biomed Opt Express. 2016; 7(9): 3747-3755. PMCID: PMC5030047Imaging depth in turbid media by two-photon fluorescence microscopy depends on the ability of the optical system to detect weak fluorescence signals. We have shown that use of a wide area detector in transmission geometry allows increasing imaging depth in turbid media due to efficient photon collection. Compared to the conventional epi-detection scheme used in most commercial microscopes, the transmission detector was found to be 2–3 orders of magnitude more sensitive when used for in depth imaging in scattering samples simulating brain optical properties.
Di Rienzo C, Gratton E, Beltram F, Cardarelli F.
Spatiotemporal fluctuation analysis: a powerful tool for the future nanoscopy of molecular processes.
Biophys J. 2016; 111(4): 679-685. PMCID: PMC5002078The enormous wealth of information available today from optical microscopy measurements on living samples is often underexploited. We argue that spatiotemporal analysis of fluorescence fluctuations using multiple detection channels can enhance the performance of current nanoscopy methods and provide further insight into dynamic molecular processes of high biological relevance.
Di Rienzo C, Cardarelli F, Di Luca M, Beltram F, Gratton E.
Diffusion tensor analysis by two-dimensional pair correlation of fluorescence fluctuations in cells.
Biophys J. 2016; 111(4): 841-851. PMCID: PMC5002073In a living cell, the movement of biomolecules is highly regulated by the cellular organization into subcompartments that impose barriers to diffusion, can locally break the spatial isotropy, and ultimately guide these molecules to their targets. Despite the pivotal role of these processes, experimental tools to fully probe the complex connectivity (and accessibility) of the cell interior with adequate spatiotemporal resolution are still lacking. Here, we show how the heterogeneity of molecular dynamics and the location of barriers to molecular motion can be mapped in live cells by exploiting a two-dimensional (2D) extension of the pair correlation function (pCF) analysis. Starting from a time series of images collected for the same field of view, the resulting 2D pCF is calculated in the proximity of each point for each time delay and allows us to probe the spatial distribution of the molecules that started from a given pixel. This 2D pCF ... [truncated at 150 words]
Sobrino A, Phan DTT, Datta R, Wang X, Hachey SJ, Romero-López M, Gratton E, Lee AP, George SC, Hughes CCW.
3D microtumors in vitro supported by perfused vascular networks.
Sci Rep. 2016; 6, 31589. PMCID: PMC4994029There is a growing interest in developing microphysiological systems that can be used to model both normal and pathological human organs in vitro. This "organs-on-chips" approach aims to capture key structural and physiological characteristics of the target tissue. Here we describe in vitro vascularized microtumors (VMTs). This "tumor-on-a-chip" platform incorporates human tumor and stromal cells that grow in a 3D extracellular matrix and that depend for survival on nutrient delivery through living, perfused microvessels. Both colorectal and breast cancer cells grow vigorously in the platform and respond to standard-of-care therapies, showing reduced growth and/or regression. Vascular-targeting agents with different mechanisms of action can also be distinguished, and we find that drugs targeting only VEGFRs (Apatinib and Vandetanib) are not effective, whereas drugs that target VEGFRs, PDGFR and Tie2 (Linifanib and Cabozantinib) do regress the vasculature. Tumors in the VMT show strong metabolic heterogeneity when imaged using NADH Fluorescent Lifetime Imaging ... [truncated at 150 words]
Ranjit S, Dvornikov A, Levi M, Furgeson S, Gratton E.
Characterizing fibrosis in UUO mice model using multiparametric analysis of phasor distribution from FLIM images.
Biomed Opt Express. 2016; 7(9): 3519-3530. PMCID: PMC5030029Phasor approach to fluorescence lifetime microscopy is used to study development of fibrosis in the unilateral ureteral obstruction model (UUO) of kidney in mice. Traditional phasor analysis has been modified to create a multiparametric analysis scheme that splits the phasor points in four equidistance segments based on the height of peak of the phasor distribution and calculates six parameters including average phasor positions, the shape of each segment, the angle of the distribution and the number of points in each segment. These parameters are used to create a spectrum of twenty four points specific to the phasor distribution of each sample. Comparisons of spectra from diseased and healthy tissues result in quantitative separation and calculation of statistical parameters including AUC values, positive prediction values and sensitivity. This is a new method in the evolving field of analyzing phasor distribution of FLIM data and provides further insights. Additionally, the progression of ... [truncated at 150 words]
Scipion L, Gratton E, Diaspro A, Lanzanò L.
Phasor analysis of local ICS detects heterogeneity in size and number of intracellular vesicles.
Biophys J. 2016; 111(3): 619-629. PMCID: PMC4982927Organelles represent the scale of organization immediately below that of the cell itself, and their composition, size, and number are tailored to their function. Monitoring the size and number of organelles in live cells is relevant for many applications but can be challenging due to their highly heterogeneous properties. Image correlation spectroscopy is a well-established analysis method capable of extracting the average size and number of particles in images. However, when image correlation spectroscopy is applied to a highly heterogeneous system, it can fail to retrieve, from a single correlation function, the characteristic size and the relative amount associated to each subspecies. Here, we describe a fast, unbiased, and fit-free algorithm based on the phasor analysis of multiple local image correlation functions, capable of mapping the sizes of elements contained in a heterogeneous system. The method correctly provides the size and number of separate subspecies, which otherwise would be hidden ... [truncated at 150 words]
Szymanska AF, Heylman C, Datta R, Gratton E, Nenadic Z.
Automated detection and analysis of depolarization events in human cardiomyocytes using MaDEC.
Comput Biol Med. 2016; 75: 109-117. PMCID: PMC4938768Optical imaging-based methods for assessing the membrane electrophysiology of in vitro human cardiac cells allow for non-invasive temporal assessment of the effect of drugs and other stimuli. Automated methods for detecting and analyzing the depolarization events (DEs) in image-based data allow quantitative assessment of these different treatments. In this study, we use 2-photon microscopy of fluorescent voltage-sensitive dyes (VSDs) to capture the membrane voltage of actively beating human induced pluripotent stem cell-derived cardiomyocytes (hiPS-CMs). We built a custom and freely available Matlab software, called MaDEC, to detect, quantify, and compare DEs of hiPS-CMs treated with the β-adrenergic drugs, propranolol and isoproterenol. The efficacy of our software is quantified by comparing detection results against manual DE detection by expert analysts, and comparing DE analysis results to known drug-induced electrophysiological effects. The software accurately detected DEs with true positive rates of 98–100% and false positive rates of 1–2%, at signal-to-noise ratios (SNRs) ... [truncated at 150 words]
Noutsi P, Gratton E, Chaieb S.
Assessment of membrane fluidity fluctuations during cellular development reveals time and cell type specificity.
PLoS One. 2016; 11(6): e0158313. PMCID: PMC4928918Cell membrane is made up of a complex structure of lipids and proteins that diffuse laterally giving rise to what we call membrane fluidity. During cellular development, such as differentiation cell membranes undergo dramatic fluidity changes induced by proteins such as ARC and Cofilin among others. In this study we used the generalized polarization (GP) property of fluorescent probe Laurdan using two-photon microscopy to determine membrane fluidity as a function of time and for various cell lines. A low GP value corresponds to a higher fluidity and a higher GP value is associated with a more rigid membrane. Four different cell lines were monitored such as hN2, NIH3T3, HEK293 and L6 cells. Membrane fluidity was measured at 12h, 72h and 92 h. Our results show significant changes in membrane fluidity among all cell types at different time points. GP values tend to increase significantly within 92 h in hN2 cells ... [truncated at 150 words]
Clark NM, Hinde E, Winter CM, Fisher AP, Crosti G, Blilou I, Gratton E, Benfey PN, Sozzani R.
Tracking transcription factor mobility and interaction in Arabidopsis roots with fluorescence correlation spectroscopy.
eLife. 2016; 5: e14770. PMCID: PMC4946880To understand complex regulatory processes in multicellular organisms, it is critical to be able to quantitatively analyze protein movement and protein-protein interactions in time and space. During Arabidopsis development, the intercellular movement of SHORTROOT (SHR) and subsequent interaction with its downstream target SCARECROW (SCR) control root patterning and cell fate specification. However, quantitative information about the spatio-temporal dynamics of SHR movement and SHR-SCR interaction is currently unavailable. Here, we quantify parameters including SHR mobility, oligomeric state, and association with SCR using a combination of Fluorescent Correlation Spectroscopy (FCS) techniques. We then incorporate these parameters into a mathematical model of SHR and SCR, which shows that SHR reaches a steady state in minutes, while SCR and the SHR-SCR complex reach a steady-state between 18 and 24 hours. Our model reveals the timing of SHR and SCR dynamics and allows us to understand how protein movement and protein-protein stoichiometry contribute to development.
Ma N, Digman MA, Malacrida L, Gratton E.
Measurements of absolute concentrations of NADH in cells using the phasor FLIM method.
Biomed Opt Express. 2016; 7(7): 2441-2452. PMCID: PMC4948605We 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 of a solution of NADH at a known concentration. The phasor representation of the fluorescence decay accounts for the differences in quantum yield of the free and bound form of NADH, pixel by pixel of an image. The concentration of NADH in every pixel in a cell is obtained after adding to each pixel in the phasor plot a given amount of unmodulated light which causes a shift of the phasor towards the origin by an amount that depends on the intensity at the pixel and the fluorescence lifetime at the pixel. The absolute concentration of NADH is obtained by comparison of the shift obtained at each pixel of an image with the shift of the calibrated solution.
Cardarelli F, Digiacomo L, Marchini C, Amici A, Salomone F, Fiume G, Rossetta A, Gratton E, Pozzi D, Caracciolo G.
The intracellular trafficking mechanism of Lipofectamine-based transfection reagents and its implication for gene delivery.
Sci Rep. 2016; 11(6): 25879. PMCID: PMC4863168Lipofectamine reagents are widely accepted as "gold-standard" for the safe delivery of exogenous DNA or RNA into cells. Despite this, a satisfactory mechanism-based explanation of their superior efficacy has remained mostly elusive thus far. Here we apply a straightforward combination of live cell imaging, single-particle tracking microscopy, and quantitative transfection-efficiency assays on live cells to unveil the intracellular trafficking mechanism of Lipofectamine/DNA complexes. We find that Lipofectamine, contrary to alternative formulations, is able to efficiently avoid active intracellular transport along microtubules, and the subsequent entrapment and degradation of the payload within acidic/digestive lysosomal compartments. This result is achieved by random Brownian motion of Lipofectamine-containing vesicles within the cytoplasm. We demonstrate here that Brownian diffusion is an efficient route for Lipofectamine/DNA complexes to avoid metabolic degradation, thus leading to optimal transfection. By contrast, active transport along microtubules results in DNA degradation and subsequent poor transfection. Intracellular trafficking, endosomal escape and lysosomal ... [truncated at 150 words]
Lin S, Hedde PN, Venugopalan V, Gratton E, Khine M.
Multi-scale silica structures for improved HIV-1 Capsid (p24) antigen detection.
Analyst. 2016; 141(13): 4181-4188. PMCID: PMC4924925Silica (SiO2) micro- and nanostructures fabricated with pre-stressed thermoplastic shrink wrap film have been shown to yield far-field fluorescence signal enhancements over their planar or wrinkled counterparts. The SiO2 structures have previously been used for improved detection of fluorescently labelled proteins and DNA. In this work, we probe the mechanism responsible for the dramatic increases in fluorescence signal intensity. Optical characterization studies attribute the fluorescence signal enhancements to increased surface density and light scattering from the rough SiO2 structures. Using this information, we come up with a theoretical approximation for the enhancement factor based off the scattering effects alone. We show that increased deposition thickness of SiO2 yields improved fluorescence signal enhancements, with an optimal SiO2 thin layer achieved at 20 nm. Finally, we show that the SiO2 substrates serve as a suitable platform for disease diagnostics, and show improved limits of detection (LOD) for the human immunodeficiency virus type ... [truncated at 150 words]
Borrego SL, Fahrmann J, Datta R, Stringari C, Grapov D, Zeller M, Chen Y, Wang P, Baldi P, Gratton E, Fiehn O, Kaiser P.
Metabolic changes associated with methionine stress sensitivity in MDA-MB-468 breast cancer cells.
Cancer Metab. 2016; 4: 9. PMCID: PMC4852440BACKGROUND: The majority of cancer cells have a unique metabolic requirement for methionine that is not observed in normal, non-tumorigenic cells. This phenotype is described as "methionine dependence" or "methionine stress sensitivity" in which cancer cells are unable to proliferate when methionine has been replaced with its metabolic precursor, homocysteine, in cell culture growth media. We focus on the metabolic response to methionine stress in the triple negative breast cancer cell line MDA-MB-468 and its methionine insensitive derivative cell line MDA-MB-468res-R8.
RESULTS: Using a variety of techniques including fluorescence lifetime imaging microscopy (FLIM) and extracellular flux assays, we identified a metabolic down-regulation of oxidative phosphorylation in both MDA-MB-468 and MDA-MB-468res-R8 cell types when cultured in homocysteine media. Untargeted metabolomics was performed by way of gas chromatography/time-of-flight mass spectrometry on both cell types cultured in homocysteine media over a period of 2 to 24 h. We determined unique metabolic responses between the ... [truncated at 150 words]
Alfonso-García A, Smith TD, Datta R, Luu TU, Gratton E, Potma EO, Liu WF.
Label-free identification of macrophage phenotype by fluorescence lifetime imaging microscopy.
J Biomed Opt. 2016; 21(4): 46005. PMCID: PMC4833856Macrophages adopt a variety of phenotypes that are a reflection of the many functions they perform as part of the immune system. In particular, metabolism is a phenotypic trait that differs between classically activated, proinflammatory macrophages, and alternatively activated, prohealing macrophages. Inflammatory macrophages have a metabolism based on glycolysis while alternatively activated macrophages generally rely on oxidative phosphorylation to generate chemical energy. We employ this shift in metabolism as an endogenous marker to identify the phenotype of individual macrophages via live-cell fluorescence lifetime imaging microscopy (FLIM). We demonstrate that polarized macrophages can be readily discriminated with the aid of a phasor approach to FLIM, which provides a fast and model-free method for analyzing fluorescence lifetime images.
Sosnik J, Zheng L, Rackauckas CV, Digman MA, Gratton E, Nie Q, Schilling TF.
Noise modulation in retinoic acid signaling sharpens segmental boundaries of gene expression in the embryonic zebrafish hindbrain.
eLife. 2016; 5: e14034. PMCID: PMC4829421Morphogen gradients induce sharply defined domains of gene expression in a concentration-dependent manner, yet how cells interpret these signals in the face of spatial and temporal noise remains unclear. Using fluorescence lifetime imaging microscopy (FLIM) and phasor analysis to measure endogenous retinoic acid (RA) directly in vivo, we have investigated the amplitude of noise in RA signaling, and how modulation of this noise affects patterning of hindbrain segments (rhombomeres) in the zebrafish embryo. We demonstrate that RA forms a noisy gradient during critical stages of hindbrain patterning and that cells use distinct intracellular binding proteins to attenuate noise in RA levels. Increasing noise disrupts sharpening of rhombomere boundaries and proper patterning of the hindbrain. These findings reveal novel cellular mechanisms of noise regulation, which are likely to play important roles in other aspects of physiology and disease.
Datta R, Heylman C, George SC, Gratton E.
Label-free imaging of metabolism and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes.
Biomed Opt Express. 2016; 7(5): 1690-701. PMCID: PMC4871074In this work we demonstrate a label-free optical imaging technique to assess metabolic status and oxidative stress in human induced pluripotent stem cell-derived cardiomyocytes by two-photon fluorescence lifetime imaging of endogenous fluorophores. Our results show the sensitivity of this method to detect shifts in metabolism and oxidative stress in the cardiomyocytes upon pathological stimuli of hypoxia and cardiotoxic drugs. This non-invasive imaging technique could prove beneficial for drug development and screening, especially for in vitro cardiac models created from stem cell-derived cardiomyocytes and to study the pathogenesis of cardiac diseases and therapy.
Hinde E, Pandžić E, Yang Z, Ng IHW, Jans DA, Bogoyevitch MA, Gratton E, Gaus K.
Quantifying the dynamics of the oligomeric transcription factor STAT3 by pair correlation of molecular brightness.
Nat Commun. 2016; 7: 11047. PMCID: PMC4820838Oligomerization of transcription factors controls their translocation into the nucleus and DNA-binding activity. Here we present a fluorescence microscopy analysis termed pCOMB (pair correlation of molecular brightness) that tracks the mobility of different oligomeric species within live cell nuclear architecture. 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. We use this method to demonstrate a dependence of signal transducer and activator of transcription 3 (STAT3) mobility on oligomeric state. We find that on 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 that chromatin accessibility modulates STAT3 tetramer formation. Thus, the pCOMB approach is suitable for mapping the impact oligomerization on transcription factor dynamics.
Brenner MD, Zhou R, Conway DE, Lanzanò L, Gratton E, Schwartz MA, Ha T.
Spider silk peptide is a compact, linear nano-spring ideal for intracellular tension sensing.
Nano Lett. 2016; 16(3): 2096-2102. PMCID: PMC4851340Recent development and applications of calibrated, FRET-based tension sensors have led to a new understanding of single molecule mechanotransduction in a number of biological systems. To expand the range of accessible forces, we systematically measured FRET vs. force trajectories for 25, 40 and 50 amino acid peptide repeats derived from spider silk. Single molecule fluorescence-force spectroscopy showed that the peptides behaved as linear springs instead of the nonlinear behavior expected for a disordered polymer. Our data are consistent with a compact, rod-like structure that measures 0.26 nm per 5 amino acid repeat that can stretch by 500% while maintaining linearity, suggesting that the remarkable elasticity of spider silk proteins may in part derive from the properties of individual chains. We found the shortest peptide to have the widest range of force sensitivity: between 2 pN and 11 pN. Live cell imaging of the three tension sensor constructs inserted into vinculin ... [truncated at 150 words]
Wang B, Rong X, Duerr MA, Hermanson DJ, Hedde PN, Wong JS, de Vallim TQA, Cravatt BF, Gratton E, Ford DA, Tontonoz P.
Intestinal phospholipid remodeling is required for dietary-lipid uptake and survival on a high-fat diet.
Cell Metab. 2016; 23: 1-13. PMCID: PMC4785086Phospholipids are important determinants of membrane biophysical properties, but the impact of membrane acyl chain composition on dietary-lipid absorption is unknown. Here we demonstrate that the LXR-responsive phospholipid-remodeling enzyme Lpcat3 modulates intestinal fatty acid and cholesterol absorption and is required for survival on a high-fat diet. Mice lacking Lpcat3 in the intestine thrive on carbohydrate-based chow but lose body weight rapidly and become moribund on a triglyceride-rich diet. Lpcat3-dependent incorporation of polyunsaturated fatty acids into phospholipids is required for the efficient transport of dietary lipids into enterocytes. Furthermore, loss of Lpcat3 amplifies the production of gut hormones, including GLP-1 and oleoylethanolamide, in response to high-fat feeding, contributing to the paradoxical cessation of food intake in the setting of starvation. These results reveal that membrane phospholipid composition is a gating factor in passive lipid absorption and implicate LXR-Lpcat3 signaling in a gut-brain feedback loop that couples absorption to food intake.
Annibale P, Dvornikov A, Gratton E.
Optical measurement of focal offset in tunable lenses.
Opt Express. 2016; 24(2): 1031-1036. PMCID: PMC4741313Electrically tunable lenses are becoming a widely used optical tool, and have brought significant innovation to microscopy methods. One current limitation of such systems is the difficulty of directly monitor the focal change in real time. Affordable and reliable feedback for such lenses, compatible with any microscopy setup, represents a much-needed improvement that is still not widely available. We discuss here the implementation and technical performance of an optical device to measure with a high frequency response the displacement of the focal offset of a commercial tunable lens with a precision in the range of the axial Point Spread Function (PSF) of the microscope. The technology presented is cost effective and can be employed on any microscopy setup.
Moore JS, Xantheas SS, Grate JW, Wietsma TW, Gratton E, Vasdekis AE.
Modular polymer biosensors by solvent immersion imprint lithography.
J Polym Sci B Polym Phys. 2016; 54(1): 98-103. PMCID: PMC5111810We recently demonstrated Solvent Immersion Imprint Lithography (SIIL), a rapid benchtop microsystem prototyping technique, including polymer functionalization, imprinting and bonding. Here, we focus on the realization of planar polymer sensors using SIIL through simple solvent immersion without imprinting. We describe SIIL's impregnation characteristics, including an inherent mechanism that not only achieves practical doping concentrations, but their unexpected 2-fold enhancement compared to the immersion solution. Subsequently, we developed and characterized optical sensors for detecting molecular O2. To this end, a substantially high dynamic range is reported, including its control through the immersion duration, a manifestation of SIIL's modularity. Overall, SIIL exhibits the potential of improving the operating characteristics of polymer sensors, while significantly accelerating their prototyping, as it requires a few seconds of processing and no need for substrates or dedicated instrumentation. These are critical for O2 sensing as probed by way of example here, as well as any polymer permeable ... [truncated at 150 words]
Heylman C, Datta R, Sobrino A, George S, Gratton E.
Supervised machine learning for classification of the electrophysiological effects of chronotropic drugs on human induced pluripotent stem cell-derived cardiomyocytes.
PLoS One. 2015; 10(12): e0144572. PMCID: PMC4690607Supervised machine learning can be used to predict which drugs human cardiomyocytes have been exposed to. Using electrophysiological data collected from human cardiomyocytes with known exposure to different drugs, a supervised machine learning algorithm can be trained to recognize and classify cells that have been exposed to an unknown drug. Furthermore, the learning algorithm provides information on the relative contribution of each data parameter to the overall classification. Probabilities and confidence in the accuracy of each classification may also be determined by the algorithm. In this study, the electrophysiological effects of β-adrenergic drugs, propranolol and isoproterenol, on cardiomyocytes derived from human induced pluripotent stem cells (hiPS-CM) were assessed. The electrophysiological data were collected using high temporal resolution 2-photon microscopy of voltage sensitive dyes as a reporter of membrane voltage. The results demonstrate the ability of our algorithm to accurately assess, classify, and predict hiPS-CM membrane depolarization following exposure to chronotropic ... [truncated at 150 words]
Chen H, Holst G, Gratton E.
Modulated CMOS camera for fluorescence lifetime microscopy.
Microsc Res Tech. 2015; 78(12): 1075-1081. PMCID: PMC4770881Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime of entire images. However, the complexity and high costs involved in construction of such a system limit the extensive use of this technique. PCO AG recently released the first luminescence lifetime imaging camera based on a high frequency modulated CMOS image sensor, QMFLIM2. Here we tested and provide operational procedures to calibrate the camera and to improve the accuracy using corrections necessary for image analysis. With its flexible input/output options, we are able to use a modulated laser diode or a 20 MHz pulsed white supercontinuum laser as the light source. The output of the camera consists of a stack of modulated images that can be analyzed by the SimFCS software using the phasor approach. The nonuniform system response across the image sensor must be calibrated at the pixel level. This pixel ... [truncated at 150 words]
Lakhani VV, Hinde E, Gratton E, Elston TC.
Spatio-temporal regulation of Rac1 mobility by actin islands.
PLoS One. 2015; 10(11): e0143753. PMCID: PMC4659588Rho GTPases play important roles in many aspects of cell migration, including polarity establishment and organizing actin cytoskeleton. In particular, the Rho GTPase Rac1 has been associated with the generation of protrusions at leading edge of migrating cells. Previously we showed the mobility of Rac1 molecules is not uniform throughout a migrating cell (Hinde E et. al. PNAS 2013). Specifically, the closer a Rac1 molecule is to the leading edge, the slower the molecule diffuses. Because actin-bound Rac1 diffuses slower than unbound Rac1, we hypothesized that regions of high actin concentration, called "actin islands", act as diffusive traps and are responsible for the non-uniform diffusion observed in vivo. Here, in silico model simulations demonstrate that equally spaced actin islands can regulate the time scale for Rac1 diffusion in a manner consistent with data from live-cell imaging experiments. Additionally, we find this mechanism is robust; different patterns of Rac1 mobility can ... [truncated at 150 words]
Malacrida L, Gratton E, Jameson DM.
Model-free methods to study membrane environmental probes: a comparison of the spectral phasor and generalized polarization approaches.
Methods Appl Fluoresc. 2015; 3(4): 047001. PMCID: PMC4862737In this note, we present a discussion of the advantages and scope of model-free analysis methods applied to the popular solvatochromic probe LAURDAN, which is widely used as an environmental probe to study dynamics and structure in membranes. In particular, we compare and contrast the generalized polarization approach with the spectral phasor approach. To illustrate our points we utilize several model membrane systems containing pure lipid phases and, in some cases, cholesterol or surfactants. We demonstrate that the spectral phasor method offers definitive advantages in the case of complex systems.
Zhang K, Kang DK, Ali MM, Liu L, Labanieh L, Lu M, Riazifar H, Nguyen TN, Zell JA, Digman MA, Gratton E, Li J, Zhao W.
Digital quantification of miRNA directly in plasma using integrated comprehensive droplet digital detection.
Lab Chip. 2015; 15(21): 4217-4226. PMCID: PMC4631652Quantification of miRNAs in blood can be potentially used for early disease detection, surveillance monitoring and drug response evaluation. However, quantitative and robust measurement of miRNAs in blood is still a major challenge in large part due to their low concentration and complicated sample preparation processes typically required in conventional assays. Here, we present the 'Integrated Comprehensive Droplet Digital Detection' (IC 3D) system where the plasma sample containing target miRNAs is encapsulated into microdroplets, enzymatically amplified and digitally counted using a novel, high-throughput 3D particle counter. Using Let-7a as a target, we demonstrate that IC 3D can specifically quantify target miRNA directly from blood plasma at extremely low concentrations ranging from 10s to 10 000 copies per mL in ≤3 hours without the need for sample processing such as RNA extraction. Using this new tool, we demonstrate that target miRNA content in colon cancer patient blood is significantly higher than that ... [truncated at 150 words]
Tan Z, Dai W, van Erp TGM, Overman J, Demuro A, Digman MA, Hatami A, Albay R, Sontag EM, Potkin KT, Ling S, Macciardi F, Bunney WE, Long JD, Paulsen JS, Ringman JM, Parker I, Glabe C, Thompson LM, Chiu W, Potkin SG.
Huntington’s disease cerebrospinal fluid seeds aggregation of mutant huntingtin.
Mol Psychiatry. 2015; 20(11): 1286-1293. PMCID: PMC4718563Huntington's disease (HD), a progressive neurodegenerative disease, is caused by an expanded CAG triplet repeat producing a mutant huntingtin protein (mHTT) with a polyglutamine-repeat expansion. Onset of symptoms in mutant huntingtin gene-carrying individuals remains unpredictable. We report that synthetic polyglutamine oligomers and cerebrospinal fluid (CSF) from BACHD transgenic rats and from human HD subjects can seed mutant huntingtin aggregation in a cell model and its cell lysate. Our studies demonstrate that seeding requires the mutant huntingtin template and may reflect an underlying prion-like protein propagation mechanism. Light and cryo-electron microscopy show that synthetic seeds nucleate and enhance mutant huntingtin aggregation. This seeding assay distinguishes HD subjects from healthy and non-HD dementia controls without overlap (blinded samples). Ultimately, this seeding property in HD patient CSF may form the basis of a molecular biomarker assay to monitor HD and evaluate therapies that target mHTT.
Adu-Gyamfi E, Johnson KA, Fraser ME, Scott JL, Soni SP, Jones KR, Digman MA, Gratton E, Tessier CR, Stahelin RV.
Host cell plasma membrane phosphatidylserine regulates the assembly and budding of the Ebola virus.
J Virol. 2015; 89(18): 9440-9453. PMCID: PMC4542376Lipid enveloped viruses replicate and bud from the host cell where they acquire their lipid coat. The Ebola virus, which buds from the plasma membrane of the host cell causes viral hemorrhagic fever and has a high fatality rate. To date little is known about how budding and egress of the Ebola virus is mediated at the plasma membrane. We have found that the lipid phosphatidylserine (PS) regulates the assembly of the Ebola virus matrix protein VP40. VP40 binds PS containing membranes with nanomolar affinity, and binding of PS regulates VP40 localization and oligomerization on the plasma membrane inner leaflet. Further, alteration of PS levels in mammalian cells inhibits assembly and egress of VP40. Notably, interactions of VP40 with the plasma membrane induced exposure of PS on the outer leaflet of the plasma membrane at sites of egress; whereas PS is typically only on the inner leaflet. Together, we present ... [truncated at 150 words]
Mieruszynski S, Digman MA, Gratton E, Jones MR.
Characterization of exogenous DNA mobility in live cells through fluctuation correlation spectroscopy.
Sci Rep. 2015; 5: 13848. PMCID: PMC4564760The spatial-temporal dynamics of delivered DNA is a critical aspect influencing successful gene delivery. A comprehensive model of DNA lipoplex trafficking through live cells has yet to be demonstrated. Here the bioimaging approaches Raster Image Correlation Spectroscopy (RICS) and image-Means Square Displacement (iMSD) were applied to quantify DNA mechanical dynamics in live cells. DNA lipoplexes formed from DNA with a range of 21 bp to 5.5 kbp exhibited a similar range of motion within the cytoplasm of myoblast cells regardless of size. However, the rate of motion was dictated by the intracellular location, and DNA cluster size. This analysis demonstrated that the different transport mechanisms either had a size dependent mobility, including random diffusion, whereas other mechanisms were not influenced by the DNA size such as active transport. The transport mechanisms identified followed a spatial dependence comparable to viral trafficking of non-active transport mechanism upon cellular entry, active transport within the cytoplasm ... [truncated at 150 words]
Hedde PN, Ranjit S, Gratton E.
3D fluorescence anisotropy imaging using selective plane illumination microscopy.
Opt Express. 2015; 23(17): 22308-22317. PMCID: PMC4646523Fluorescence anisotropy imaging is a popular method to visualize changes in organization and conformation of biomolecules within cells and tissues. In such an experiment, depolarization effects resulting from differences in orientation, proximity and rotational mobility of fluorescently labeled molecules are probed with high spatial resolution. Fluorescence anisotropy is typically imaged using laser scanning and epifluorescence-based approaches. Unfortunately, those techniques are limited in either axial resolution, image acquisition speed, or by photobleaching. In the last decade, however, selective plane illumination microscopy has emerged as the preferred choice for three-dimensional time lapse imaging combining axial sectioning capability with fast, camera-based image acquisition, and minimal light exposure. We demonstrate how selective plane illumination microscopy can be utilized for three-dimensional fluorescence anisotropy imaging of live cells. We further examined the formation of focal adhesions by three-dimensional time lapse anisotropy imaging of CHO-K1 cells expressing an EGFP-paxillin fusion protein.
Ranjit S, Dvornikov A, Stakic M, Hong SH, Levi M, Evans RM, Gratton E.
Imaging fibrosis and separating collagens using second harmonic generation and phasor approach to fluorescence lifetime imaging.
Sci Rep. 2015; 5: 13378. PMCID: PMC4543938In this paper we have used second harmonic generation (SHG) and phasor approach to auto fluorescence lifetime imaging (FLIM) to obtain fingerprints of different collagens and then used these fingerprints to observe bone marrow fibrosis in the mouse femur. This is a label free approach towards fast automatable detection of fibrosis in tissue samples. FLIM has previously been used as a method of contrast in different tissues and in this paper phasor approach to FLIM is used to separate collagen I from collagen III, the markers of fibrosis, the largest groups of disorders that are often without any effective therapy. Often characterized by an increase in collagen content of the corresponding tissue, the samples are usually visualized by histochemical staining, which is pathologist dependent and cannot be automated.
Hendrix J, Baumgärtel V, Schrimpf W, Ivanchenko S, Digman MA, Gratton E, Kräusslich HG, Müller B, Lamb DC.
Live-cell observation of cytosolic HIV-1 assembly onset reveals RNA-interacting Gag oligomers.
J Cell Biol. 2015; 210(4): 629-646. PMCID: PMC4539982Assembly of the Gag polyprotein into new viral particles in infected cells is a crucial step in the retroviral replication cycle. Currently, little is known about the onset of assembly in the cytosol. In this paper, we analyzed the cytosolic HIV-1 Gag fraction in real time in live cells using advanced fluctuation imaging methods and thereby provide detailed insights into the complex relationship between cytosolic Gag mobility, stoichiometry, and interactions. We show that Gag diffuses as a monomer on the subsecond timescale with severely reduced mobility. Reduction of mobility is associated with basic residues in its nucleocapsid (NC) domain, whereas capsid (CA) and matrix (MA) domains do not contribute significantly. Strikingly, another diffusive Gag species was observed on the seconds timescale that oligomerized in a concentration-dependent manner. Both NC- and CA-mediated interactions strongly assist this process. Our results reveal potential nucleation steps of cytosolic Gag fractions before membrane-assisted Gag assembly.
Hinde E, Cardarelli F, Gratton E.
Spatiotemporal regulation of Heterochromatin Protein 1- alpha oligomerization and dynamics in live cells.
Sci Rep. 2015; 5: 12001. PMCID: PMC4523856Heterochromatin protein 1 (HP1) is a central factor in establishing and maintaining the heterochromatin state. As consequence of playing a structural role in heterochromatin, HP1 proteins can have both an activating as well as repressive function in gene expression. Here we probe how oligomerisation of the HP1-α isoform modulates interaction with chromatin, by spatially resolved fluorescence correlation spectroscopy (FCS). We find from fluctuation analysis of HP1-α dynamics that this isoform exists as a dimer around the periphery of heterochromatin foci and these foci locally rotate with characteristic turn rates that range from 5-100ms. From inhibition of HP1-α homo-oligomerization we find the slow turn rates (20-100 ms) are dimer dependent. From treatment with drugs that disrupt or promote chromatin compaction, we find that HP1-α dimers spatially redistribute to favor fast (5-10 ms) or slow (20-100 ms) turn rates. Collectively our results demonstrate HP1-α oligomerization is critical to the maintenance of heterochromatin and the tunable ... [truncated at 150 words]
Ito A, Hong C, Rong X, Zhu X, Tarling EJ, Hedde PN, Gratton E, Parks J, Tontonoz P.
LXRs link metabolism to inflammation through Abca1-dependent regulation of membrane composition and TLR signaling.
eLife. 2015; 4: e08009. PMCID: PMC4517437The liver X receptors (LXRs) are transcriptional regulators of lipid homeostasis that also have potent anti-inflammatory effects. The molecular basis for their anti-inflammatory effects is incompletely understood, but has been proposed to involve the indirect tethering of LXRs to inflammatory gene promoters. Here we demonstrate that the ability of LXRs to repress inflammatory gene expression in cells and mice derives primarily from their ability to regulate lipid metabolism through transcriptional activation and can occur in the absence of SUMOylation. Moreover, we identify the putative lipid transporter Abca1 as a critical mediator of LXR's anti-inflammatory effects. Activation of LXR inhibits signaling from TLRs 2, 4 and 9 to their downstream NF-κB and MAPK effectors through Abca1-dependent changes in membrane lipid organization that disrupt the recruitment of MyD88 and TRAF6. These data suggest that a common mechanism-direct transcriptional activation-underlies the dual biological functions of LXRs in metabolism and inflammation.
Plotegher N, Stringari C, Jahid S, Veronesi M, Girotto S, Gratton E, Bubacco L.
NADH fluorescence lifetime is an endogenous reporter of α-synuclein aggregation in live cells.
FASEB J. 2015; 29(6): 2484-2494. PMCID: PMC4447231α-Synuclein (aS) aggregation has been amply investigated for its involvement in Parkinson's disease because its amyloid fibrils are the main constituent of Lewy bodies, one of the hallmarks of the disease. aS aggregation was studied here in vitro and in cellular models to correlate aggregation products with toxicity mechanisms. Independent results published elsewhere suggested that aS overexpression and/or aggregation may impair cellular metabolism and cause mitochondrial damage. In this context, we report the characterization of changes in NADH fluorescence properties in vitro and in human embryonic kidney 293 cells upon aS aggregation. The application of the phasor approach to study NADH fluorescence lifetime and emission allowed us to identify changes that correlate with aS aggregation. In particular, the fraction of bound NADH, characterized by longer lifetimes in comparison to free NADH, is increased, and the maximum of the NADH emission is shifted toward shorter wavelengths in the presence of aggregating ... [truncated at 150 words]
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Active focus stabilization for upright selective plane illumination microscopy.
Opt Express. 2015; 23(11): 14707-14714. PMCID: PMC4523369Due to its sectioning capability, large field of view, and minimal light exposure, selective plane illumination microscopy has become the preferred choice for 3D time lapse imaging. Single cells in a dish can be conveniently imaged using an upright/inverted configuration. However, for measurements on long time scales (hours to days), mechanical drift is a problem; especially for studies of mammalian cells that typically require heating to 37°C which causes a thermal gradient across the instrument. Since the light sheet diverges towards the edges of the field of view, such a drift leads to a decrease in axial resolution over time. Or, even worse, the specimen could move out of the imaging volume. Here, we present a simple, cost-effective way to stabilize the axial position using the microscope camera to track the sample position. Thereby, sample loss is prevented and an optimal axial resolution is maintained by keeping the sample at ... [truncated at 150 words]
Mieruszynski S, Briggs C, Digman MA, Gratton E, Jones MR.
Live cell characterization of DNA aggregation delivered through lipofection.
Sci Rep. 2015; 5: 10528. PMCID: PMC4444954DNA trafficking phenomena, such as information on where and to what extent DNA aggregation occurs, have yet to be fully characterised in the live cell. Here we characterise the aggregation of DNA when delivered through lipofection by applying the Number and Brightness (N&B) approach. The N&B analysis demonstrates extensive aggregation throughout the live cell with DNA clusters in the extremity of the cell and peri-nuclear areas. Once within the nucleus aggregation had decreased 3-fold. In addition, we show that increasing serum concentration of cell media results in greater cytoplasmic aggregation. Further, the effects of the DNA fragment size on aggregation was explored, where larger DNA constructs exhibited less aggregation. This study demonstrates the first quantification of DNA aggregation when delivered through lipofection in live cells. In addition, this study has presents a model for alternative uses of this imaging approach, which was originally developed to study protein oligomerization and aggregation.
Annibale P, Dvornikov A, Gratton E.
Electrically tunable lens speeds up 3D orbital tracking.
Biomed Opt Express. 2015; 6(6): 2181-2190. PMCID: PMC44737523D orbital particle tracking is a versatile and effective microscopy technique that allows following fast moving fluorescent objects within living cells and reconstructing complex 3D shapes using laser scanning microscopes. We demonstrated notable improvements in the range, speed and accuracy of 3D orbital particle tracking by replacing commonly used piezoelectric stages with Electrically Tunable Lens (ETL) that eliminates mechanical movement of objective lenses. This allowed tracking and reconstructing shape of structures extending 500 microns in the axial direction. Using the ETL, we tracked at high speed fluorescently labeled genomic loci within the nucleus of living cells with unprecedented temporal resolution of 8ms using a 1.42NA oil-immersion objective. The presented technology is cost effective and allows easy upgrade of scanning microscopes for fast 3D orbital tracking.
Datta R, Alfonso-García A, Cinco R, Gratton E.
Fluorescence lifetime imaging of endogenous biomarker of oxidative stress.
Sci Rep. 2015; 5: 9848. PMCID: PMC4438616Presence of reactive oxygen species (ROS) in excess of normal physiological level results in oxidative stress. This can lead to a range of pathological conditions including inflammation, diabetes mellitus, cancer, cardiovascular and neurodegenerative disease. Biomarkers of oxidative stress play an important role in understanding the pathogenesis and treatment of these diseases. A number of fluorescent biomarkers exist. However, a non-invasive and label-free identification technique would be advantageous for in vivo measurements. In this work we establish a spectroscopic method to identify oxidative stress in cells and tissues by fluorescence lifetime imaging (FLIM). We identified an autofluorescent, endogenous species with a characteristic fluorescent lifetime distribution as a probe for oxidative stress. To corroborate our hypothesis that these species are products of lipid oxidation by ROS, we correlate the spectroscopic signals arising from lipid droplets by combining FLIM with THG and CARS microscopy which are established techniques for selective lipid body imaging. ... [truncated at 150 words]
Anzalone A, Gabriel M, Estrada LC, Gratton E.
Spectral properties of single gold nanoparticles in close proximity to biological fluorophores excited by 2-photon excitation.
PLoS One. 2015; 10(4): e0124975. PMCID: PMC4409109Metallic nanoparticles (NPs) are able to modify the excitation and emission rates (plasmonic enhancement) of fluorescent molecules in their close proximity. In this work, we measured the emission spectra of 20 nm Gold Nanoparticles (AuNPs) fixed on a glass surface submerged in a solution of different fluorophores using a spectral camera and 2-photon excitation. While on the glass surface, we observed the presence in the emission at least 3 components: i) second harmonic signal (SHG), ii) a broad emission from AuNPS and iii) fluorescence arising from fluorophores nearby. When on the glass surface, we found that the 3 spectral components have different relative intensities when the incident direction of linear polarization was changed indicating different physical origins for these components. Then we measured by fluctuation correlation spectroscopy (FCS) the scattering and fluorescence signal of the particles alone and in a solution of 100 nM EGFP using the spectral camera or ... [truncated at 150 words]
Olivera-Couto A, Salzman V, Mailhos M, Digman MA, Gratton E, Aguilar PS.
Eisosomes are dynamic plasma membrane domains showing Pil1-Lsp1 heteroligomer binding equilibrium.
Biophys J. 2015; 108(7): 1633-1644. PMCID: PMC4390835Eisosomes are plasma membrane domains concentrating lipids, transporters, and signaling molecules. In the budding yeast Saccharomyces cerevisiae, these domains are structured by scaffolds composed mainly by two cytoplasmic proteins Pil1 and Lsp1. Eisosomes are immobile domains, have relatively uniform size, and encompass thousands of units of the core proteins Pil1 and Lsp1. In this work we used fluorescence fluctuation analytical methods to determine the dynamics of eisosome core proteins at different subcellular locations. Using a combination of scanning techniques with autocorrelation analysis, we show that Pil1 and Lsp1 cytoplasmic pools freely diffuse whereas an eisosome-associated fraction of these proteins exhibits slow dynamics that fit with a binding-unbinding equilibrium. Number and brightness analysis shows that the eisosome-associated fraction is oligomeric, while cytoplasmic pools have lower aggregation states. Fluorescence lifetime imaging results indicate that Pil1 and Lsp1 directly interact in the cytoplasm and within the eisosomes. These results support a model where ... [truncated at 150 words]
Lanzanò L, Hernández IC, Castello M, Gratton E, Diaspro A, Vicidomini G.
Encoding and decoding spatio-temporal information for super-resolution microscopy.
Nat Commun. 2015; 6, 6701. PMCID: PMC4384168The 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 a scanning microscope can be encoded within the fluorescence dynamics and decoded by resolving the signal into its dynamics components. Here we present a robust and general method to decode this information using phasor analysis. As an example of the application of this method, we optically generate spatially controlled gradients in the fluorescence lifetime by stimulated emission. Spatial resolution can be increased indefinitely by increasing the number of resolved dynamics components up to a maximum determined by the amount of noise. We demonstrate that the proposed method provides nanoscale imaging of subcellular structures, opening new routes in super-resolution microscopy based on the encoding/decoding of spatial information through manipulation of ... [truncated at 150 words]
Rong X, Wang B, Dunham MM, Hedde PN, Wong JS, Gratton E, Young SG, Ford DA, Tontonoz P.
Lpcat3-dependent production of arachidonoyl phospholipids is a key determinant of triglyceride secretion.
eLife. 2015; 4: e06557. PMCID: PMC4400582The role of specific phospholipids in lipid transport has been difficult to assess due to an inability to selectively manipulate membrane composition in vivo. Here we show that the phospholipid remodeling enzyme lysophosphatidylcholine acyltransferase 3 (Lpcat3) is a critical determinant of triglyceride secretion due to its unique ability to catalyze the incorporation of arachidonate into membranes. Mice lacking Lpcat3 in the intestine fail to thrive during weaning and exhibit enterocyte lipid accumulation and reduced plasma triglycerides. Mice lacking Lpcat3 in the liver show reduced plasma triglycerides, hepatosteatosis, and secrete lipid-poor VLDL lacking arachidonoyl phospholipids. Mechanistic studies indicate that Lpcat3 activity impacts membrane lipid mobility in living cells, suggesting a biophysical basis for the requirement of arachidonoyl phospholipids in lipidating lipoprotein particles. These data identify Lpcat3 as a key factor in lipoprotein production and illustrate how manipulation of membrane composition can be used as a regulatory mechanism to control metabolic pathways.
Moens PDJ, Digman MA, Gratton E.
Modes of diffusion of Cholera toxin bound to GM1 on live cell membrane by image mean square displacement analysis.
Biophys J. 2015; 108(6): 1448-1458. PMCID: PMC4375681The image-mean square displacement technique applies the calculation of the mean square displacement commonly used in single-molecule tracking to images without resolving single particles. The image-mean square displacement plot obtained is similar to the mean square displacement plot obtained using the single-particle tracking technique. This plot is then used to reconstruct the protein diffusion law and to identify whether the labeled molecules are undergoing pure isotropic, restricted, corralled, transiently confined, or directed diffusion. In our study total internal reflection fluorescence microscopy images were taken of Cholera toxin subunit B (CtxB) membrane-labeled NIH 3T3 mouse fibroblasts and MDA 231 MB cells. We found a population of CTxB undergoing purely isotropic diffusion and one displaying restricted diffusion with corral sizes ranging from 150 to ∼1800 nm. We show that the diffusion rate of CTxB bound to GM1 is independent of the size of the confinement, suggesting that the mechanism of confinement is ... [truncated at 150 words]
Perumal V, Krishnan K, Gratton E, Dharmarajan AM, Fox SA.
Number and brightness analysis of sFRP4 domains in live cells demonstrates vesicle association signal of the NLD domain and dynamic intracellular responses to Wnt3a.
Int J Biochem Cell Biol. 2015; 64: 91-96. PMCID: PMC4464842The Wnts are secreted, lipidated glycoproteins that play a role in cellular processes of differentiation, proliferation, migration, survival, polarity and stem cell self-renewal. The majority of Wnts biological effects are through binding to specific frizzled (Fzd) receptor complexes leading to activation of downstream pathways. Secreted Frizzled-related proteins (sFRPs) were first identified as antagonists of Wnt signalling by binding directly to Wnts. They comprise two domains, a Fzd-like cysteine rich domain (CRD) and a netrin-like domain (NLD). Subsequently sFRPs have been shown to also interact with Fzd receptors and more diverse functions have been identified, including potentiation of Wnt signalling. Many aspects of the biology of this family remain to be elucidated. We used the number and brightness (N&B) method, a technique based on fluorescence fluctuation analysis, to characterise the intracellular aggregation and trafficking of sFRP4 domains. We expressed sFRP4 and its’ domains as EGFP fusions and then characterised the effect ... [truncated at 150 words]
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Single cell visualization of transcription kinetics variance of highly mobile identical genes using 3D nanoimaging.
Sci Rep. 2015; 5, 9258. PMCID: PMC4365385Multi-cell biochemical assays and single cell fluorescence measurements revealed that the elongation rate of Polymerase II (PolII) in eukaryotes varies largely across different cell types and genes. However, there is not yet a consensus whether intrinsic factors such as the position, local mobility or the engagement by an active molecular mechanism of a genetic locus could be the determinants of the observed heterogeneity. Here by employing high-speed 3D fluorescence nanoimaging techniques we resolve and track at the single cell level multiple, distinct regions of mRNA synthesis within the model system of a large transgene array. We demonstrate that these regions are active transcription sites that release mRNA molecules in the nucleoplasm. Using fluctuation spectroscopy and the phasor analysis approach we were able to extract the local PolII elongation rate at each site as a function of time. We measured a four-fold variation in the average elongation between identical copies of ... [truncated at 150 words]
Chen H, Gratton E, Digman MA.
Spectral properties and dynamics of gold nanorods revealed by EMCCD-based spectral phasor method.
Microsc Res Tech. 2015; 78(4): 283-293. PMCID: PMC4404027Gold nanorods (NRs) with tunable plasmon-resonant absorption in the near-infrared region have considerable advantages over organic fluorophores as imaging agents due to their brightness and lack of photobleaching. However, the luminescence spectral properties of NRs have not been fully characterized at the single particle level due to lack of proper analytic tools. Here, we present a spectral phasor analysis method that allows investigations of NRs' spectra at single particle level showing the spectral variance and providing spatial information during imaging. The broad phasor distribution obtained by the spectral phasor analysis indicates that spectra of NRs are different from particle to particle. NRs with different spectra can be identified in images with high spectral resolution. The spectral behaviors of NRs under different imaging conditions, for example, different excitation powers and wavelengths, were revealed by our laser-scanning multiphoton microscope using a high-resolution spectrograph with imaging capability. Our results prove that the spectral ... [truncated at 150 words]
Stringari C, Wang H, Geyfman M, Crosignani V, Kumar V, Takahashi JS, Andersen B, Gratton E.
In vivo single-cell detection of metabolic oscillations in stem cells.
Cell Rep. 2015; 10(1): 1-7. PMCID: PMC4340841Through the use of bulk measurements in metabolic organs, the circadian clock was shown to play roles in organismal energy homeostasis. However, the relationship between metabolic and circadian oscillations has not been studied in vivo at a single-cell level. Also, it is unknown whether the circadian clock controls metabolism in stem cells. We used a sensitive, noninvasive method to detect metabolic oscillations and circadian phase within epidermal stem cells in live mice at the single-cell level. We observe a higher NADH/NAD+ ratio, reflecting an increased glycolysis/oxidative phosphorylation ratio during the night compared to the day. Furthermore, we demonstrate that single-cell metabolic heterogeneity within the basal cell layer correlates with the circadian clock and that diurnal fluctuations in NADH/NAD+ ratio are Bmal1 dependent. Our data show that, in proliferating stem cells, the circadian clock coordinates activities of oxidative phosphorylation and glycolysis with DNA synthesis, perhaps as a protective mechanism against genotoxicity.
di Rienzo C, Piazza V, Gratton E, Beltram F, Cardarelli F.
Probing short-range protein Brownian motion in the cytoplasm of living cells.
Nat Commun. 2014; 5: 5891. PMCID: PMC4281647The translational motion of molecules in cells deviates from what is observed in dilute solutions. Theoretical models provide explanations for this effect but with predictions that drastically depend on the nanoscale organization assumed for macromolecular crowding agents. A conclusive test of the nature of the translational motion in cells is missing owing to the lack of techniques capable of probing crowding with the required temporal and spatial resolution. Here we show that fluorescence-fluctuation analysis of raster scans at variable timescales can provide this information. By using green fluorescent proteins in cells, we measure protein motion at the unprecedented timescale of 1 μs, unveiling unobstructed Brownian motion from 25 to 100 nm, and partially suppressed diffusion above 100 nm. Furthermore, experiments on model systems attribute this effect to the presence of relatively immobile structures rather than to diffusing crowding agents. We discuss the implications of these results for intracellular processes.
Ranjit S, Dvornikov AS, Holland D, Reinhart GD, Jameson DM, Gratton E.
Application of three-photon excitation FCS to the study of protein oligomerization.
J Phys Chem B. 2014; 118(50): 14627-14631. PMCID: PMC4275161Three-photon excitation fluorescence correlation spectroscopy was used to detect oligomerization equilibria of rat liver phosphofructokinase. The fluorescence intensity produced by the three photon excitation of tryptophan was collected using the DIVER microscope. In this home-built upright microscope, a large area photomultiplier, placed directly below the sample, is used as the detector. The lack of optical elements in the microscope detection path results in a significantly improved detection efficiency in the UV region down to about 300 nm, which encompasses the fluorescence emission from tryptophan. The three-photon excitation autocorrelation decays obtained for phosphofructokinase in presence of F6P showed the presence of large oligomers. Substitution of F6P with ATP in the buffer medium results in dissociation of the large oligomers, which is reported by the decreased autocorrelation amplitude. The three photon excitation process was verified from the slope of the log-log plot of intensity against laser power.
Ranjit S, Lanzanò L, Gratton E.
Mapping diffusion in a living cell via the phasor approach.
Biophys J. 2014; 107(12): 2775-2785. PMCID: PMC4269802Diffusion of a fluorescent protein within a cell has been measured using either fluctuation-based techniques (fluorescence correlation spectroscopy (FCS) or raster-scan image correlation spectroscopy) or particle tracking. However, none of these methods enables us to measure the diffusion of the fluorescent particle at each pixel of the image. Measurement using conventional single-point FCS at every individual pixel results in continuous long exposure of the cell to the laser and eventual bleaching of the sample. To overcome this limitation, we have developed what we believe to be a new method of scanning with simultaneous construction of a fluorescent image of the cell. In this believed new method of modified raster scanning, as it acquires the image, the laser scans each individual line multiple times before moving to the next line. This continues until the entire area is scanned. This is different from the original raster-scan image correlation spectroscopy approach, where data ... [truncated at 150 words]
Hedde PN, Stakic M, Gratton E.
Rapid measurement of molecular transport and interaction inside living cells using single plane illumination.
Sci Rep. 2014; 4: 7048. PMCID: PMC4231332The ability to measure biomolecular dynamics within cells and tissues is very important to understand fundamental physiological processes including cell adhesion, signalling, movement, division or metabolism. Usually, such information is obtained using particle tracking methods or single point fluctuation spectroscopy. We show that image mean square displacement analysis, applied to single plane illumination microscopy data, is a faster and more efficient way of unravelling rapid, three-dimensional molecular transport and interaction within living cells. From a stack of camera images recorded in seconds, the type of dynamics such as free diffusion, flow or binding can be identified and quantified without being limited by current camera frame rates. Also, light exposure levels are very low and the image mean square displacement method does not require calibration of the microscope point spread function. To demonstrate the advantages of our approach, we quantified the dynamics of several different proteins in the cyto- and nucleoplasm ... [truncated at 150 words]
Kang DK, Ali MM, Zhang K, Huang SS, Peterson E, Digman MA, Gratton E, Zhao W.
Rapid detection of single bacteria in unprocessed blood using Integrated Comprehensive Droplet Digital Detection.
Nat Commun. 2014; 5: 5427. PMCID: PMC4243214Blood stream infection or sepsis is a major health problem worldwide, with extremely high mortality, which 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 can selectively detect bacteria directly from milliliters of diluted blood at single-cell sensitivity in a one-step, culture- and amplification-free process within 1.5–4 h. The IC 3D integrates real-time, DNAzyme-based sensors, droplet microencapsulation and a high-throughput 3D particle counter system. Using Escherichia coli as a target, we demonstrate that the IC 3D can provide absolute quantification of both stock and clinical isolates of E. coli in spiked blood within a broad range of extremely low concentration from 1 to 10,000 bacteria per ml with exceptional robustness and limit of detection in the single digit regime.
Bonaventura G, Barcellona ML, Golfetto O, Nourse JL, Flanagan LA, Gratton E.
Laurdan monitors different lipids content in eukaryotic membrane during embryonic neural development.
Cell Biochem Biophys. 2014; 70(2): 785-794. PMCID: PMC4228983We describe a method based on fluorescence-lifetime imaging microscopy (FLIM) to assess the fluidity of various membranes in neuronal cells at different stages of development [day 12 (E12) and day 16 (E16) of gestation]. For the FLIM measurements, we use the Laurdan probe which is commonly used to assess membrane water penetration in model and in biological membranes using spectral information. Using the FLIM approach, we build a fluidity scale based on calibration with model systems of different lipid compositions. In neuronal cells, we found a marked difference in fluidity between the internal membranes and the plasma membrane, being the plasma membrane the less fluid. However, we found no significant differences between the two cell groups, E12 and E16. Comparison with NIH3T3 cells shows that the plasma membranes of E12 and E16 cells are significantly more fluid than the plasma membrane of the cancer cells.
Adu-Gyamfi E, Soni SP, Jee CS, Digman MA, Gratton E, Stahelin RV.
A loop region in the N-terminal domain of Ebola virus VP40 is important in viral assembly, budding, and egress.
Viruses. 2014; 6(10): 3837-3854. PMCID: PMC4213565Ebola virus (EBOV) causes viral hemorrhagic fever in humans and can have clinical fatality rates of ~60%. The EBOV genome consists of negative sense RNA that encodes seven proteins including viral protein 40 (VP40). VP40 is the major Ebola virus matrix protein and regulates assembly and egress of infectious Ebola virus particles. It is well established that VP40 assembles on the inner leaflet of the plasma membrane of human cells to regulate viral budding where VP40 can produce virus like particles (VLPs) without other Ebola virus proteins present. The mechanistic details, however, of VP40 lipid-interactions and protein-protein interactions that are important for viral release remain to be elucidated. Here, we mutated a loop region in the N-terminal domain of VP40 (Lys127, Thr129, and Asn130) and find that mutations (K127A, T129A, and N130A) in this loop region reduce plasma membrane localization of VP40. Additionally, using total internal reflection fluorescence microscopy and ... [truncated at 150 words]
di Rienzo C, Gratton E, Beltram F, Cardarelli F.
From fast fluorescence imaging to molecular diffusion law on live cell membranes in a commercial microscope.
J Vis Exp. 2014; (92), e51994. PMCID: PMC4461222It has become increasingly evident that the spatial distribution and the motion of membrane components like lipids and proteins are key factors in the regulation of many cellular functions. However, due to the fast dynamics and the tiny structures involved, a very high spatio-temporal resolution is required to catch the real behavior of molecules. Here we present the experimental protocol for studying the dynamics of fluorescently-labeled plasma-membrane proteins and lipids in live cells with high spatiotemporal resolution. Notably, this approach doesn't need to track each molecule, but it calculates population behavior using all molecules in a given region of the membrane. The starting point is a fast imaging of a given region on the membrane. Afterwards, a complete spatio-temporal autocorrelation function is calculated correlating acquired images at increasing time delays, for example each 2, 3, n repetitions. It is possible to demonstrate that the width of the peak of the ... [truncated at 150 words]
Anzalone A, Annibale P, Gratton E.
3D orbital tracking in a modified two-photon microscope: an application to the tracking of intracellular vesicles.
J Vis Exp. 2014; (92), e51794. PMCID: PMC4274936The objective of this video protocol is to discuss how to perform and analyze a three-dimensional fluorescent orbital particle tracking experiment using a modified two-photon microscope. As opposed to conventional approaches (raster scan or wide field based on a stack of frames), the 3D orbital tracking allows to localize and follow with a high spatial (10 nm accuracy) and temporal resolution (50 Hz frequency response) the 3D displacement of a moving fluorescent particle on length-scales of hundreds of microns. The method is based on a feedback algorithm that controls the hardware of a two-photon laser scanning microscope in order to perform a circular orbit around the object to be tracked: the feedback mechanism will maintain the fluorescent object in the center by controlling the displacement of the scanning beam. To demonstrate the advantages of this technique, we followed a fast moving organelle, the lysosome, within a living cell. Cells were ... [truncated at 150 words]
Nicolai E, Garau S, Favalli C, D’Agostini C, Gratton E, Motolese G, Rosato N.
Evaluation of BiesseBioscreen as a new methodology for bacteriuria screening.
New Microbiol. 2014; 37(4): 495-501. PMCID: PMC4273739Urinary tract infection is a common disease diagnosed from symptoms and clinical signs, and bacterial count per volume of urine. This study have evaluated the BiesseBioscreen analyzer as a new way to analyze urine samples en- abling fast screening of urine, prior to reference standard methods currently utilized in microbiology analysis labo- ratory. We analyzed 962 urine samples from outpatients and inpatients of the Tor Vergata (TV) University Hospital of the University of Rome "Tor Vergata". All samples were processed both with the BiesseBioscreen and with the standard methodology adopted by the clinical microbiology laboratory of TV Hospital and the results were com- pared. Of the samples analyzed 54.9% were concordant negative with the reference method and 21.6% concordant positive, 23.3% resulted false positive and 0.2% false negative. The results obtained from BiesseBioscreen showed a sensitivity of 99.0%, indicating it as a system suitable to rule out urinary tract infection. ... [truncated at 150 words]
Subashchandrabose SR, Krishnan K, Gratton E, Mallavarapu M, Naidu R.
Potential of fluorescence imaging techniques to monitor mutagenic PAH uptake by microalga.
Environ Sci Technol. 2014; 48(16): 9152-9160. PMCID: PMC4140530Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is one of the major environmental pollutants that causes mutagenesis and cancer. BaP has been shown to accumulate in phytoplankton and zooplankton. We have studied the localization and aggregation of BaP in Chlorella sp., a microalga that is one of the primary producers in the food chain, using fluorescence confocal microscopy and fluorescence lifetime imaging microscopy with the phasor approach to characterize the location and the aggregation of BaP in the cell. Our results show that BaP accumulates in the lipid bodies of Chlorella sp. and that there is Förster resonance energy transfer between BaP and photosystems of Chlorella sp., indicating the close proximity of the two molecular systems. The lifetime of BaP fluorescence was measured to be 14 ns in N,N-dimethylformamide, an average of 7 ns in Bold’s basal medium, and 8 ns in Chlorella cells. Number and brightness analysis suggests that ... [truncated at 150 words]
Bachir AI, Zareno J, Moissoglu K, Plow EF, Gratton E, Horwitz AR.
Integrin-associated complexes form hierarchically with variable stoichiometry in nascent adhesions.
Curr Biol. 2014; 24(16): 1845-1853. PMCID: PMC4138543Background: A complex network of putative molecular interactions underlies the architecture and function of cell-matrix adhesions. Most of these interactions are implicated from coimmunoprecipitation studies using expressed components, but few have been demonstrated or characterized functionally in living cells.
Results: We introduce fluorescence fluctuation methods to determine, at high spatial and temporal resolution, “when” and “where” molecular complexes form and their stoichiometry in nascent adhesions (NAs). We focus on integrin-associated molecules implicated in integrin activation and in the integrin-actin linkage in NAs and show that these molecules form integrin-containing complexes hierarchically within the adhesion itself. Integrin and kindlin reside in a molecular complex as soon as adhesions are visible; talin, although also present early, associates with the integrin-kindlin complex only after NAs have formed and in response to myosin II activity. Furthermore, talin and vinculin association precedes the formation of the integrin-talin complex. Finally, α-actinin enters NAs periodically and in clusters ... [truncated at 150 words]
Pate KT, Stringari C, Sprowl-Tanio S, Wang K, TeSlaa T, Hoverter NP, McQuade MM, Garner C, Digman MA, Teitell MA, Edwards RA, Gratton E, Waterman ML.
Wnt signaling directs a metabolic program of glycolysis and angiogenesis in colon cancer.
EMBO J. 2014; 33(13): 1454-73. PMCID: PMC4194089Much of the mechanism by which Wnt signaling drives proliferation during oncogenesis is attributed to its regulation of the cell cycle. Here, we show how Wnt/β‐catenin signaling directs another hallmark of tumorigenesis, namely Warburg metabolism. Using biochemical assays and fluorescence lifetime imaging microscopy (FLIM) to probe metabolism in vitro and in living tumors, we observe that interference with Wnt signaling in colon cancer cells reduces glycolytic metabolism and results in small, poorly perfused tumors. We identify pyruvate dehydrogenase kinase 1 (PDK1) as an important direct target within a larger gene program for metabolism. PDK1 inhibits pyruvate flux to mitochondrial respiration and a rescue of its expression in Wnt‐inhibited cancer cells rescues glycolysis as well as vessel growth in the tumor microenvironment. Thus, we identify an important mechanism by which Wnt‐driven Warburg metabolism directs the use of glucose for cancer cell proliferation and links it to vessel delivery of oxygen and ... [truncated at 150 words]
Hinde E, Kong X, Yokomori K, Gratton E.
Chromatin dynamics during DNA repair revealed by pair correlation analysis of molecular flow in the nucleus.
Biophys J. 2014; 107(1): 55-65. PMCID: PMC4119266Chromatin dynamics modulate DNA repair factor accessibility throughout the DNA damage response. The spatiotemporal scale upon which these dynamics occur render them invisible to live cell imaging. Here we present a believed novel assay to monitor the in vivo structural rearrangements of chromatin during DNA repair. By pair correlation analysis of EGFP molecular flow into chromatin before and after damage, this assay measures millisecond variations in chromatin compaction with submicron resolution. Combined with laser microirradiation we employ this assay to monitor the real-time accessibility of DNA at the damage site. We find from comparison of EGFP molecular flow with a molecule that has an affinity toward double-strand breaks (Ku-EGFP) that DNA damage induces a transient decrease in chromatin compaction at the damage site and an increase in compaction to adjacent regions, which together facilitate DNA repair factor recruitment to the lesion with high spatiotemporal control.
Chiu CL, Aguilar JS, Tsai CY, Wu G, Gratton E, Digman MA.
Nanoimaging of focal adhesion dynamics in 3D.
PLoS One. 2014; 9(6): e99896. PMCID: PMC4069057Organization and dynamics of focal adhesion proteins have been well characterized in cells grown on two-dimensional (2D) cell culture surfaces. However, much less is known about the dynamic association of these proteins in the 3D 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 scaning-fluorescence correlation spectroscopy (sFCS) and the number and molecular brightness (N&B) methods to investigate paxillin and actin dynamics at focal adhesions in 3D. 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 sFCS we found higher percentage of slow diffusing proteins at these focal spots, suggesting assembling/disassembling processes. In addition, the ... [truncated at 150 words]
Plotegher N, Gratton E, Bubacco L.
Number and Brightness analysis of alpha-synuclein oligomerization and the associated mitochondrial morphology alterations in live cells.
Biochim Biophys Acta. 2014; 1840(6): 2014-2024. PMCID: PMC4088272BACKGROUND: Alpha-synuclein oligomerization is associated to Parkinson's disease etiopathogenesis. The study of alpha-synuclein oligomerization properties in live cell and the definition of their effects on cellular viability are among fields expected to provide the knowledge required to unravel the mechanism(s) of toxicity that lead to the disease.
METHODS: We used Number and Brightness method, which is a method based on fluorescence fluctuation analysis, to monitor alpha-synuclein tagged with EGFP aggregation in living SH-SY5Y cells. The presence of alpha-synuclein oligomers detected with this method was associated with intracellular structure conditions, evaluated by fluorescence confocal imaging.
RESULTS: Cells overexpressing alpha-synuclein-EGFP present a heterogeneous ensemble of oligomers constituted by less than 10 monomers, when the protein approaches a threshold concentration value of about 90 nM in the cell cytoplasm. We show that the oligomeric species are partially sequestered by lysosomes and that the mitochondria morphology is altered in cells presenting oligomers, suggesting that these mitochondria ... [truncated at 150 words]
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Orbital single particle tracking on a commercial confocal microscope using piezoelectric stage feedback.
Methods Appl Fluoresc. 2014; 2(2): 024010. PMCID: PMC4235585Single Particle Tracking (SPT) is a technique used to locate fluorescent particles with nanometer precision. 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. Here we describe a SPT setup based on a feedback approach implemented with minimal modification of a commercially available confocal laser scanning microscope, the Zeiss LSM 510, in combination with an external piezoelectric stage scanner. The commercial microscope offers the advantage of a user-friendly software interface and pre-calibrated hardware components. The use of an external piezo-scanner allows the addition of feedback into the system but also represents a limitation in terms of its mechanical response. We describe in detail this implementation of the orbital tracking method and discuss advantages and ... [truncated at 150 words]
Crosignani V, Jahid S, Dvornikov AS, Gratton E.
A deep tissue fluorescence imaging system with enhanced SHG detection capabilities.
Microsc Res Tech. 2014; 77(5): 368-373. PMCID: PMC4009694We describe a novel two-photon fluorescence microscopy system capable of producing high-quality second harmonic generation (SHG) images in thick turbid media by using an innovative detection system. This novel detection system is capable of detecting photons from a very large surface area. This system has proven effective in providing images of thick turbid samples, both biological and artificial. Due to its transmission detection geometry, the system is particularly suitable for detecting SHG signals, which are generally forward directed. In this article, we present comparative data acquired simultaneously on the same sample with the forward and epidetection schemes.
Sharma H, Digman MA, Felsinger N, Gratton E, Khine M.
Enhanced emission of fluorophores on shrink-induced wrinkled composite structures.
Optical Materials Express. 2014; 4(4): 753-763. PMCID: PMC4220269We introduce a manufacturable and scalable method for creating tunable wrinkled ferromagnetic-metallic structures to enhance fluorescence signals. Thin layers of nickel (Ni) and gold (Au) were deposited onto a pre-stressed thermoplastic (shrink wrap film) polymer. Heating briefly forced the metal films to buckle when the thermoplastic retracted, resulting in multi-scale composite ‘wrinkles’. This is the first demonstration of leveraging the plasmons in such hybrid nanostructures by metal enhanced fluorescence (MEF) in the near-infrared wavelengths. We observed more than three orders of magnitude enhancement in the fluorescence signal of a single molecule of goat anti-mouse immunoglobulin G (IgG) antibody conjugated to fluorescein isothiocyanate, FITC, (FITC-IgG) by two-photon excitation with these structures. These large enhancements in the fluorescence signal at the nanoscale gaps between the composite wrinkles corresponded to shortened lifetimes due to localized surface plasmons. To characterize these structures, we combined fluctuation correlation spectroscopy (FCS), fluorescence lifetime imaging microscopy (FLIM), and ... [truncated at 150 words]
Jaureguiberry MS, Tricerri MA, Sánchez SA, Finarelli GS, Montanaro MA, Prieto ED, Rimoldi OJ.
Role of plasma membrane lipid composition on cellular homeostasis: learning from cell line models expressing fatty acid desaturases.
Acta Biochim Biophys Sin (Shanghai). 2014; 46(4): 273-282. PMCID: PMC3967655Experimental evidence has suggested that plasma membrane (PM)-associated signaling and hence cell metabolism and viability depend on lipid composition and organization. The aim of the present work is to develop a cell model to study the endogenous polyunsaturated fatty acids (PUFAs) effect on PM properties and analyze its influence on cholesterol (Chol) homeostasis. We have previously shown that by using a cell line over-expressing stearoyl-CoA-desaturase, membrane composition and organization coordinate cellular pathways involved in Chol efflux and cell viability by different mechanisms. Now, we expanded our studies to a cell model over-expressing both Δ5 and Δ6 desaturases, which resulted in a permanently higher PUFA content in PM. Furthermore, this cell line showed increased PM fluidity, Chol storage, and mitochondrial activity. In addition, human apolipoprotein A-I-mediated Chol removal was less efficient in these cells than in the corresponding control. Taken together, our results suggested that the cell functionality is preserved by ... [truncated at 150 words]
Paladino S, Lebreton S, Tivodar S, Formiggini F, Ossato G, Gratton E, Tramier M, Coppey-Moisan M, Zurzolo C.
Golgi sorting regulates organization and activity of GPI proteins at apical membranes.
Nat Chem Biol. 2014; 10(5): 350-357. PMCID: PMC4027978Here we combined classical biochemistry with new biophysical approaches to study the organization of glycosylphosphatidylinositol (GPI)-anchored proteins (GPI-APs) with high spatial and temporal resolution at the plasma membrane of polarized epithelial cells. We show that in polarized MDCK cells, after sorting in the Golgi, each GPI-AP reaches the apical surface in homoclusters. Golgi-derived homoclusters are required for their subsequent plasma membrane organization into cholesterol-dependent heteroclusters. By contrast, in nonpolarized MDCK cells, GPI-APs are delivered to the surface as monomers in an unpolarized manner and are not able to form heteroclusters. We further demonstrate that this GPI-AP organization is regulated by the content of cholesterol in the Golgi apparatus and is required to maintain the functional state of the protein at the apical membrane. Thus, in contrast to fibroblasts, in polarized epithelial cells, a selective cholesterol-dependent sorting mechanism in the Golgi regulates both the organization and function of GPI-APs at the ... [truncated at 150 words]
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Advanced fluorescence microscopy methods for the real-time study of transcription and chromatin dynamics.
Transcription. 2014; 5(2), e28425. PMCID: PMC4214231In this contribution we provide an overview of the recent advances allowed by the use of fluorescence microscopy methods in the study of transcriptional processes and their interplay with the chromatin architecture in living cells. Although the use of fluorophores to label nucleic acids dates back at least to about half a century ago,1 two recent breakthroughs have effectively opened the way to use fluorescence routinely for specific and quantitative probing of chromatin organization and transcriptional activity in living cells: namely, the possibility of labeling first the chromatin loci and then the mRNA synthesized from a gene using fluorescent proteins. In this contribution we focus on methods that can probe rapid dynamic processes by analyzing fast fluorescence fluctuations.
Pozzi D, Marchini C, Cardarelli F, Salomone F, Coppola S, Montani M, Zabaleta ME, Digman MA, Gratton E, Colapicchioni V, Caracciolo G.
Mechanistic evaluation of the transfection barriers involved in lipid-mediated gene delivery: Interplay between nanostructure and composition.
Biochim Biophys Acta. 2014; 1838(3): 957-967. PMCID: PMC3940149Here we present a quantitative mechanism-based investigation aimed at comparing the cell uptake, intracellular trafficking, endosomal escape and final fate of lipoplexes and lipid-protamine/DNA (LPD) nanoparticles (NPs) in living Chinese hamster ovary (CHO) cells. As a model, two lipid formulations were used for comparison. The first formulation is made of the cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and the zwitterionic lipid dioleoylphosphocholine (DOPC), while the second mixture is made of the cationic 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol (DC-Chol) and the zwitterionic helper lipid dioleoylphosphatidylethanolamine (DOPE). Our findings indicate that lipoplexes are efficiently taken up through fluid-phase macropinocytosis, while a less efficient uptake of LPD NPs occurs through a combination of both macropinocytosis and clathrin-dependent pathways. Inside the cell, both lipoplexes and LPD NPs are actively transported towards the cell nucleus, as quantitatively addressed by spatio-temporal image correlation spectroscopy (STICS). For each lipid formulation, LPD NPs escape from endosomes more efficiently than lipoplexes. When cells were ... [truncated at 150 words]
Hinde E, Yokomori K, Gaus K, Hahn KM, Gratton E.
Fluctuation-based imaging of nuclear Rac1 activation by protein oligomerisation.
Sci Rep. 2014; 4, 4219. PMCID: PMC3936235Here we describe a fluctuation-based method to quantify how protein oligomerisation modulates signalling activity of a multifunctional protein. By recording fluorescence lifetime imaging microscopy (FLIM) data of a FRET biosensor in a format that enables concomitant phasor and cross Number and Brightness (cN&B) analysis, we measure the nuclear dynamics of a Rac1 FRET biosensor and assess how Rac1 homo-oligomers (N&B) regulate Rac1 activity (hetero-oligomerisation with the biosensor affinity reagent, PBD, by FLIM-FRET) or interaction with an unknown binding partner (cN&B). The high spatiotemporal resolution of this method allowed us to discover that upon DNA damage monomeric and active Rac1 in the nucleus is segregated from dimeric and inactive Rac1 in the cytoplasm. This reorganisation requires Rac1 GTPase activity and is associated with an importin-α2 redistribution. Only with this multiplexed approach can we assess the oligomeric state a molecular complex must form in order to regulate a complex signalling network.
O’Carrigan A, Hinde E, Lu N, Xu XQ, Duan H, Huang G, Maka M, Bellottia B, Chen ZH.
Effects of light irradiance on stomatal regulation and growth of tomato.
Environ Exp Bot. 2014; 98: 65-73.Light is not only a primary energy source for photosynthesis but also a vital regulator of numerous processes in plants. However, high light intensity always poses a dilemma for plants: to grow or to suffer. Combining physiological techniques at plant, tissue, and cellular levels, we investigated the regulation of stomatal behaviour and cytosolic Ca2+ concentration ([Ca2+]cyt) on growth of tomato plants under different light irradiance. Overall, plants exhibited a distinct short-term (days) and a long-term (weeks) response to high light by significantly increasing shoot biomass, leaf number, leaf temperature, vapour pressure deficit, stomatal index, aperture length and guard cell length. However, most physiological parameters were significantly reduced upon high light treatment, indicating a strong negative impact of high light on photosynthesis and stomatal opening. For instance, Short- and long-term exposure to high light significantly reduced stomatal aperture width by 31.7% and 46.3%, respectively. Moreover, high light treatments significantly decreased [Ca2+]cyt ... [truncated at 150 words]
James NG, Digman MA, Ross JA, Barylko B, Wang L, Li J, Chen Y, Mueller JD, Gratton E, Albanesi JP, Jameson DM.
A mutation associated with centronuclear myopathy enhances the size and stability of dynamin 2 complexes in cells.
Biochim Biophys Acta. 2014; 1840(1): 315-321. PMCID: PMC3859711BACKGROUND: Dynamin 2 (Dyn2) is a ~100kDa GTPase that assembles around the necks of nascent endocytic and Golgi vesicles and catalyzes membrane scission. Mutations in Dyn2 that cause Centronuclear Myopathy (CNM) have been shown to stabilize Dyn2 polymers against GTP-dependent disassembly in vitro. Precisely timed regulation of assembly and disassembly is believed to be critical for Dyn2 function in membrane vesiculation, and the CNM mutations interfere with this regulation by shifting the equilibrium toward the assembled state.
METHODS: In this study we use two fluorescence fluctuation spectroscopy (FFS) approaches to show that a CNM mutant form of Dyn2 also has a greater propensity to self-assemble in the cytosol and on the plasma membrane of living cells.
RESULTS: Results obtained using brightness analysis indicate that unassembled wild-type Dyn2 is predominantly tetrameric in the cytosol, although different oligomeric species are observed, depending on the concentration of expressed protein. In contrast, an R369W mutant identified ... [truncated at 150 words]
Gehricke JG, Polzonetti CM, Caburian C, Gratton E.
Prefrontal hemodynamic changes during cigarette smoking in young adult smokers with and without ADHD.
Pharmacol Biochem Behav. 2013; 112: 78-81. PMCID: PMC3854671Individuals with Attention-Deficit/Hyperactivity Disorder (ADHD) have elevated smoking prevalence and reduced cessation rates compared to the general population. However, the effects of cigarette smoking on underlying brain activity in smokers with ADHD are not well characterized. Non-invasive Near-Infrared Spectroscopy (NIRS) was used to characterize how cigarette smoking affects prefrontal brain hemodynamics in smokers with and without ADHD. Prefrontal changes of oxy- and deoxyhemoglobin (HbO2 and HHb) were measured in six male adult smokers with ADHD and six age- and gender-matched control smokers. NIRS measurements were separated into four sequential time intervals, i.e., before smoking, during smoking, after smoking, and during a breath hold. Prefrontal HbO2 was lower during smoking in smokers with ADHD compared to control smokers. More specifically, smokers with ADHD showed decreased prefrontal HbO2 during smoking compared to breath hold, before and after smoking periods. In contrast, control smokers showed increased prefrontal HbO2 from before smoking to breath ... [truncated at 150 words]
Chiu CL, Digman MA, Gratton E.
Measuring actin flow in 3D cell protrusions.
Biophys J. 2013; 105(8): 1746-1755. PMCID: PMC3797595Actin dynamics is important in determining cell shape, tension, and migration. Methods such as fluorescent speckle microscopy and spatial temporal image correlation spectroscopy have been used to capture high-resolution actin turnover dynamics within cells in two dimensions. However, these methods are not directly applicable in 3D due to lower resolution and poor contrast. Here, we propose to capture actin flow in 3D with high spatial-temporal resolution by combining nanoscale precise imaging by rapid beam oscillation and fluctuation spectroscopy techniques. To measure the actin flow along cell protrusions in cell expressing actin-eGFP cultured in a type I collagen matrix, the laser was orbited around the protrusion and its trajectory was modulated in a clover-shaped pattern perpendicularly to the protrusion. Orbits were also alternated at two positions closely spaced along the protrusion axis. The pair cross-correlation function was applied to the fluorescence fluctuation from these two positions to capture the flow of ... [truncated at 150 words]
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Axial super resolution topography of focal adhesion by confocal microscopy.
Microsc Res Tech. 2013; 76(10): 1070-1078. PMCID: PMC3871876The protein organization within focal adhesions has been studied by state-of-the-art super resolution methods because of 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, limiting 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 with about 4,000 photon counts collected along a z-scan, axial localization precision close to 10 nm is achievable. We show that, with within 10 nm, the axial location of paxillin, FAK, and talin is similar at focal adhesion sites, while F-actin shows a sharp increase in height towards the cell center. We further demonstrated the live imaging capability of this method. With the advantage of simple data acquisition and no special instrument requirement, ... [truncated at 150 words]
Leproux A, Durkin A, Compton M, Cerussi AE, Gratton E, Tromberg BJ.
Assessing tumor contrast in radiographically dense breast tissue using Diffuse Optical Spectroscopic Imaging (DOSI).
Breast Cancer Res. 2013; 15(5): R89. PMCID: PMC3979060INTRODUCTION: Radiographic density adversely affects the performance of x-ray mammography and can be particularly problematic in younger and high-risk women. Because of this limitation, there is signficant ongoing effort to develop alternative cancer screening and detection strategies for this population. This pilot study evaluates the potential of Diffuse Optical Spectroscopic Imaging (DOSI) to image known tumors in dense breast tissue.
METHODS: We performed a retrospective analysis on 24 radiographically dense breast cancer subjects measured with DOSI over a 4 year period (Bi-RADS category 3 and 4, average age = 39 +/- 7.6, average maximum size 31 +/- 17 mm). Two previously-described DOSI contrast functions, the tissue optical index (TOI), and the specific tumor component (STC), which are based upon the concentrations and spectral signatures of hemoglobin, water and lipids, respectively, were used to form 2D optical images of breast tumors.
RESULTS: Using TOI and STC, 21 out of 24 breast tumors were ... [truncated at 150 words]
Cutrale F, Salih A, Gratton E.
Spectral Phasor approach for fingerprinting of photo-activatable fluorescent proteins Dronpa, Kaede and KikGR.
Method Appl Fluoresc. 2013; 1(3): 35001. PMCID: PMC3771857The phasor global analysis algorithm is common for fluorescence lifetime applications, but has only been recently proposed for spectral analysis. Here the phasor representation and fingerprinting is exploited in its second harmonic to determine the number and spectra of photo-activated states as well as their conversion dynamics. We follow the sequence of photo-activation of proteins over time by rapidly collecting multiple spectral images. The phasor representation of the cumulative images provides easy identification of the spectral signatures of each photo-activatable protein.
Chiu CL, Digman MA, Gratton E.
Cell matrix remodeling ability shown by image spatial correlation.
J Biophys. 2013; 2013, 532030. PMCID: PMC3725897Extracellular matrix (ECM) remodeling is a critical step of many biological and pathological processes. However, most of the studies to date lack a quantitative method to measure ECM remodeling at a scale comparable to cell size. Here, we applied image spatial correlation to collagen second harmonic generation (SHG) images to quantitatively evaluate the degree of collagen remodeling by cells. We propose a simple statistical method based on spatial correlation functions to determine the size of high collagen density area around cells. We applied our method to measure collagen remodeling by two breast cancer cell lines (MDA-MB-231 and MCF-7), which display different degrees of invasiveness, and a fibroblast cell line (NIH/3T3). We found distinct collagen compaction levels of these three cell lines by applying the spatial correlation method, indicating different collagen remodeling ability. Furthermore, we quantitatively measured the effect of Latrunculin B and Marimastat on MDA-MB-231 cell line collagen remodeling ability ... [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.
Proc Natl Acad Sci USA. 2013; 110(30): 12307/12. PMCID: PMC3725058Spatial distribution and dynamics of plasma-membrane proteins are thought to be modulated by lipid composition and by the underlying cytoskeleton, which forms transient barriers to diffusion. So far this idea was probed by single-particle tracking of membrane components in which gold particles or antibodies were used to individually monitor the molecules of interest. Unfortunately, the relatively large particles needed for single-particle tracking can in principle alter the very dynamics under study. Here, we use a method that makes it possible to investigate plasma-membrane proteins by means of small molecular labels, specifically single GFP constructs. First, fast imaging of the region of interest on the membrane is performed. For each time delay in the resulting stack of images the average spatial correlation function is calculated. We show that by fitting the series of correlation functions, the actual protein "diffusion law" can be obtained directly from imaging, in the form of a ... [truncated at 150 words]
Skinner JP, Swift KM, Ruan Q, Perfetto S, Gratton E, Tetin S.
Simplified confocal microscope for counting particles at low concentrations.
Rev Sci Instrum. 2013; 84(7): 074301. PMCID: PMC3724729We describe a compact scanning confocal fluorescence microscope capable of detecting particles concentrations less than 100 particles/ml in similar to 15 min. The system mechanically moves a cuvette containing similar to 3 ml of sample. A relatively large confocal volume is observed within the cuvette using a 1 mm pinhole in front of a detection PMT. Due to the motion of the sample, particles traverse the confocal volume quickly, and analysis by pattern recognition qualifies spikes in the emission intensity data and counts them as events. We show linearity of detection as a function of concentration and also characterize statistical behavior of the instrument. We calculate a detection sensitivity of the system using 3 mu m fluorescent microspheres to be 5 particles/ml. Furthermore, to demonstrate biological application, we performed a dilution series to quantify stained E. coli and yeast cells. We counted E. coli cells at a concentration as low ... [truncated at 150 words]
Altamore I, Lanzanò L, Gratton E.
Dual channel detection of ultra low concentration of bacteria in real time by scanning fluorescence correlation spectroscopy.
Meas Sci Technol. 2013; 24(6): 065702. PMCID: PMC3770197We describe a novel method to detect very low concentrations of bacteria in water. Our device consists of a portable horizontal geometry small confocal microscope with large pinhole and a holder for cylindrical cuvettes containing the sample. Two motors provide fast rotational and slow vertical motion of the cuvette so the device looks like a simplified flow cytometer without flow. To achieve high sensitivity, the design has two detection channels. Bacteria are stained by two different nucleic acid dyes and excited with two different lasers. Data are analyzed with a correlation filter based on particle passage pattern recognition. The passage of a particle through the illumination volume is compared with a Gaussian pattern in both channels. The width of the Gaussian correlates with the time of passage of the particle so one particle is counted when the algorithm finds a match with a Gaussian in both channels. The concentration of ... [truncated at 150 words]
Conway DE, Breckenridge MT, Hinde E, Gratton E, Chen CS, Schwartz MA.
Fluid shear stress on endothelial cells modulates mechanical tension across VE-Cadherin and PECAM-1.
Curr Biol. 2013; 23(11): 1024-1030. PMCID: PMC3676707Fluid shear stress (FSS) from blood flow acting on the endothelium critically regulates vascular morphogenesis, blood pressure, and atherosclerosis [1]. FSS applied to endothelial cells (ECs) triggers signaling events including opening of ion channels, activation of signaling pathways, and changes in gene expression. Elucidating how ECs sense flow is important for understanding both normal vascular function and disease. EC responses to FSS are mediated in part by a junctional mechanosensory complex consisting of VE-cadherin, PECAM-1, and VEGFR2 [2]. Previous work suggested that flow increases force on PECAM-1, which initiates signaling [2-4]. Deletion of PECAM-1 blocks responses to flow in vitro and flow-dependent vascular remodeling in vivo [2, 5]. To understand this process, we developed and validated FRET-based tension sensors for VE-cadherin and PECAM-1 using our previously developed FRET tension biosensor [6]. FRET measurements showed that in static culture, VE-cadherin in cell-cell junctions bears significant myosin-dependent tension, whereas there was no ... [truncated at 150 words]
Alexander BS, Gelb AW, Mantulin WW, Cerussi AE, Tromberg BJ, Yu Z, Lee C, Meng L.
Impact of stepwise hyperventilation on cerebral tissue oxygen saturation in anesthetized patients: a mechanistic study.
Acta Anaesthesiol Scand. 2013; 57(5): 604-612. PMCID: PMC3992996Background: While the decrease in blood carbon dioxide (CO2) secondary to hyperventilation is generally accepted to play a major role in the decrease of cerebral tissue oxygen saturation (SctO2), it remains unclear if the associated systemic hemodynamic changes are also accountable.
Methods: Twenty-six patients (American Society of Anesthesiologists I–II) undergoing nonneurosurgical procedures were anesthetized with either propofol-remifentanil (n = 13) or sevoflurane (n = 13). During a stable intraoperative period, ventilation was adjusted stepwise from hypoventilation to hyperventilation to achieve a progressive change in end-tidal CO2 (ETCO2) from 55 to 25 mmHg. Minute ventilation, SctO2, ETCO2, mean arterial pressure (MAP), and cardiac output (CO) were recorded.
Results: Hyperventilation led to a SctO2 decrease from 78 ± 4% to 69 ± 5% (Δ = −9 ± 4%, P < 0.001) in the propofol-remifentanil group and from 81 ± 5% to 71 ± 7% (Δ = −10 ± 3%, P < 0.001) in the sevoflurane group. The decreases in SctO2 were not statistically different between these two groups (P = 0.5). SctO2 correlated significantly with ETCO2 in both groups (P < 0.001). SctO2 ... [truncated at 150 words]
Torno K, Wright BK, Jones MR, Digman MA, Gratton E, Phillips M.
Real-time analysis of metabolic activity within lactobacillus acidophilus by phasor fluorescence lifetime imaging microscopy of NADH.
Curr Microbiol. 2013; 66(4): 365-367.Nicotinamide adenine dinucleotide (NADH) is an endogenous fluorescent molecule commonly used as a metabolic biomarker. Fluorescence lifetime imaging microscopy (FLIM) is a method in which the fluorescence decay is measured at each pixel of an image. While the fluorescence spectrum of free and protein-bound NADH is very similar, free and protein-bound NADH display very different decay profiles. Therefore, FLIM can provide a way to distinguish free/bound NADH at the level of single bacteria within biological samples. The phasor technique is a graphical method to analyse the entire image and to produce a histogram of pixels with different decay profile. In this study, NADH fluorescence decay profiles within Lactobacillus acidophilus samples treated using different protocols indicated discernible variations. Clear distinctions between fluorescence decay profiles of NADH in samples of artificially heightened metabolic activity in comparison to those of samples lacking an accessible carbon source were obtained.
Golfetto O, Hinde E, Gratton E.
Laurdan fluorescence lifetime discriminates Cholesterol content from changes in fluidity in living cell membranes.
Biophys J. 2013; 104(6): 1238-1247. PMCID: PMC3602759Detection of the fluorescent properties of Laurdan has been proven to be an efficient tool to investigate membrane packing and ordered lipid phases in model membranes and living cells. Traditionally the spectral shift of Laurdan’s emission from blue in the ordered lipid phase of the membrane (more rigid) toward green in the disordered lipid phase (more fluid) is quantified by the generalized polarization function. Here, we investigate the fluorescence lifetime of Laurdan at two different emission wavelengths and find that when the dipolar relaxation of Laurdan’s emission is spectrally isolated, analysis of the fluorescence decay can distinguish changes in membrane fluidity from changes in cholesterol content. Using the phasor representation to analyze changes in Laurdan’s fluorescence lifetime we obtain two different phasor trajectories for changes in polarity versus changes in cholesterol content. This gives us the ability to resolve in vivo membranes with different properties such as water content and ... [truncated at 150 words]
Krasieva TB, Stringari C, Liu F, Sun CH, Kong Y, Balu M, Meyskens FL, Gratton E, Tromberg BJ.
Two-photon excited fluorescence lifetime imaging and spectroscopy of melanins in vitro and in vivo.
J Biomed Opt. 2013; 18(3): 31107. PMCID: PMC3595716Changes in the amounts of cellular eumelanin and pheomelanin have been associated with carcinogenesis. The goal of this work is to develop methods based on two-photon-excited-fluorescence (TPEF) for measuring relative concentrations of these compounds. We acquire TPEF emission spectra (λex=1000 nm) of melanin in vitro from melanoma cells, hair specimens, and in vivo from healthy volunteers. We find that the pheomelanin emission peaks at approximately 615 to 625 nm and eumelanin exhibits a broad maximum at 640 to 680 nm. Based on these data we define an optical melanin index (OMI) as the ratio of fluorescence intensities at 645 and 615 nm. The measured OMI for the MNT-1 melanoma cell line is 1.6±0.22 while the Mc1R gene knockdown lines MNT-46 and MNT-62 show substantially greater pheomelanin production (OMI=0.5±0.05 and 0.17±0.03, respectively). The measured values are in good agreement with chemistry-based melanin extraction methods. In order to better separate melanin fluorescence from ... [truncated at 150 words]
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A practical implementation of multifrequency widefield frequency-domain fluorescence lifetime imaging microscopy.
Microsc Res Tech. 2013; 76(3): 282-289. PMCID: PMC3742114Widefield frequency-domain fluorescence lifetime imaging microscopy (FD-FLIM) is a fast and accurate method to measure the fluorescence lifetime, especially in kinetic studies in biomedical researches. However, the small range of modulation frequencies available in commercial instruments makes this technique limited in its applications. Herein, we describe a practical implementation of multifrequency widefield FD-FLIM using a pulsed supercontinuum laser and a direct digital synthesizer. In this instrument we use a pulse to modulate the image intensifier rather than the more conventional sine-wave modulation. This allows parallel multifrequency FLIM measurement using the Fast Fourier Transform and the cross-correlation technique, which permits precise and simultaneous isolation of individual frequencies. In addition, the pulse modulation at the cathode of image intensifier restores the loss of optical resolution caused by the defocusing effect when the cathode is sinusoidally modulated. Furthermore, in our implementation of this technique, data can be graphically analyzed by the phasor method ... [truncated at 150 words]
Dobrinskikh E, Lanzano L, Rachelson J, Cranston D, Moldovan R, Lei T, Gratton E, Doctor RB.
Shank2 contributes to the apical retention and intracellular redistribution of NaPiIIa in OK cells.
Am J Physiol Cell Physiol. 2013; 304(6): C561-573. PMCID: PMC3674434In renal proximal tubule (PT) cells, sodium-phosphate co-transporter IIa (NaPiIIa) is normally concentrated within the apical membrane where it reabsorbs ~70% of luminal phosphate (Pi). NaPiIIa activity is acutely regulated by moderating its abundance within the apical membrane. Under low Pi conditions, NaPiIIa is retained within the apical membrane. Under high Pi conditions, NaPiIIa is retrieved from the apical membrane and trafficked to the lysosomes for degradation. The present study investigates the role of Shank2 in regulating the distribution of NaPiIIa. In opossum kidney (OK) cells, a PT cell model, knockdown of Shank2 in cells maintained in low Pi media resulted in a marked decrease in NaPiIIa abundance. After transferring into high phosphate media, live cell imaging showed that mRFP-Shank2E and GFP-NaPiIIa underwent endocytosis and trafficked together through the sub-apical domain. Fluorescence cross-correlation spectroscopy demonstrated that GFP-NaPiIIa and mRFP-Shank2 have indistinguishable diffusion coefficients and migrated through the sub-apical domain in ... [truncated at 150 words]
Andrews LM, Jones MR, Digman MA, Gratton E.
Detecting Pyronin Y labeled RNA transcripts in live cell microenvironments by phasor-FLIM analysis.
Methods Appl Fluoresc. 2013; 1(1): 015001. PMCID: PMC3929300Pyronin Y is an environment-sensitive probe which labels all double-stranded RNA in live cells. Methods to determine which RNA species Pyronin Y may be labeling are limited due to the lack of studies aimed at determining whether this probe has different spectroscopic properties when bound to specific transcripts. A major issue is that transcripts are difficult to isolate and study individually. We detected transcripts directly in their biological environment allowing us to identify RNA species on the basis of their location in the cell. We show that the phasor approach to lifetime analysis has the sensitivity to determine at least six different RNA species in live fibroblast cells. The detected lifetime differences were consistent among cells. To our knowledge this is the first application of a spectroscopic technique aimed at identifying Pyronin Y labeled RNA subtypes in living cells.
Coppola S, Pozzi D, de Sanctis SC, Digman MA, Gratton E, Caracciolo G.
Quantitative measurement of intracellular transport of nanocarriers by spatio-temporal image correlation spectroscopy.
Methods Appl Fluoresc. 2013; 1(1): 015005. PMCID: PMC3871877Spatio-temporal image correlation spectroscopy (STICS) is a powerful technique for assessing the nature of particle motion in complex systems although it has been rarely used to investigate the intracellular dynamics of nanocarriers so far. Here we introduce a method for characterizing the mode of motion of nanocarriers and for quantifying their transport parameters on different length scales from single-cell to subcellular level. Using this strategy we were able to study the mechanisms responsible for the intracellular transport of DOTAP–DOPC/DNA (DOTAP: 1,2-dioleoyl-3-trimethylammonium-propane; DOPC: dioleoylphosphocholine) and DC-Chol–DOPE/DNA (DC-Chol: 3β-[N-(N,N-dimethylaminoethane)-carbamoyl] cholesterol; DOPE: dioleoylphosphatidylethanolamine) lipoplexes in CHO-K1 (CHO: Chinese hamster ovary) live cells. Measurement of both diffusion coefficients and velocity vectors (magnitude and direction) averaged over regions of the cell revealed the presence of distinct modes of motion. Lipoplexes diffused slowly on the cell surface (diffusion coefficient: D ≈ 0.003 μm2 s−1). In the cytosol, the lipoplexes' motion was characterized by active transport with ... [truncated at 150 words]
Adu-Gyamfi E, Soni SP, Xue Y, Digman MA, Gratton E, Stahelin RV.
The Ebola virus matrix protein penetrates into the plasma membrane: A key step in VP40 oligomerization and viral egress.
J Biol Chem. 2013; 288(8): 5779-5789. PMCID: PMC3581432Ebola, a fatal virus in humans and non-human primates has no FDA approved vaccines or therapeutics. The virus from the Filoviridae family causes hemorrhagic fever, which rapidly progresses and in some cases has a fatality rate near 90%. The Ebola genome encodes seven genes the most abundantly expressed of which is viral protein 40 (VP40) the major Ebola matrix protein that regulates assembly and egress of the virus. It is well established that VP40 assembles on the inner leaflet of the PM; however, the mechanistic details of plasma membrane association by VP40 are not well understood. In this study, we used an array of biophysical experiments and cellular assays along with mutagenesis of VP40 to investigate the role of membrane penetration in VP40 assembly and egress. Here we demonstrate that VP40 is able to penetrate specifically into the plasma membrane through an interface enriched in hydrophobic residues in its C-terminal ... [truncated at 150 words]
Coppola S, Cardarelli F, Pozzi D, Estrada LC, Digman MA, Gratton E, Bifone A, Marianecci C, Caracciolo G.
The role of cytoskeleton networks on lipid-mediated delivery of DNA.
Ther Deliv. 2013; 4(2): 191-202. PMCID: PMC3771858Background: Lipid-mediated delivery of DNA is hindered by extracellular and intracellular barriers that significantly reduce the transfection efficiency of synthetic nonviral vectors.
Results: In this study we investigated the role of the actin and microtubule networks on the uptake and cytoplasmic transport of multicomponent cationic liposome–DNA complexes in CHO-K1 live cells by means of confocal laser scanning microscopy and 3D single particle tracking. Treatment with actin (latrunculin B)- and microtubule-disrupting (nocodazole) reagents indicated that intracellular trafficking of complexes predominantly involves microtubule-dependent active transport. We found that the actin network has a major effect on the initial uptake of complexes, while the microtubule network is mainly responsible for the subsequent active transportation to the lysosomes.
Conclusion: Collectively, a strategy to improve the efficiency of lipid gene vectors can be formulated. We could find a lipid formulation that allows the nanoparticles to avoid the microtubule pathway to lysosomes.
Li K, Markosyan RM, Zheng YM, Golfetto O, Bungart B, Li M, Ding S, He Y, Liang C, Lee JC, Gratton E, Cohen FS, Liu SL.
IFITM Proteins restrict viral membrane hemifusion.
PLoS Pathog. 2013; 9(1): e1003124. PMCID: PMC3554583The interferon-inducible transmembrane (IFITM) protein family represents a new class of cellular restriction factors that block early stages of viral replication; the underlying mechanism is currently not known. Here we provide evidence that IFITM proteins restrict membrane fusion induced by representatives of all three classes of viral membrane fusion proteins. IFITM1 profoundly suppressed syncytia formation and cell-cell fusion induced by almost all viral fusion proteins examined; IFITM2 and IFITM3 also strongly inhibited their fusion, with efficiency somewhat dependent on cell types. Furthermore, treatment of cells with IFN also markedly inhibited viral membrane fusion and entry. By using the Jaagsiekte sheep retrovirus envelope and influenza A virus hemagglutinin as models for study, we showed that IFITM-mediated restriction on membrane fusion is not at the steps of receptor- and/or low pH-mediated triggering; instead, the creation of hemifusion was essentially blocked by IFITMs. Chlorpromazine (CPZ), a chemical known to promote the transition from ... [truncated at 150 words]
Hinde E, Digman MA, Hahn KM, Gratton E.
Millisecond spatiotemporal dynamics of FRET biosensors by the pair correlation function and the phasor approach to FLIM.
Proc Natl Acad Sci USA. 2013; 110(1): 135-140. PMCID: PMC3538204Here we present a fluctuation-based approach to biosensor Förster resonance energy transfer (FRET) detection that can measure the molecular flow and signaling activity of proteins in live cells. By simultaneous use of the phasor approach to fluorescence lifetime imaging microscopy (FLIM) and cross-pair correlation function (pCF) analysis along a line scanned in milliseconds, we detect the spatial localization of Rho GTPase activity (biosensor FRET signal) as well as the diffusive route adopted by this active population. In particular we find, for Rac1 and RhoA, distinct gradients of activation (FLIM-FRET) and a molecular flow pattern (pCF analysis) that explains the observed polarized GTPase activity. This multiplexed approach to biosensor FRET detection serves as a unique tool for dissection of the mechanism(s) by which key signaling proteins are spatially and temporally coordinated.
Andrews LM, Jones MR, Digman MA, Gratton E.
Spectral phasor analysis of Pyronin Y labeled RNA microenvironments in living cells.
Biomed Opt Express. 2013; 4(1): 171-177. PMCID: PMC3539199We show that the spectral phasor approach of the fluorescent dye Pyronin Y (PY) can be used to identify specific RNA subspecies of ribonuclear proteins complexes in live cells. We applied spectral phasors to isolate intracellular RNA species with similar spectral properties. We identified at least 4 different PY labeled species in live cells and further spatially mapped their presence at the pixel level. Most notable were transcripts in the nucleoli which were spectrally similar to RNA clusters in the cytoplasm. We propose that these species represent ribosomal RNA and clustered ribonucleoprotein complexes. Further, we observed within this cluster Cajal bodies in the proximity of the nucleolus. In addition, transcripts in the cytoplasm undertook a filamentous morphology composed of multiple puncti structures which individually localized along and close to mitochondria but were distinct from mitochondria.
de Torre DL, Gordon EA, Digman MA, Stakic M, Häberlein H, Gratton E, Caballero-George C.
Raster Image Correlation Spectroscopy in Live cells expressing endothelin ETA receptor.
Current Trends in Biotechnology and Pharmacy. 2013; 7(1): 499-504.Fluorescence spectroscopy is the most common non-radioactive technique used to study GPCR interactions with their ligands. Raster image correlation spectroscopy (RICS) exploits spatio-temporal correlation functions rather than the simple temporal correlations of conventional fluorescence correlation spectroscopy. In this paper we describe the use of RICS and the number and brightness method to determine the diffusion of a construct of endothelin ETA receptor with EGFP and the aggregation state in the cytoplasm. Our construct seems to locate mainly in the cytoplasm where it undergoes diffusion and it appears to be monomeric. Although our construct could not fully represent the native protein, we believe that the methodology we describe in this paper could be used by anyone in this field.
Hinde E, Cardarelli F, Chen A, Khine M, Gratton E.
Tracking the mechanical dynamics of human embryonic stem cell chromatin.
Epigenetics Chromatin. 2012; 5(1): 20. PMCID: PMC3570407BACKGROUND: 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 high frequency chromatin movement and quantify the mechanical dynamics of chromatin in live cells
RESULTS:
We use this method to study how the mechanical properties of chromatin movement in human embryonic stem cells (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.
CONCLUSIONS:
We thus propose that the measured chromatin high frequency movements in hESCs may represent a hallmark of ... [truncated at 150 words]
Whiteside MD, Digman MA, Gratton E, Treseder KK.
Organic nitrogen uptake by arbuscular mycorrhizal fungi in a boreal forest.
Soil Biol Biochem. 2012; 55: 7-13. PMCID: PMC3871874The breakdown of organic nitrogen in soil is a potential rate-limiting step in nitrogen cycling. Arbuscular mycorrhizal (AM) fungi are root symbionts that might improve the ability of plants to compete for organic nitrogen products against other decomposer microbes. However, AM uptake of organic nitrogen, especially in natural systems, has traditionally been difficult to test. We developed a novel quantitative nanotechnological technique to determine in situ that organic nitrogen uptake by AM fungi can occur to a greater extent than has previously been assumed. Specifically, we found that AM fungi acquired recalcitrant and labile forms of organic nitrogen. Moreover, N enrichment of soil reduced plot-scale uptake of these compounds. Since most plants host AM fungi, AM use of organic nitrogen could widely influence plant productivity, especially where N availability is relatively low.
Wright BK, Andrews LM, Jones MR, Stringari C, Digman MA, Gratton E.
Phasor-flim analysis of NADH distribution and localization in the nucleus of live progenitor myoblast cells.
Microsc Res Tech. 2012; 75(12): 1717-1722. PMCID: PMC3635844Analysis of the cellular distributions of coenzymes including NADH may aid in understanding a cells metabolic status. We altered serum concentration (0, 2, and 10%) to induce living myoblast cells to undergo the early stages of differentiation. Through microscopy and phasor-FLIM, we spatially mapped and identified variations in the distribution of free and bound NADH. Undifferentiated cells displayed abundant free NADH within the nucleus along with specific regions of more bound NADH. Complete serum starvation dramatically increased the fraction of bound NADH in the nucleus, indicating heightened requirement for transcriptional processes. In comparison, cells exposed to 2% serum exhibited intermediate free nuclear NADH fraction. Overall our results suggest an order of events in which a cell metabolic status alters significantly during the early stages of serum induced differentiation.
Wigal SB, Polzonetti CM, Stehli A, Gratton E.
Phase synchronization of oxygenation waves in the frontal areas of children with attention-deficit hyperactivity disorder detected by optical diffusion spectroscopy correlates with medication.
J Biomed Opt. 2012; 17(12): 127002. PMCID: PMC3518849The beneficial effects of pharmacotherapy on children with attention-deficit hyperactivity disorder (ADHD) are well documented. We use near-infrared spectroscopy (NIRS) methodology to determine reorganization of brain neurovascular properties following the medication treatment. Twenty-six children with ADHD (ages six through 12) participated in a modified laboratory school protocol to monitor treatment response with lisdexamfetamine dimesylate (LDX; Vyvanse®, Shire US Inc.). All children refrained from taking medication for at least two weeks (washout period). To detect neurovascular reorganization, we measured changes in synchronization of oxy (HbO2) and deoxy (HHb) hemoglobin waves between the two frontal lobes. Participants without medication displayed average baseline HbO2 phase difference at about -7-deg. and HHb differences at about 240-deg.. This phase synchronization index changed after pharmacological intervention. Medication induced an average phase changes of HbO2 after first medication to 280-deg. and after medication optimization to 242-deg.. Instead first medication changed of the average HHb phase difference at ... [truncated at 150 words]
Crosignani V, Dvornikov AS, Aguilar JS, Stringari C, Edwards R, Mantulin WW, Gratton E.
Deep tissue fluorescence imaging and in vivo biological applications.
J Biomed Opt. 2012; 17(11): 116023. PMCID: PMC3494495We describe a novel technical approach with enhanced fluorescence detection capabilities in two-photon microscopy that achieves deep tissue imaging, while maintaining micron resolution. Compared to conventional two-photon microscopy, greater imaging depth is achieved by more efficient harvesting of fluorescence photons propagating in multiple-scattering media. The system maintains the conventional two-photon microscopy scheme for excitation. However, for fluorescence collection the detection system harvests fluorescence photons directly from a wide area of the turbid sample. The detection scheme relies on a wide area detector, minimal optical components and an emission path bathed in a refractive-index-matching fluid that minimizes emission photon losses. This detection scheme proved to be very efficient, allowing us to obtain high resolution images at depths up to 3 mm. This technique was applied to in vivo imaging of the murine small intestine (SI) and colon. The challenge is to image normal and diseased tissue in the whole live animal, ... [truncated at 150 words]
Adu-Gyamfi E, Digman MA, Gratton E, Stahelin RV.
Single-particle tracking demonstrates that actin coordinates the movement of the Ebola virus matrix protein.
Biophys J. 2012; 103(9): L41-L43. PMCID: PMC3491695The Ebola virus causes severe hemorrhagic fever and has a mortality rate that can be as high as 90%, yet no vaccines or approved therapeutics, to our knowledge, are available. To replicate and egress the infected host cell the Ebola virus uses VP40, its major matrix protein to assemble at the inner leaflet of the plasma membrane. The assembly and budding of VP40 from the plasma membrane of host cells seem still poorly understood. We investigated the assembly and egress of VP40 at the plasma membrane of human cells using single-particle tracking. Our results demonstrate that actin coordinates the movement and assembly of VP40, a critical step in viral egress. These findings underscore the ability of single-molecule techniques to investigate the interplay of VP40 and host proteins in viral replication.
Stringari C, Nourse JL, Flanagan LA, Gratton E.
Phasor fluorescence lifetime microscopy of free and protein-bound NADH reveals neural stem cell differentiation potential.
PLoS One. 2012; 7(11): e48014. PMCID: PMC3489895In the stem cell field there is a lack of non invasive and fast methods to identify stem cell's metabolic state, differentiation state and cell-lineage commitment. Here we describe a label-free method that uses NADH as an intrinsic biomarker and the Phasor approach to Fluorescence Lifetime microscopy to measure the metabolic fingerprint of cells. We show that different metabolic states are related to different cell differentiation stages and to stem cell bias to neuronal and glial fate, prior the expression of lineage markers. Our data demonstrate that the NADH FLIM signature distinguishes non-invasively neurons from undifferentiated neural progenitor and stem cells (NPSCs) at two different developmental stages (E12 and E16). NPSCs follow a metabolic trajectory from a glycolytic phenotype to an oxidative phosphorylation phenotype through different stages of differentiation. NSPCs are characterized by high free/bound NADH ratio, while differentiated neurons are characterized by low free/bound NADH ratio. We demonstrate that ... [truncated at 150 words]
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Inclined selective plane illumination microscopy adaptor for conventional microscopes.
Microsc Res Tech. 2012; 75(11): 1461-1466. PMCID: PMC3742098Driven by the biological sciences, there is an increased need for imaging modalities capable of live cell imaging with high spatial and temporal resolution. To achieve this goal in a comprehensive manner, three-dimensional acquisitions are necessary. Ideal features of a modern microscope system should 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. Given the importance of collecting data in live sample further increases the technical challenges required to solve these issues. This work presents a practical version of a microscopy method, Selective Plane Illumination Microscopy re-introduced by Huisken et al. (Science2004,305,1007-1009). This method is gaining importance in the biomedical field, but its use is limited by difficulties associated with unconventional microscope design which employs two objectives and a particular kind of sample preparation needed to insert the sample between the objectives. Based on the selective ... [truncated at 150 words]
Giral H, Cranston D, Lanzanò L, Caldas Y, Sutherland E, Rachelson J, Dobrinskikh E, Weinman EJ, Doctor RB, Gratton E, Levi M.
NHE3 regulatory factor 1 (NHERF1) modulates intestinal Na-dependent phosphate transporter (NaPi-2b) expression in apical microvilli.
J Biol Chem. 2012; 287(42): 35047-35056. PMCID: PMC3471691Pi uptake in the small intestine occurs predominantly through the NaPi-2b (SLC34a2) co-transporter. NaPi-2b is regulated by changes in dietary Pi but the mechanisms underlying this regulation are largely undetermined. Sequence analyses show NaPi-2b has a PDZ binding motif at its C-terminus. Immunofluorescence imaging shows NaPi-2b and two PDZ domain containing proteins, NHERF1 and PDZK1, are expressed in the apical microvillar domain of rat small intestine enterocytes. Co-immunoprecipitation studies in rat enterocytes show that NHERF1 associates with NaPi-2b but not PDZK1. In HEK co-expression studies, GFP-NaPi-2b co-precipitates with Flag-NHERF1. This interaction is markedly diminished when the C-terminal four amino acids are truncated from NaPi-2b. FLIM-FRET analyses using tagged proteins in CACO-2BBE cells show a distinct phasor shift between NaPi-2b and NHERF1 but not between NaPi-2b and PDZK1 pair. This shift demonstrates NaPi-2b and NHERF1 reside within 10 nm of each other. NHERF1 (-/-) mice, but not PDZK1 (-/-) mice, had ... [truncated at 150 words]
Arnesano C, Santoro Y, Gratton E.
Digital parallel frequency-domain spectroscopy for tissue imaging.
J Biomed Opt. 2012; 17(9): 096014. PMCID: PMC3442105Near-infrared (NIR) (650 to 1000 nm) optical properties of turbid media can be quantified accurately and noninvasively using methods based on diffuse reflectance or transmittance, such as frequency-domain photon migration (FDPM). Conventional FDPM techniques based on white-light steady-state (SS) spectral measurements in conjunction with the acquisition of frequency-domain (FD) data at selected wavelengths using laser diodes are used to measure broadband NIR scattering-corrected absorption spectra of turbid media. These techniques are limited by the number of wavelength points used to obtain FD data and by the sweeping technique used to collect FD data over a relatively large range. We have developed a method that introduces several improvements in the acquisition of optical parameters, based on the digital parallel acquisition of a comb of frequencies and on the use of a white laser as a single light source for both FD and SS measurements. The source, due to the high brightness, ... [truncated at 150 words]
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Measurement of distance with the nanoscale precise imaging by rapid beam oscillation method.
Microsc Res Tech. 2012; 75(9): 1253-1264. PMCID: PMC3426651We discuss here the principles of a novel optical method in which the scanning of a laser spot around a fluorescent object is used to determine its shape, orientation, and fluorophore distribution. The scanning pattern is adapted to the shape of the object according to a feedback principle based on intensity modulation measurements. The modulation of the intensity with respect to the angular coordinate is used to keep the orbit centered on the object. The modulation induced by rapid oscillations of the orbit radius is used to measure the local distance from the surface with nanometer precision. We provide a model to describe the fundamental relationship between modulation and distance and discuss the range of validity of several approximate expressions. According to this model, the distance can be measured with a precision dependent on the steepness of the point spread function and the total number of detected photons. To test ... [truncated at 150 words]
Coppola S, Estrada LC, Digman MA, Pozzi D, Cardarelli F, Gratton E, Caracciolo G.
Intracellular trafficking of cationic liposome-DNA complexes in living cells.
Soft Matter. 2012; 8(30): 7919-7927. PMCID: PMC4138718Three-dimensional single-particle tracking (SPT) was used to calculate the mean square displacement (MSD) and the diffusion coefficients of multicomponent cationic liposome–DNA complexes (lipoplexes) in CHO-K1 living cells. In untreated (NT) control cells, we found that the intracellular lipoplex motion was either directed or Brownian with active transportation being definitely more frequent (more than 70%) than Brownian diffusion. The MSD analysis was supported by the calculation of the three-dimensional asphericity, A3, which was close to unity, denoting the preponderant 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). When cells were treated with inhibitors, the lipoplex movement tended towards a random walk at the expense of directed motion. The disassembly of microtubules had a stronger effect on the reduction of directional movement than that of actin ... [truncated at 150 words]
Stringari C, Edwards RA, Pate KT, Waterman ML, Donovan PJ, Gratton E.
Metabolic trajectory of cellular differentiation in small intestine by phasor fluorescence lifetime microscopy of NADH.
Sci Rep. 2012; 2: 568. PMCID: PMC3416911There is a lack of fast and high resolution methods to measure metabolic activity of single cells in their native environment. Here we develop a straightforward, non-invasive and sensitive method to measure metabolic phenotype of single cells in a live tissue. By using NADH as optical biomarker and the phasor approach to Fluorescence Lifetime microscopy (FLIM) we identify cellular metabolic fingerprints related to different rates of oxidative phosphorylation and glycolysis. For the first time we measure a three dimensional metabolic gradient in the small intestine (SI) epithelia that appears tightly associated with epithelial cell proliferation, differentiation and the Wnt gradient. The highest free/bound NADH ratios are measured at the base of the crypt within the highly proliferative stem cells, indicating high levels of glycolysis. For the first time mouse small intestinal stem cells in intact live crypts are identified within the tissue by their metabolic fingerprint.
Wright BK, Andrews LM, Markham J, Jones MR, Stringar C, Digman MA, Gratton E.
NADH distribution in live progenitor stem cells by phasor-fluorescence lifetime image microscopy.
Biophys J. 2012; 103(1): L7-L9. PMCID: PMC3388207NADH is a naturally fluorescent metabolite associated with cellular respiration. Exploiting the different fluorescence lifetime of free and bound NADH has the potential to quantify the relative amount of bound and free NADH, enhancing understanding of cellular processes including apoptosis, cancer pathology, and enzyme kinetics. We use the phasor- fluorescence lifetime image microscopy approach to spatially map NADH in both the free and bound forms of live undifferentiated and differentiated myoblast cells. The phasor approach graphically depicts the change in lifetime at a pixel level without the requirement for fitting the decay. Comparison of the spatial distribution of NADH in the nucleus of cells induced to differentiate through serum starvation and undifferentiated cells show differing distributions of bound and free NADH. Undifferentiated cells displayed a short lifetime indicative of free NADH in the nucleus and a longer lifetime attributed to the presence of bound NADH outside of the nucleus. Differentiating ... [truncated at 150 words]
Cardarelli F, Lanzanò L, Gratton E.
Capturing directed molecular motion in the nuclear pore complex of live cells.
Proc Natl Acad Sci USA. 2012; 109(25): 9863-9868. PMCID: PMC3382504Nuclear pore complexes (NPCs) are gateways for nucleocytoplasmic exchange. Intrinsically disordered nucleoporins (Nups) form a selective filter inside the NPC, taking a central role in the vital nucleocytoplasmic transport mechanism. How such intricate meshwork relates to function and gives rise to a transport mechanism is still unclear. Here we set out to tackle this issue in intact cells by an established combination of fluorescence correlation spectroscopy and real-time tracking of the center of mass of single NPCs. We find the dynamics of nucleoporin Nup153 to be regulated so as to produce rapid, discrete exchange between two separate positions within the NPC. A similar behavior is also observed for both karyopherinβ1 transport-receptor and cargoes destined to nuclear import. Thus, we argue that directed Nup-mediated molecular motion may represent an intrinsic feature of the overall selective gating through intact NPCs.
Adu-Gyamfi E, Digman MA, Gratton E, Stahelin RV.
Investigation of Ebola VP40 assembly and oligomerization in live cells using number and brightness analysis.
Biophys J. 2012; 102(11): 2517-2525. PMCID: PMC3368128Ebola virus assembles and buds from the inner leaflet of the plasma membrane of mammalian cells, which is primarily attributed to its major matrix protein VP40. Oligomerization of VP40 has been shown to be essential to the life cycle of the virus including formation of virions from infected cells. To date, VP40 oligomerization has mainly been assessed by chemical cross-linking following cell fractionation studies with VP40 transfected cells. This has made it difficult to discern the spatial and temporal dynamics of VP40 oligomerization. To gain a better understanding of the VP40 assembly and oligomerization process in live cells, we have employed real-time imaging of enhanced green fluorescent protein tagged VP40. Here, we use both confocal and total internal reflection microscopy coupled with number and brightness analysis to show that VP40 oligomers are localized on the plasma membrane and are highly enriched at sites of membrane protrusion, consistent with sites of ... [truncated at 150 words]
James NG, Digman MA, Gratton E, Barylko B, Ding X, Albanesi JP, Goldberg MS, Jameson DM.
Number and brightness analysis of LRRK2 oligomerization in live cells.
Biophys J. 2012; 102(11): L41-L43. PMCID: PMC3368136Leucine-rich repeat kinase 2 (LRRK2) is a large multidomain protein that contains enzymatically functional GTPase and kinase domains. Several noncoding LRRK2 gene polymorphisms have been associated with susceptibility to Parkinson's disease (PD), Crohn's disease, and leprosy. Many LRRK2 coding polymorphisms have been associated with or causally linked to PD. The G2019S point mutation within the LRRK2 kinase domain is the most common cause of familial PD. The G2019S mutation appears to alter LRRK2 kinase activity. Some but not all studies have reported that LRRK2 kinase activity is dependent upon LRRK2 dimerization and membrane localization. It is important to define the oligomeric state(s) of LRRK2 in living cells, which to date have only been characterized in vitro. Here we use confocal and total internal reflection microscopy coupled with number and brightness analysis to study the oligomeric states of LRRK2 within the cytosol and on the plasma membrane of live CHO-K1 cells. ... [truncated at 150 words]
Battisti A, Digman MA, Gratton E, Storti B, Beltram F, Bizzarri R.
Intracellular pH measurements made simple by fluorescent protein probes and the phasor approach to fluorescence lifetime imaging.
Chem Commun (Camb). 2012; 48(42): 5127-5129. PMCID: PMC3742097A versatile pH-dependent fluorescent protein was applied to intracellular pH measurements by means of the phasor approach to fluorescence lifetime imaging. By this fit-less method we obtain intracellular pH maps under resting or altered physiological conditions by single-photon confocal or two-photon microscopy.
Stringari C, Gratton E, Sierra R, Donovan PJ.
Label-free separation of human embryonic stem cells and their differentiating progenies by phasor fluorescence lifetime microscopy.
J Biomed Opt. 2012; 17(7): 046012. PMCID: PMC3381030We develop a label-free optical technique to image and discriminate undifferentiated human embryonic stem cells (hESCs) from their differentiating progenies in vitro. Using intrinsic cellular fluorophores, we perform fluorescence lifetime microscopy (FLIM) and phasor analysis to obtain hESC metabolic signatures. We identify two optical biomarkers to define the differentiation status of hESCs: Nicotinamide adenine dinucleotide (NADH) and lipid droplet-associated granules (LDAGs). These granules have a unique lifetime signature and could be formed by the interaction of reactive oxygen species and unsaturated metabolic precursor that are known to be abundant in hESC. Changes in the relative concentrations of these two intrinsic biomarkers allow for the discrimination of undifferentiated hESCs from differentiating hESCs. During early hESC differentiation we show that NADH concentrations increase, while the concentration of LDAGs decrease. These results are in agreement with a decrease in oxidative phosphorylation rate. Single-cell phasor FLIM signatures reveal an increased heterogeneity in the metabolic ... [truncated at 150 words]
Sánchez SA, Tricerri MA, Gratton E.
Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo.
Proc Natl Acad Sci USA. 2012; 109(19): 7314-7319. PMCID: PMC3358851Cellular membranes are heterogeneous in composition, and the prevailing theory holds that the structures responsible for this heterogeneity in vivo are small structures (10–200 nm), sterol- and sphingolipid-enriched, of different sizes, highly dynamic denominated rafts. Rafts are postulated to be platforms, which by sequestering different membrane components can compartmentalize cellular processes and regulate signaling pathways. Despite an enormous effort in this area, the existence of these domains is still under debate due to the characteristics of the structures itself: small in size and highly mobile, which from the technical point of view implies using techniques with high spatial and temporal resolution. In this report we measured rapid fluctuations of the normalized ratio of the emission intensity at two wavelengths of Laurdan, a membrane fluorescent dye sensitive to local membrane packing. We observed generalized polarization fluctuations in the plasma membrane of intact rabbit erythrocytes and Chinese hamster ovary cells that can ... [truncated at 150 words]
Montecinos-Franjola F, Ross JA, Sánchez SA, Brunet JE, Lagos R, Jameson DM, Monasterio O.
Studies on the dissociation and urea-induced unfolding of FtsZ support the dimer nucleus polymerization mechanism.
Biophys J. 2012; 102(9): 2176-2185. PMCID: PMC3341561FtsZ is a major protein in bacterial cytokinesis that polymerizes into single filaments. A dimer has been proposed to be the nucleating species in FtsZ polymerization. To investigate the influence of the self-assembly of FtsZ on its unfolding pathway, we characterized its oligomerization and unfolding thermodynamics. We studied the assembly using size-exclusion chromatography and fluorescence spectroscopy, and the unfolding using circular dichroism and two-photon fluorescence correlation spectroscopy. The chromatographic analysis demonstrated the presence of monomers, dimers, and tetramers with populations dependent on protein concentration. Dilution experiments using fluorescent conjugates revealed dimer-to-monomer and tetramer-to-dimer dissociation constants in the micromolar range. Measurements of fluorescence lifetimes and rotational correlation times of the conjugates supported the presence of tetramers at high protein concentrations and monomers at low protein concentrations. The unfolding study demonstrated that the three-state unfolding of FtsZ was due to the mainly dimeric state of the protein, and that the monomer unfolds ... [truncated at 150 words]
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Scanning image correlation spectroscopy.
Bioessays. 2012; 34(5): 377-385. PMCID: PMC3635842Molecular interactions are at the origin of life. How molecules get at different locations in the cell and how they locate their partners is a major and partially unresolved question in biology that is paramount to signaling. Spatio-temporal correlations of fluctuating fluorescently tagged molecules reveal how they move, interact, and bind in the different cellular compartments. Methods based on fluctuations represent a remarkable technical advancement in biological imaging. Here we discuss image analysis methods based on spatial and temporal correlation of fluctuations, raster image correlation spectroscopy, number and brightness, and spatial cross-correlations that give us information about how individual molecules move in cells and interact with partners at the single molecule level. These methods can be implemented with a standard laser scanning microscope and produce a cellular level spatio-temporal map of molecular interactions.
Meng L, Gelb AW, Alexander BS, Cerussi AE, Tromberg BJ, Yu Z, Mantulin WW.
Impact of phenylephrine administration on cerebral tissue oxygen saturation and blood volume is modulated by carbon dioxide in anaesthetized patients.
Br J Anaesth. 2012; 108(5): 815-822. PMCID: PMC3325051BACKGROUND: Multiple studies have shown that cerebral tissue oxygen saturation () is decreased after phenylephrine treatment. We hypothesized that the negative impact of phenylephrine administration on is affected by arterial blood carbon dioxide partial pressure () because CO(2) is a powerful modulator of cerebrovascular tone.
METHODS: In 14 anaesthetized healthy patients, i.v. phenylephrine bolus was administered to increase the mean arterial pressure ∼20-30% during hypocapnia, normocapnia, and hypercapnia. and cerebral blood volume (CBV) were measured using frequency domain near-infrared spectroscopy, a quantitative technology. Data collection occurred before and after each treatment.
RESULTS: Phenylephrine caused a significant decrease in during hypocapnia [=-3.4 (1.5)%, P<0.001], normocapnia [=-2.4 (1.5)%, P<0.001], and hypercapnia [=-1.4 (1.5)%, P<0.01]. Decreases in were significantly different between hypocapnia, normocapnia, and hypercapnia (P<0.001). Phenylephrine also caused a significant decrease in CBV during hypocapnia (P<0.01), but not during normocapnia or hypercapnia.
CONCLUSION: The negative impact of phenylephrine treatment on and CBV is intensified during ... [truncated at 150 words]
Zhou S, Lo WC, Suhalim JL, Digman MA, Gratton E, Nie Q, Lander AD.
Free extracellular diffusion creates the Dpp morphogen gradient of the Drosophila wing disc.
Curr Biol. 2012; 22(8): 668-675. PMCID: PMC3338872BACKGROUND: How morphogen gradients form has long been a subject of controversy. The strongest support for the view that morphogens do not simply spread by free diffusion has come from a variety of studies of the Decapentaplegic (Dpp) gradient of the Drosophila larval wing disc.
RESULTS: In the present study, we initially show how the failure, in such studies, to consider the coupling of transport to receptor-mediated uptake and degradation has led to estimates of transport rates that are orders of magnitude too low, lending unwarranted support to a variety of hypothetical mechanisms, such as "planar transcytosis" and "restricted extracellular diffusion." Using several independent dynamic methods, we obtain data that are inconsistent with such models and show directly that Dpp transport occurs by simple, rapid diffusion in the extracellular space. We discuss the implications of these findings for other morphogen systems in which complex transport mechanisms have been proposed.
CONCLUSIONS: We believe ... [truncated at 150 words]
Kniazeva E, Weidling JW, Singh R, Botvinick EL, Digman MA, Gratton E, Putnam AJ.
Quantification of local matrix deformations and mechanical properties during capillary morphogenesis in 3D.
Integr Biol (Camb). 2012; 4(4): 431-439. PMCID: PMC3771856Reciprocal mechanical interactions between cells and the extracellular matrix (ECM) are thought to play important instructive roles in branching morphogenesis. However, most studies to date have failed to characterize these interactions on a length scale relevant to cells, especially in three-dimensional (3D) matrices. Here we utilized two complementary methods, spatio-temporal image correlation spectroscopy (STICS) and laser optical tweezers-based active microrheology (AMR), to quantify endothelial cell (EC)-mediated deformations of individual ECM elements and the local ECM mechanical properties, respectively, during the process of capillary morphogenesis in a 3D cell culture model. In experiments in which the ECM density was systematically varied, STICS revealed that the rate at which ECs deformed individual ECM fibers on the microscale positively correlated with capillary sprouting on the macroscale. ECs expressing constitutively active V14-RhoA displaced individual matrix fibers at significantly faster rates and displayed enhanced capillary sprouting relative to wild-type cells, while those expressing dominant-negative N19-RhoA ... [truncated at 150 words]
Meng L, Mantulin WW, Alexander BS, Cerussi AE, Tromberg BJ, Yu Z, Laning K, Kain ZN, Cannesson M, Gelb AW.
Head-up tilt and hyperventilation produce similar changes in cerebral oxygenation and blood volume: an observational comparison study using frequency-domain near-infrared spectroscopy.
Can J Anaesth. 2012; 59(4): 357-365. PMCID: PMC3371769PURPOSE: During anesthesia, maneuvers which cause the least disturbance of cerebral oxygenation with the greatest decrease in intracranial pressure would be most beneficial to patients with intracranial hypertension. Both head-up tilt (HUT) and hyperventilation are used to decrease brain bulk, and both may be associated with decreases in cerebral oxygenation. In this observational study, our null hypothesis was that the impact of HUT and hyperventilation on cerebral tissue oxygen saturation (SctO(2)) and cerebral blood volume (CBV) are comparable.
METHODS: Surgical patients without neurological disease were anesthetized with propofol-remifentanil. Before the start of surgery, frequency-domain near-infrared spectroscopy was used to measure SctO(2) and CBV at the supine position, at the 30° head-up and head-down positions, as well as during hypoventilation and hyperventilation.
RESULTS: Thirty-three patients were studied. Both HUT and hyperventilation induced small decreases in SctO(2) [3.5 (2.6)%; P < 0.001 and 3.0 (1.8)%; P < 0.001, respectively] and in CBV [0.05 (0.07) ... [truncated at 150 words]
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Spectroscopic properties of gold nanoparticles at the single-particle level in biological environments.
Chemphyschem. 2012; 13(4): 1087-1092. PMCID: PMC4245151Labeling 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 undesirable photophysical/photochemical processes, such as blinking and photobleaching, limiting their utilization. The main challenges in fluorescent probe design are to improve their absorption/emission properties, and to provide higher stability against photobleaching. In the last few years, metallic nanoparticles (NPs) of various sizes, shapes, and compositions have been used as a new alternative 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. These characteristics make them attractive contrast agents for cell imaging both in vitro and in vivo. For these reasons, the emission of metallic NPs in bulk ... [truncated at 150 words]
Hinde E, Digman MA, Welch C, Hahn KM, Gratton E.
Biosensor Förster resonance energy transfer detection by the phasor approach to fluorescence lifetime imaging microscopy.
Microsc Res Tech. 2012; 75(3): 271-281. PMCID: PMC3523109We present here the phasor approach to biosensor Förster resonance energy transfer (FRET) detection by fluorescence lifetime imaging microscopy (FLIM) and show that this method of data representation is robust towards biosensor design as well as the fluorescence artifacts inherent to the cellular environment. We demonstrate this property on a series of dual and single chain biosensors, which report the localization of Rac1 and RhoA activity, whilst performing concomitant ratiometric FRET analysis on the acquired FLIM data by the generalized polarization (GP) approach. We then evaluate and compare the ability of these two methods to quantitatively image biosensor FRET signal as a function of time and space. We find that with lifetime analysis in the phasor plot each molecular species is transformed into a two-dimensional coordinate system where independent mixtures of fluorophores can be distinguished from changes in lifetime due to FRET. This enables the fractional contribution of the free ... [truncated at 150 words]
Hinde E, Cardarelli F, Digman MA, Gratton E.
Changes in chromatin compaction during the cell cycle revealed by micrometer-scale measurement of molecular flow in the nucleus.
Biophys J. 2012; 102(3): 691-697. PMCID: PMC3274830We present a quantitative fluctuation-based assay to measure the degree of local chromatin compaction and investigate how chromatin density regulates the diffusive path adopted by an inert protein in dividing cells. The assay uses CHO-K1 cells coexpressing untagged enhanced green fluorescent protein (EGFP) and histone H2B tagged mCherry. We measure at the single-cell level the EGFP localization and molecular flow patterns characteristic of each stage of chromatin compaction from mitosis through interphase by means of pair-correlation analysis. We find that the naturally occurring changes in chromatin organization impart a regulation on the spatial distribution and temporal dynamics of EGFP within the nucleus. Combined with the analysis of Ca2+ intracellular homeostasis during cell division, EGFP flow regulation can be interpreted as the result of controlled changes in chromatin compaction. For the first time, to our knowledge, we were able to probe chromatin compaction on the micrometer scale, where the regulation of ... [truncated at 150 words]
Sánchez SA, Gratton E, Zanocco AL, Lemp E, Gunther G.
Sucrose monoester micelles size determined by Fluorescence Correlation Spectroscopy (FCS).
PLoS One. 2011; 6(12): e29278. PMCID: PMC3247245One of the several uses of sucrose detergents, as well as other micelle forming detergents, is the solubilization of different membrane proteins. Accurate knowledge of the micelle properties, including size and shape, are needed to optimize the surfactant conditions for protein purification and membrane characterization. We synthesized sucrose esters having different numbers of methylene subunits on the substituent to correlate the number of methylene groups with the size of the corresponding micelles. We used Fluorescence Correlation Spectroscopy (FCS) and two photon excitation to determine the translational D of the micelles and calculate their corresponding hydrodynamic radius, R(h). As a fluorescent probe we used LAURDAN (6-dodecanoyl-2-dimethylaminonaphthalene), a dye highly fluorescent when integrated in the micelle and non-fluorescent in aqueous media. We found a linear correlation between the size of the tail and the hydrodynamic radius of the micelle for the series of detergents measured.
Stafford RL, Hinde E, Knight MJ, Pennella MA, Ear J, Digman MA, Gratton E, Bowie JU.
Tandem SAM domain structure of human Caskin1: A presynaptic, self-assembling scaffold for CASK.
Structure. 2011; 19(12): 1826-1836. PMCID: PMC3240820The synaptic scaffolding proteins CASK and Caskin1 are part of the fibrous mesh of proteins that organize the active zones of neural synapses. CASK binds to a region of Caskin1 called the CASK interaction domain (CID). Adjacent to the CID, Caskin1 contains two tandem sterile α motif (SAM) domains. Many SAM domains form polymers so they are good candidates for forming the fibrous structures seen in the active zone. We show here that the SAM domains of Caskin1 form a new type of SAM helical polymer. The Caskin1 polymer interface exhibits a remarkable segregation of charged residues, resulting in a high sensitivity to ionic strength in vitro. The Caskin1 polymers can be decorated with CASK proteins, illustrating how these proteins may work together to organize the cytomatrix in active zones.
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3D nanometer images of biological fibers by directed motion of gold nanoparticles.
Nano Lett. 2011; 11(11): 4656-4660. PMCID: PMC3220937Using near-infrared femtosecond pulses we move single gold nanoparticles (AuNPs) along biological fibers such as collagen and actin filaments. While the AuNP is sliding on the fiber, its trajectory is measured in 3D with nanometer resolution providing a high resolution image of the fiber. Here, we systematically moved a single AuNP along nm-size collagen fibers and actin filament inside CHO K1 living cells mapping their 3D topography with high fidelity.
Leproux A, Cerussi AE, Tanamai VW, Durkin AF, Compton M, Tromberg BJ, Gratton E.
Impact of contralateral and ipsilateral reference tissue selection on self-referencing differential spectroscopy for breast cancer detection.
J Biomed Opt. 2011; 16(11): 116019. PMCID: PMC3223514We previously developed a self-referencing differential spectroscopic (SRDS) method to detect lesions by identifying a spectroscopic biomarker of breast cancer, i.e., the specific tumor component (STC). The SRDS method is based on the assumption of the exclusive presence of this spectroscopic biomaker in malignant disease. Although clinical results using this method have already been published, the dependence of the STC spectra on the choice of reference tissue has not yet been addressed. In this study, we explore the impact of the selection of the reference region size and location on the STC spectrum in 10 subjects with malignant breast tumors. Referencing from both contralateral and ipsilateral sides was performed. Regardless of the referencing, we are able to obtain consistent high contrast images of malignant lesions using the STC with less than 13% deviation. These results suggest that the STC measurements are independent of any type, location, and amount of normal ... [truncated at 150 words]
Lanzanò L, Lei T, Okamura K, Giral H, Caldas YA, Masihzadeh O, Gratton E, Levi M, Blaine J.
Differential modulation of the molecular dynamics of the type IIa and IIc sodium phosphate cotransporters by parathyroid hormone.
Am J Physiol Cell Physiol. 2011; 301(4): C850-861. PMCID: PMC3191571The kidney is a key regulator of phosphate homeostasis. There are two predominant renal sodium phosphate cotransporters, NaPi2a and NaPi2c. Both are regulated by parathyroid hormone (PTH) which decreases the abundance of the NaPi cotransporters in the apical membrane of renal proximal tubule cells. The time course of PTH-induced removal of the two cotransporters from the apical membrane however is markedly different for NaPi2a compared to NaPi2c. In animals and in cell culture PTH treatment results in almost complete removal of NaPi2a from the BB within one hour whereas for NaPi2c this process in not complete until 4 to 8 hours after PTH treatment. The reason for this is poorly understood. We have previously shown that the unconventional myosin motor myosin VI is required for PTH-induced removal of NaPi2a from the proximal tubule BB. Here we demonstrate that myosin VI is also necessary for PTH-induced removal of NaPi2c from the ... [truncated at 150 words]
Labeed FH, Lu J, Mulhall HJ, Marchenko SA, Hoettges KF, Estrada LC, Lee AP, Hughes MP, Flanagan LA.
Biophysical characteristics reveal neural stem cell differentiation potential.
PLoS One. 2011; 6(9): e25458. PMCID: PMC3184132BACKGROUND:
Distinguishing human neural stem/progenitor cell (huNSPC) populations that will predominantly generate neurons from those that produce glia is currently hampered by a lack of sufficient cell type-specific surface markers predictive of fate potential. This limits investigation of lineage-biased progenitors and their potential use as therapeutic agents. A live-cell biophysical and label-free measure of fate potential would solve this problem by obviating the need for specific cell surface markers.
METHODOLOGY/PRINCIPAL FINDINGS:
We used dielectrophoresis (DEP) to analyze the biophysical, specifically electrophysiological, properties of cortical human and mouse NSPCs that vary in differentiation potential. Our data demonstrate that the electrophysiological property membrane capacitance inversely correlates with the neurogenic potential of NSPCs. Furthermore, as huNSPCs are continually passaged they decrease neuron generation and increase membrane capacitance, confirming that this parameter dynamically predicts and negatively correlates with neurogenic potential. In contrast, differences in membrane conductance between NSPCs do not consistently correlate with the ability of the ... [truncated at 150 words]
Crosignani V, Dvornikov AS, Gratton E.
Enhancement of imaging depth in turbid media using a wide area detector.
J Biophotonics. 2011; 4(9): 592-599. PMCID: PMC4245154The depth of two-photon fluorescence imaging in turbid media can be significantly enhanced by the use of the here described fluorescence detection method that allows to efficiently collect scattered fluorescence photons from a wide area of the turbid sample. By using this detector we were able to perform imaging of turbid samples, simulating brain tissue, at depths up to 3 mm, where the two-photon induced fluorescence signal is too weak to be detected by means used in conventional two-photon microscopy.
Santoro Y, Leproux A, Cerussi AE, Tromberg BJ, Gratton E.
Breast cancer spatial heterogeneity in near-infrared spectra and the prediction of neoadjuvant chemotherapy response.
J Biomed Opt. 2011; 16(9): 097007. PMCID: PMC3203125We describe an algorithm to calculate an index that characterizes spatial differences in broadband near-infrared [(NIR), 650-1000 nm] absorption spectra of tumor-containing breast tissue. Patient-specific tumor spatial heterogeneities are visualized through a heterogeneity spectrum function (HS). HS is a biomarker that can be attributed to different molecular distributions within the tumor. To classify lesion heterogeneities, we built a heterogeneity index (HI) derived from the HS by weighing the HS in specific NIR absorption bands. It is shown that neoadjuvant chemotherapy (NAC) response is potentially related to the tumor heterogeneity. Therefore, we correlate the heterogeneity index obtained prior to treatment with the final response to NAC. From a pilot study of 15 cancer patients treated with NAC, pathological complete responders (pCR) were separated from non-pCR according to their HI (-44 ± 12 and 43 ± 17, p = 3 × 10(-8), respectively). We conclude that the HS function is a biomarker ... [truncated at 150 words]
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Measuring the flow of molecules in cells.
Biophys Rev. 2011; 3(3): 119-129.No methods proposed thus far have the capability to measure molecular flow in live cells at the single molecule level. Here, we review the potentiality of a newly established method based on the spatial correlation of fluorescence fluctuations at a pair of points in the sample (pair correlation method). The pair correlation function (pCF) offers a unique tool to probe the directionality of intracellular traffic, by measuring the accessibility of the cellular landscape and its role in determining the diffusive routes adopted by molecules. The sensitivity of the pCF method toward detection of barriers means that different structural elements of the cell can be tested in terms of penetrability and mechanisms of regulation imparted on molecular flow. This has been recently demonstrated in a series of studies looking at molecular transport inside live cells. Here, we will review the theory behind detection of barriers to molecular flow, the rules to ... [truncated at 150 words]
Wolf U, Toronov VY, Choi JH, Gupta R, Michalos A, Gratton E, Wolf M.
Correlation of functional and resting state connectivity of cerebral oxy-, deoxy-, and total hemoglobin concentration changes measured by near-infrared spectrophotometry.
J Biomed Opt. 2011; 16(8): 087013. PMCID: PMC3170400The aim is to study cerebral vascular functional connectivity during motor tasks and resting state using multichannel frequency-domain near-infrared spectrophotometry. Maps of 5.7 × 10.8 cm size displaying changes in cerebral oxyhemoglobin (O(2)Hb), deoxyhemoglobin (HHb), and total hemoglobin (tHb) concentrations were measured in the motor cortex in 12 subjects (mean age of 28.8±12.7 yrs) during resting state and during two palm squeezing tasks with different timing. For each condition, phase plane plots, cross correlation functions, and connectivity indices were generated for O(2)Hb, HHb, and tHb. The amplitude of the concentration changes in O(2)Hb and HHb depends on the age of the subject. We found large regions of connectivity, which were similar for resting state and task conditions. This means the spatial relationships during resting state, when changes in O(2)Hb, HHb, and tHb corresponded to spontaneous oscillations, were correlated to the spatial patterns during the activation tasks, when changes in O(2)Hb, ... [truncated at 150 words]
Cardarelli F, Lanzanò L, Gratton E.
Fluorescence correlation spectroscopy of intact nuclear pore complexes.
Biophys J. 2011; 101(4): L27-29. PMCID: PMC3175086No methods proposed thus far have the sensitivity to measure the transport of single molecules through single nuclear pore complexes (NPCs) in intact cells. Here we demonstrate that fluorescence correlation spectroscopy (FCS) combined with real-time tracking of the center of mass of single NPCs in live, unperturbed cells allows us to detect the transport of single molecules in a reference system of a pore with high temporal (millisecond) and spatial (limited by diffraction) resolution. We find that the transport of the classical receptor karyopherin-β1 (Kapβ1) is regulated so as to produce a peculiar distribution of characteristic times at the NPC. This regulation, which is spatially restricted to the pore, depends on the properties and metabolic energy of Kapβ1. As such, this method provides a powerful tool for studying nucleocytoplasmic shuttling at the nanometer scale under physiological conditions.
Stringari C, Cinquin A, Cinquin O, Digman MA, Donovan PJ, Gratton E.
Phasor approach to fluorescence lifetime microscopy distinguishes different metabolic states of germ cells in a live tissue.
Proc Natl Acad Sci USA. 2011; 108(33): 13582-13587. PMCID: PMC3158156We describe a label-free imaging method to monitor stem-cell metabolism that discriminates different states of stem cells as they differentiate in living tissues. In this method we use intrinsic fluorescence biomarkers and the phasor approach to fluorescence lifetime imaging microscopy in conjunction with image segmentation, which we use to introduce the concept of the cell phasor. In live tissues we are able to identify intrinsic fluorophores, such as collagen, retinol, retinoic acid, porphyrin, flavins, and free and bound NADH. We have exploited the cell phasor approach to detect a trend in metabolite concentrations along the main axis of the Caenorhabditis elegans germ line. This trend is consistent with known changes in metabolic states during differentiation. The cell phasor approach to lifetime imaging provides a label-free, fit-free, and sensitive method to identify different metabolic states of cells during differentiation, to sense small changes in the redox state of cells, and may ... [truncated at 150 words]
Marchini C, Pozzi D, Montani M, Alfonsi C, Amici A, de Sanctis SC, Digman MA, Sánchez SA, Gratton E, Amenitsch H, Fabbretti A, Gualerzi CO, Caracciolo G.
Role of temperature-independent lipoplex-cell membrane interactions in the efficiency boost of multicomponent lipoplexes.
Cancer Gene Ther. 2011; 18(8): 543-552. PMCID: PMC3940159Multicomponent lipoplexes have recently emerged as especially promising transfection candidates, as they are from 10 to 100 times more efficient than binary complexes usually employed for gene delivery purposes. Previously, we investigated a number of chemical-physical properties of DNA-lipid complexes that were proposed to affect transfection efficiency (TE) of lipoplexes, such as nanoscale structure, size, surface potential, DNA-protection ability and DNA release from complexes upon interaction with cellular lipids. Although some minor differences between multicomponent and binary lipoplexes were found, they did not correlate clearly with efficiency. Instead, here we show that a marked difference between the cell internalization mechanism of binary and multicomponent lipoplexes does exist. Multicomponent lipoplexes significantly transfect cells at 4 °C, when endocytosis does not take place suggesting that they can enter cells via a temperature-independent mechanism. Confocal fluorescence microscopy experiments showed the existence of a correlation between endosomal escape and TE. Multicomponent lipoplexes exhibited a distinctive ... [truncated at 150 words]
Ramella NA, Rimoldi OJ, Prieto ED, Schinella GR, Sánchez SA, Jaureguiberry MS, Vela ME, Ferreira ST, Tricerri MA.
Human apolipoprotein A-I-derived amyloid: its association with atherosclerosis.
PLoS One. 2011; 6(7): e22532. PMCID: PMC3139661Amyloidoses constitute a group of diseases in which soluble proteins aggregate and deposit extracellularly in tissues. Nonhereditary apolipoprotein A-I (apoA-I) amyloid is characterized by deposits of nonvariant protein in atherosclerotic arteries. Despite being common, little is known about the pathogenesis and significance of apoA-I deposition. In this work we investigated by fluorescence and biochemical approaches the impact of a cellular microenvironment associated with chronic inflammation on the folding and pro-amyloidogenic processing of apoA-I. Results showed that mildly acidic pH promotes misfolding, aggregation, and increased binding of apoA-I to extracellular matrix elements, thus favoring protein deposition as amyloid like-complexes. In addition, activated neutrophils and oxidative/proteolytic cleavage of the protein give rise to pro amyloidogenic products. We conclude that, even though apoA-I is not inherently amyloidogenic, it may produce non hereditary amyloidosis as a consequence of the pro-inflammatory microenvironment associated to atherogenesis.
Sánchez SA, Bakás L, Gratton E, Herlax V.
Alpha Hemolysin induces an increase of Erythrocytes Calcium: A FLIM 2-photon phasor analysis approach.
PLoS One. 2011; 6(6): e21127. PMCID: PMC3116868α-hemolysin (HlyA) from Escherichia coli is considered as the prototype of a family of toxins called RTX (repeat in toxin), a group 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 cause the lysis of several cells such as erythrocytes, granulocytes, monocytes, endothelial and renal epithelial cells of different species. At low concentrations it induces the production of cytokines and apoptosis. Since many of the subcytolytic effects in other cells have been reported to be triggered by the increase of intracellular calcium, we followed the calcium concentration inside the erythrocytes while incubating with sublytic concentrations of HlyA. Calcium concentration was monitored using the calcium indicator Green 1, 2-photon excitation, and fluorescence lifetime imaging microscopy (FLIM). Data were analyzed using the phasor representation. In this report, we present evidence ... [truncated at 150 words]
Itano MS, Neumann AK, Liu P, Zhang F, Gratton E, Parak WJ, Thompson NL, Jacobson KA.
DC-SIGN and influenza Hemagglutinin dynamics in plasma membrane microdomains are markedly different.
Biophys J. 2011; 100(11): 2662-2670. PMCID: PMC3117154DC-SIGN, a Ca(2+)-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 in microdomains were explored with several fluorescence microscopy methods and compared with dynamics 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. Fluorescence recovery after photobleaching measurements showed that neither full-length nor cytoplasmically truncated DC-SIGN in microdomains appreciably exchanged with like molecules in other microdomains and the membrane surround, whereas HA in microdomains exchanged almost completely. Line-scan fluorescence correlation spectroscopy indicated an essentially undetectable lateral mobility for DC-SIGN but an appreciable mobility for HA within their respective domains. Single-particle tracking with defined-valency quantum dots confirmed that ... [truncated at 150 words]
Meng L, Cannesson M, Alexander BS, Yu Z, Kain ZN, Cerussi AE, Tromberg BJ, Mantulin WW.
Effect of phenylephrine and ephedrine bolus treatment on cerebral oxygenation in anaesthetized patients.
Br J Anaesth. 2011; 107(2): 209-217. PMCID: PMC3136202BACKGROUND. How phenylephrine and ephedrine treatments affect global and regional haemodynamics is of major clinical relevance. Cerebral tissue oxygen saturation (SctO2)-guided management may improve postoperative outcome. The physiological variables responsible for SctO2 changes induced by phenylephrine and ephedrine bolus treatment in anaesthetized patients need to be defined.
METHODS. A randomized two-treatment cross-over trial was conducted: one bolus dose of phenylephrine (100–200 mg) and one bolus dose of ephedrine (5–20 mg) were given to 29 ASA I–III patients anaesthetized with propofol and remifentanil. SctO2 , mean arterial pressure (MAP), cardiac output (CO), and other physiological variables were recorded before and after treatments. The associations of changes were analysed using linear-mixed models.
RESULTS. The CO decreased significantly after phenylephrine treatment [△CO=-22.1 (1.4) litre min^-1, P,0.001], but was preserved after ephedrine treatment [△CO=0.5 (1.4) litre min^-1, P.0.05]. The SctO2 was significantly decreased after phenylephrine treatment [△SctO2=-23.2 (3.0)%, P,0.01] but preserved after ephedrine treatment [△SctO2=0.04 (1.9)%, ... [truncated at 150 words]
Lanzanò L, Digman MA, Fwu P, Giral H, Levi M, Gratton E.
Nanometer-scale imaging by the modulation tracking method.
J Biophotonics. 2011; 4(6): 415-424. PMCID: PMC3393040We developed an optical imaging method based on a feedback principle in which the specific scan pattern is adapted according to the shape of the sample. The feedback approach produces nanometer-resolved 3D images of very small and moving features in live cells in seconds. We show images of microvilli in live cultured opossum kidney cells expressing NaPi co-transporter proteins with different GFP constructs and images of cell protrusions in a collagen matrix with a resolution of about 20 nm. We found that in the microvilli the NaPi proteins can be found clustered. Along cell protrusions in 3D we identified cellular adhesions to the extracellular matrix. Our approach to super-resolution and to 3D nanoimaging is different than other proposed methods that break the diffraction limit using non-linear effects or are based on single molecule localization.
Kotlarchyk MA, Shreim SG, Alvarez-Elizondo MB, Estrada LC, Singh R, Valdevit L, Kniazeva E, Gratton E, Putnam AJ, Botvinick EL.
Concentration independent modulation of local micromechanics in a fibrin gel.
PLoS One. 2011; 6(5): e20201. PMCID: PMC3100350Methods for tuning extracellular matrix (ECM) mechanics in 3D cell culture that rely on increasing the concentration of either protein or cross-linking molecules fail to control important parameters such as pore size, ligand density, and molecular diffusivity. Alternatively, ECM stiffness can be modulated independently from protein concentration by mechanically loading the ECM. We have developed a novel device for generating stiffness gradients in naturally derived ECMs, where stiffness is tuned by inducing strain, while local mechanical properties are directly determined by laser tweezers based active microrheology (AMR). Hydrogel substrates polymerized within 35 mm diameter Petri dishes are strained non-uniformly by the precise rotation of an embedded cylindrical post, and exhibit a position-dependent stiffness with little to no modulation of local mesh geometry. Here we present the device in the context of fibrin hydrogels. First AMR is used to directly measure local micromechanics in unstrained hydrogels of increasing fibrin concentration. Changes ... [truncated at 150 words]
Caracciolo G, Pozzi D, Capriotti AL, Marianecci C, Carafa M, Marchini C, Montani M, Amici A, Amenitsch H, Digman MA, Gratton E, Sánchez SA, Laganà A.
Factors determining the superior performance of lipid/DNA/protammine nanoparticles over lipoplexes.
J Med Chem. 2011; 54(12): 4160-4171.The utility of using a protammine/DNA complex coated with a lipid envelope made of cationic 1,2-dioleoyl-3-trimethylammonium propane (DOTAP) for transfecting CHO (Chinese hamster ovary cells), HEK293 (human embryonic kidney cells), NIH 3T3 (mouse embryonal cells), and A17 (murine cancer cells) cells was examined. The widely used DOTAP/DNA lipoplex was employed as a reference. In all the tested cell lines lipid/protamine/DNA (LPD) nanoparticles were more efficient in transfecting cells than lipoplexes even though the lipid composition of the lipid envelope was the same in both devices. Physical–chemical properties were found to control the ability of nanocarriers to release DNA upon interaction with cellular membranes. LPD complexes easily release their DNA payload, while lipoplexes remain largely intact and accumulate at the cell nucleus. Collectively, these data explain why LPD nanoparticles often exhibit superior performances compared to lipoplexes in trasfecting cells and represent a promising class of nanocarriers for gene delivery.
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Lessons in fluctuation correlation spectroscopy.
Ann Rev Phys Chem. 2011; 62: 645-668. PMCID: PMC3576135Molecular diffusion and transport processes are fundamental in physical, chemical and biological systems. Current approaches to measure molecular transport in cells and tissues based on perturbation methods, e.g. fluorescence recovery after photobleaching (FRAP) are invasive, single-point fluctuation correlation methods are local and single particle tracking requires observation of isolated particles for relatively long periods of time. We discuss here detecting molecular transport by exploiting spatiotemporal correlations measured among points at large distance (>1 µm). We illustrate the evolution of the conceptual framework that started with single point fluorescence fluctuation analysis based on the transit of fluorescent molecules through a small volume of illumination. This idea has evolved to include measurement of fluctuations at many locations in the sample using microscopy imaging methods. Image fluctuation analysis has become a rich and powerful technique that can be used to extract information about spatial distribution of molecular concentration and transport in cells and ... [truncated at 150 words]
Sánchez SA, Gunther G, Tricerri MA, Gratton E.
Methyl-β-Cyclodextrins preferentially remove cholesterol from the liquid disordered phase in giant unilamellar vesicles.
J Membr Biol. 2011; 241(1): 1-10. PMCID: PMC3082695Methyl-β-cyclodextrins (MβCDs) are molecules that are extensively used to remove and to load cholesterol (Chol) from artificial and natural membranes; however, the mechanism of Chol extraction by MβCD from pure lipids or from complex mixtures is not fully understood. One of the outstanding questions in this field is the capability of MβCD to remove Chol from lipid domains having different packing. Here, we investigated the specificity of MβCD to remove Chol from coexisting macrodomains with different lipid packing. We used giant unilamellar vesicles (GUVs) made of 1,2-dioleoylphosphatidylcholine:1,2-dipalmitoylphatidylcholine:free cholesterol, 1:1:1 molar ratio at 27°C. Under these conditions, individual GUVs present Chol distributed into l ( o ) and l ( d ) phases. The two phases can be distinguished and visualized using Laurdan generalized polarization and two-photon excitation fluorescence microscopy. Our data indicate that MβCD removes Chol preferentially from the more disordered phase. The process of selective Chol removal is dependent ... [truncated at 150 words]
Giral H, Lanzanò L, Caldas Y, Blaine J, Verlander JW, Lei T, Gratton E, Levi M.
Role of PDZK1 protein in apical membrane expression of renal sodium-coupled phosphate transporters.
J Biol Chem. 2011; 286(17): 15032-15042. PMCID: PMC3083164The sodium-dependent phosphate (Na/Pi) transporters NaPi-2a and NaPi-2c play a major role in the renal reabsorption of Pi. The functional need for several transporters accomplishing the same role is still not clear. However, the fact that these transporters show differential regulation under dietary and hormonal stimuli suggests different roles in Pi reabsorption. The pathways controlling this differential regulation are still unknown, but one of the candidates involved is the NHERF family of scaffolding PDZ proteins. We propose that differences in the molecular interaction with PDZ proteins are related with the differential adaptation of Na/Pi transporters. Pdzk1−/− mice adapted to chronic low Pi diets showed an increased expression of NaPi-2a protein in the apical membrane of proximal tubules but impaired up-regulation of NaPi-2c. These results suggest an important role for PDZK1 in the stabilization of NaPi-2c in the apical membrane. We studied the specific protein-protein interactions of Na/Pi transporters with NHERF-1 ... [truncated at 150 words]
Hinde E, Cardarelli F, Digman MA, Kershner A, Kimble J, Gratton E.
The impact of mitotic versus interphase chromatin architecture on the molecular flow of EGFP by pair correlation analysis.
Biophys J. 2011; 100(7): 1829-1836. PMCID: PMC3072664Here we address the impact nuclear architecture has on molecular flow within the mitotic nucleus of live cells as compared to interphase by the pair correlation function method. The mitotic chromatin is found to allow delayed but continuous molecular flow of EGFP in and out of a high chromatin density region, which, by pair correlation function analysis, is shown as a characteristic arc shape that appears upon entry and exit. This is in contrast to interphase chromatin, which regulates flow between different density chromatin regions by means of a mechanism which turns on and off intermittently, generating discrete bursts of EGFP. We show that the interphase bursts are maintained by metabolic energy, whereas the mitotic mechanism of regulation responsible for the arc is not sensitive to ATP depletion. These two distinct routes of molecular flow were concomitantly measured in the Caenorhabditis elegans germ line, which indicates a conservation of mechanism ... [truncated at 150 words]
Moens PDJ, Gratton E, Salvemini IL.
Fluorescence correlation spectroscopy, raster image correlation spectroscopy, and number and brightness on a commercial confocal laser scanning microscope with analog detectors (Nikon C1).
Microsc Res Tech. 2011; 74(4): 377-388. PMCID: PMC3001136Fluorescence correlation spectroscopy (FCS) was developed in 1972 by Magde, Elson and Webb. Photon counting detectors and avalanche photodiodes have become standards in FCS to the point that there is a widespread belief that these detectors are essential to perform FCS experiments, despite the fact that FCS was developed using analog detectors. Spatial and temporal intensity fluctuation correlations using analog detection on a commercial Olympus Fluoview 300 microscope have been reported by Brown et al. (2008). However, each analog instrument has its own idiosyncrasies that need to be understood before using the instrument for FCS. In this work, we explore the capabilities of the Nikon C1, a low-cost confocal microscope, to obtain single point FCS, Raster-scan image correlation spectroscopy (RICS), and Number and Brightness data both in solution and incorporated into the membrane of giant unilamellar vesicles. We show that it is possible to obtain dynamic information about fluorescent molecules ... [truncated at 150 words]
Azartash K, Kwan JT, Paugh JR, Nguyen AL, Jester JV, Gratton E.
Pre-corneal tear film thickness in humans measured with a novel technique.
Mol Vis. 2011; 17: 756-767. PMCID: PMC3081798Purpose: The purpose of this work was to gather preliminary data in normals and dry eye subjects, using a new, non-invasive imaging platform to measure the thickness of pre-corneal tear film.
Methods: Human subjects were screened for dry eye and classified as dry or normal. Tear film thickness over the inferior paracentral cornea was measured using laser illumination and a complementary metal–oxide–semiconductor (CMOS) camera. A previously developed mathematical model was used to calculate the thickness of the tear film by applying the principle of spatial auto-correlation function (ACF).
Results: Mean tear film thickness values (±SD) were 3.05 μm (0.20) and 2.48 μm (0.32) on the initial visit for normals (n=18) and dry eye subjects (n=22), respectively, and were significantly different (p<0.001, 2-sample t-test). Repeatability was good between visit 1 and 2 for normals (intraclass correlation coefficient [ICC]=0.935) and dry eye subjects (ICC=0.950). Tear film thickness increased above baseline for the dry eye ... [truncated at 150 words]
Blaine J, Lanzanò L, Girala H, Caldasa Y, Levia M, Grattona E, Moldovana R, Lei T.
Dynamic imaging of the sodium phosphate cotransporters.
Adv Chronic Kidney Dis. 2011; 18(2): 145-150. PMCID: PMC3220941Although in vivo and cell culture studies have provided useful information about the regulation of the sodium phosphate (NaPi) cotransporters, such studies are unable to provide information at the molecular level about interactions between proteins. The NaPi proteins are found within both intestinal and renal brush border microvilli, and previous work has shown that these microvilli contain scaffolding proteins (PDZ proteins) and myosin motors. The recent development of several advanced imaging techniques has allowed detailed analysis of how NaPi proteins interact with scaffolding proteins and myosin motors. Using techniques such as apical total internal reflection fluorescence microscopy, fluorescence correlation spectroscopy, raster image correlation spectroscopy, and fluorescence lifetime imaging-Förster resonance energy transfer, we have found that a myosin motor is involved in trafficking of the NaPi cotransporters and also that Npt2a and Npt2c seem to have different affinities for the PDZ protein Na+/H+ exchanger regulatory factor 1. Further application of these ... [truncated at 150 words]
Behne MJ, Sánchez SA, Barry NP, Kirschner N, Meyer W, Mauro TM, Moll I, Gratton E.
Major translocation of calcium upon epidermal barrier insult: imaging and quantification via FLIM/Fourier vector analysis.
Arch Dermatol Res. 2011; 303(2): 103-115. PMCID: PMC4548958Calcium controls an array of key events in keratinocytes and epidermis: localized changes in Ca(2+) concentrations and their regulation are therefore especially important to assess when observing epidermal barrier homeostasis and repair, neonatal barrier establishment, in differentiation, signaling, cell adhesion, and in various pathological states. Yet, tissue- and cellular Ca(2+) concentrations in physiologic and diseased states are only partially known, and difficult to measure. Prior observations on the Ca(2+) distribution in skin were based on Ca(2+) precipitation followed by electron microscopy, or proton-induced X-ray emission. Neither cellular and/or subcellular localization could be determined through these approaches. In cells in vitro, fluorescent dyes have been used extensively for ratiometric measurements of static and dynamic Ca(2+) concentrations, also assessing organelle Ca(2+) concentrations. For lack of better methods, these findings together build the basis for the current view of the role of Ca(2+) in epidermis, their limitations notwithstanding. Here we report a method ... [truncated at 150 words]
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Deeper tissue imaging with total detection.
Science. 2011; 331(6020): 1016-1017.If you simply held your finger in front of a strong source of light, you would see that visible light passes through centimeters of the tissue held in front of it. Even more light passes through tissue at near-infrared wavelengths, but even if you could see it, you would still not be able to distinguish between the bone and flesh, or other internal structures. The reason is that light scatters multiple times in the tissue, and the image blurs; resolution and contrast decrease as we try to look deeper into tissue. Other instrumental methods—such as x-ray tomography, ultrasound, and magnetic resonance imaging (MRI)—can “see through” nontransparent objects and have been revolutionary in medicine and material science, yet we are still unable to use visible light and its accompanying spectroscopic information to look inside tissues. Recently, Combs et al. (1, 2) found a way to improve the amount of fluorescent light ... [truncated at 150 words]
Chen B, Estrada LC, Hellriegel C, Gratton E.
Nanometer-scale optical imaging of collagen fibers using gold nanoparticles.
Biomed Opt Express. 2011; 2(3): 511-519. PMCID: PMC3047357We describe 3D single particle tracking of gold nanoparticles (AuNPs) moving along collagen fibers in aqueous environment with two-photon excitation conditions. The photoacoustic effect at the collagen fiber caused by the irradiation with ultrashort, near-infrared laser pulses propels the particles adsorbed to the surface of the collagen fibers. We report the tracking of individual AuNPs in three dimensions with high spatial and temporal resolution, of few nanometers and milliseconds, respectively. Due to the emission signal caused by the interaction between the AuNPs and the weak chromophores in the collagen fiber, the trajectories of individual AuNPs reveal the fiber topography with nanometric resolution. The intensity along the trajectory shows that we are sensitive to the distribution of the weak chromophores on the fiber.
Ross JA, Digman MA, Wang L, Gratton E, Albanesi JP, Jameson DM.
Oligomerization state of Dynamin 2 in cell membranes using TIRF and number and brightness analysis.
Biophys J. 2011; 100(3): L15-17. PMCID: PMC3030261Dynamin 2 is an ubiquitously expressed -100 kDa GTPase involved in receptor-mediated endocytosis, Golgi budding, and cytoskeletal reorganization. Dynamin molecules assemble around the necks of budding vesicles and constrict membranes in a GTP-dependent process, resulting in vesicle release. The oligomerization state of dynamin 2 in the membrane is still controversial. We investigated dynamin 2 within the plasma membrane of live cells using total internal reflection microscopy coupled with number and brightness analysis. Our results demonstrate that dynamin 2 is primarily tetrameric throughout the entire cell membrane, aside from punctate structures that may correspond to regions of membrane vesiculation.
Choi CK, Zareno J, Digman MA, Gratton E, Horwitz AR.
Cross-correlated fluctuation analysis reveals phosphorylation-regulated Paxillin-FAK complexes in nascent adhesions.
Biophys J. 2011; 100(3): 583-592. PMCID: PMC3030238We used correlation methods to detect and quantify interactions between paxillin and focal adhesion kinase (FAK) in migrating cells. Cross-correlation raster-scan image correlation spectroscopy revealed that wild-type paxillin and the phosphorylation-inhibiting paxillin mutant Y31F-Y118F do not interact with FAK in the cytosol but a phosphomimetic mutant of paxillin, Y31E-Y118E, does. By extending cross-correlation number and brightness analysis to the total internal reflection fluorescence modality, we were able to show that tetramers of paxillin and FAK form complexes in nascent adhesions with a 1:1 stoichiometry ratio. The phosphomimetic mutations on paxillin increase the size of the complex and the assembly rate of nascent adhesions, suggesting that the physical molecular aggregation of paxillin and FAK regulates adhesion formation. In contrast, when phosphorylation is inhibited, the interaction decreases and the adhesions tend to elongate rather than turn over. These direct in vivo data show that the phosphorylation of paxillin is specific to adhesions ... [truncated at 150 words]
Vetri V, Ossato G, Militello V, Digman MA, Leone M, Gratton E.
Fluctuation methods to study protein aggregation in live cells: Concanavalin A oligomers formation.
Biophys J. 2011; 100(3): 774-783. PMCID: PMC3030242Prefibrillar oligomers of proteins are suspected to be the primary pathogenic agents in several neurodegenerative diseases. A key approach for elucidating the pathogenic mechanisms is to probe the existence of oligomers directly in living cells. In this work, we were able to monitor the process of aggregation of Concanavalin A in live cells. We used number and brightness analysis, two-color cross number and brightness analysis, and Raster image correlation spectroscopy to obtain the number of molecules, aggregation state, and diffusion coefficient as a function of time and cell location. We observed that binding of Concanavalin A to the membrane and the formation of small aggregates paralleled cell morphology changes, indicating progressive cell compaction and death. Upon protein aggregation, we observed increased membrane water penetration as reported by Laurdan generalized polarization imaging.
Kim SA, Sanabria H, Digman MA, Gratton E, Schwille P, Zipfel WR, Waxham MN.
Quantifying translational mobility in neurons: comparison between current optical techniques.
J Neurosci. 2010; 30(49): 16409-16416. PMCID: PMC3003925Translational mobility is involved in every process in neurobiology—released neurotransmitter diffuses through the synaptic cleft in search of receptor targets, membrane receptors traffic to synaptic sites in the neuron, RNA and other cargo are transported to distal dendrites, and cell signaling is mediated by circuits of diffusing proteins. However, accurate methods for quantifying translational mobility have not been widespread due to their technical demands and complicated analysis. Over the past decade, this trend has begun to reverse as major advancements have been made in the application of quantitative optical techniques, specifically fluorescence recovery after photobleaching (FRAP or FPR), fluorescence correlation spectroscopy (FCS), raster image correlation spectroscopy (RICS), and single-particle tracking (SPT), that can quantitatively analyze molecular diffusion and concentration within living cells with high spatial and temporal resolution.
Here we present a review of these optical methods to measure translational mobility in three dimensions of fluorescent molecules in neurons and address ... [truncated at 150 words]
Kuo Wj, Digman MA, Lander AD.
Heparan sulfate acts as a BMP co-receptor by facilitating ligand-induced receptor hetero-oligomerization.
Mol Biol Cell. 2010; 21(22): 4028-4041. PMCID: PMC2982130Cell surface heparan sulfate (HS) not only binds several major classes of growth factors, it sometimes potentiates their activities-an effect usually termed "coreception". A view that coreception is due to the stabilization of growth factor-receptor interactions has emerged primarily from studies of the fibroblast growth factors (FGFs). Recent in vivo studies have strongly suggested that HS also plays an important role in regulating signaling by the bone morphogenetic proteins (BMPs). Here we provide evidence that the mechanism of coreception for BMPs is markedly different from that established for FGFs. First, we demonstrate a direct, stimulatory role for cell surface HS in the immediate signaling activities of BMP2 and BMP4, and provide evidence that HS-BMP interactions are required for this effect. Next, using several independent assays of ligand binding and receptor assembly, including coimmunoprecipitation, cross-linking, and fluorescence fluctuation microscopy, we show that HS does not affect BMP binding to type I ... [truncated at 150 words]
Fu CC, Ossato G, Long M, Digman MA, Gopinathan A, Lee LP, Gratton E, Khine M.
Bimetallic nanopetals for thousand-fold fluorescence enhancements.
Appl Phys Lett. 2010; 97(20): 203101.We present a simple, ultra-rapid and robust method to create sharp nanostructures—nanopetals—in a shape memory polymer substrate demonstrating unprecedented enhancements for surface enhanced sensing over large surface areas. These bimetallic nanostructures demonstrate extremely strong surface plasmon resonance effects due to the high density multifaceted petal structures that increase the probability of forming nanogaps. We demonstrate that our nanopetals exhibit extremely strong surface plasmons, confining the emission and enhancing the fluorescence intensity of the nearby high-quantum yield fluorescein by >4000×. The enhancements are confined to the extremely small volumes at the nanopetal borders. This enables us to achieve single molecule detection at relatively high and physiological concentrations.
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Book Review: Handbook of Biomedical Nonlinear Optical Microscopy. B R Masters and P T C So. Oxford University Press, 2008.
Microsc Microanal. 2010; 16(6): 842.When you first read the title, Handbook of Biomedical Nonlinear Optical Microscopy, and the names of Barry Masters and Peter So, you know that this is serious stuff. You even may be tempted to leave the book unopened, thinking that it would be too specialized for you. However, the cover designed by Sheilah Barrett showing the familiar energy diagram for multiphoton excitation in gold over a royal blue background is so inviting that you are compelled to open the book, and then after opening it, you find the welcoming and familiar faces of the authors. At this point, you find yourself reading the first pages, and now you are at the point of no return; the magic is done; you are enchanted. This is the book for you.
Azartash K, Shy CjN, Flynn K, Jester JV, Gratton E.
Non-invasive in vivo measurement of the tear film using spatial autocorrelation in a live mammal model.
Biomed Opt Express. 2010; 1(4): 1127-1137. PMCID: PMC3018089Tear film stability and its interaction with the corneal surface play an important role in maintaining ocular surface integrity and quality of vision. We present a non-invasive technique to quantify the pre-corneal tear film thickness. A cMOS camera is used to record the interference pattern produced by the reflections from multiple layers of the tear film Principles of spatial autocorrelation are applied to extract the frequency of the periodic patterns in the images. A mathematical model is developed to obtain the thickness of the tear film from the spatial autocorrelation image. The technique is validated using micro-fabricated thin parylene films. We obtained repeatable and precise measurement on a live rabbit model (N = 6). We obtained an average value of 10.2µm and standard deviation of, SD = 0.3 (N = 4). We measured one rabbit infected with HSV-1 virus that had a baseline tear film thickness of 4.7µm.
Rossow MJ, Sasaki JM, Digman MA, Gratton E.
Raster image correlation spectroscopy in live cells.
Nat Protoc. 2010; 5(11): 1761-1774. PMCID: PMC3089972Raster image correlation spectroscopy (RICS) is a noninvasive technique to detect and quantify events in a live cell, including concentration of molecules and diffusion coefficients of molecules; in addition, by measuring changes in diffusion coefficients, RICS can indirectly detect binding. Any specimen containing fluorophores that can be imaged with a laser scanning microscope can be analyzed using RICS. There are other techniques to measure diffusion coefficients and binding; however, RICS fills a unique niche. It provides spatial information and can be performed in live cells using a conventional confocal microscope. It can measure a range of diffusion coefficients that is not accessible with any other single optical correlation–based technique. In this article we describe a protocol to obtain raster scanned images with an Olympus FluoView FV1000 confocal laser scanning microscope using Olympus FluoView software to acquire data and SimFCS software to perform RICS analysis. Each RICS measurement takes several minutes. ... [truncated at 150 words]
Presman DM, Alvarez LD, Levi V, Eduardo S, Digman MA, Martí MA, Veleiro AS, Burton G, Pecci A.
Insights on glucocorticoid receptor activity modulation through the binding of rigid steroids.
PLoS One. 2010; 5(10): e13279. PMCID: PMC2952596BACKGROUND: The glucocorticoid receptor (GR) is a transcription factor that regulates gene expression in a ligand-dependent fashion. This modular protein is one of the major pharmacological targets due to its involvement in both cause and treatment of many human diseases. Intense efforts have been made to get information about the molecular basis of GR activity.
METHODOLOGY/PRINCIPAL FINDINGS: Here, the behavior of four GR-ligand complexes with different glucocorticoid and antiglucocorticoid properties were evaluated. The ability of GR-ligand complexes to oligomerize in vivo was analyzed by performing the novel Number and Brightness assay. Results showed that most of GR molecules form homodimers inside the nucleus upon ligand binding. Additionally, in vitro GR-DNA binding analyses suggest that ligand structure modulates GR-DNA interaction dynamics rather than the receptor's ability to bind DNA. On the other hand, by coimmunoprecipitation studies we evaluated the in vivo interaction between the transcriptional intermediary factor 2 (TIF2) coactivator and different ... [truncated at 150 words]
Hinde E, Cardarelli F, Digman MA, Gratton E.
In vivo pair correlation analysis of EGFP intranuclear diffusion reveals DNA-dependent molecular flow.
Proc Natl Acad Sci USA. 2010; 107(38): 16560-16565. PMCID: PMC2944750No 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 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 flow in and between different nuclear environments. Our results suggest that there are two disconnect molecular flows throughout the nucleus associated with high and low DNA density regions. We show that different density regions of DNA form a networked channel that allows EGFP to diffuse freely throughout, however with restricted ability to traverse the channel. We also observe rare and sudden bursts of molecules traveling across DNA density regions with characteristic time of approximately ... [truncated at 150 words]
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Dynamics of lipid domain formation: fluctuation analysis.
Biochim Biophys Acta. 2010; 1798(7): 1368-1376. PMCID: PMC2883005Scanning-fluctuation correlation spectroscopy was used to detect subresolution organizational fluctuations in the lipid liquid-crystalline phase for single lipid model systems. We used the fluorescent probe Laurdan which is sensitive to the amount of water in the membrane to show that there is a spatial heterogeneity on the scale of few pixels (the size of the pixel is 50 nm). We calculated the pixel variance of the GP function and we found that the variance has a peak at the phase transition for 3 different samples made of pure lipids. The pixel variance has an abrupt change at the phase transition of the membrane and then it slowly decreases at higher temperature. The relatively large variance of the GP indicates that the liquid phase of the membrane is quite heterogeneous even several degrees higher than the phase transition temperature. We interpreted this result as evidence of an underlying microscale structure of ... [truncated at 150 words]
Sánchez SA, Tricerri MA, Ossato G, Gratton E.
Lipid packing determines protein-membrane interactions: challenges for apolipoprotein A-I and high density lipoproteins.
Biochim Biophys Acta. 2010; 1798(7): 1399-1408. PMCID: PMC2883020Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. FCS (Fluorescence Correlation Spectroscopy) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan GP (Generalized Polarization) reports on local changes in membrane water content (related to membrane fluidity) due to protein ... [truncated at 150 words]
Lim RS, Kratzer A, Barry NP, Miyazaki-Anzai S, Miyazaki M, Mantulin WW, Levi M, Potma EO, Tromberg BJ.
Multimodal CARS microscopy determination of the impact of diet on macrophage infiltration and lipid accumulation on plaque formation in ApoE-deficient mice.
J Lipid Res. 2010; 51(7): 1729-37. PMCID: PMC2882730We characterized several cellular and structural features of early stage Type II/III atherosclerotic plaques in an established model of atherosclerosis-the ApoE-deficient mouse-by using a multimodal, coregistered imaging system that integrates three nonlinear optical microscopy (NLOM) contrast mechanisms: coherent anti-Stokes Raman scattering (CARS), second harmonic generation (SHG), and two-photon excitation fluorescence (TPEF). Specifically, the infiltration of lipid-rich macrophages and the structural organization of collagen and elastin fibers were visualized by CARS, SHG, and TPEF, respectively, in thick tissue specimens without the use of exogenous labels or dyes. Label-free CARS imaging of macrophage accumulation was confirmed by histopathology using CD68 staining. A high-fat, high-cholesterol Western diet resulted in an approximate 2-fold increase in intimal plaque area, defined by CARS signals of lipid-rich macrophages. Additionally, analysis of collagen distribution within lipid-rich plaque regions revealed nearly a 4-fold decrease in the Western diet-fed mice, suggesting NLOM sensitivity to increased matrix metalloproteinase (MMP) activity and ... [truncated at 150 words]
Ossato G, Digman MA, Aiken C, Lukacsovich T, Marsh JL, Gratton E.
A two-step path to inclusion formation of huntingtin peptides revealed by number and brightness analysis.
Biophys J. 2010; 98(12): 3078-3085. PMCID: PMC2884247Protein aggregation is a hallmark of several neurodegenerative diseases including Huntington's disease. We describe the use of the recently developed number and brightness method (N&B) that uses confocal images to monitor aggregation of Huntingtin exon 1 protein (Httex1p) directly in living cells. N&B measures the molecular brightness of protein aggregates in the entire cell noninvasively based on intensity fluctuations at each pixel in an image. N&B applied to mutant Httex1p in living cells showed a two-step pathway leading to inclusion formation that is polyQ length dependent and involves four phases. An initial phase of monomer accumulation is followed by formation of small oligomers (5-15 proteins); as protein concentration increases, an inclusion is seeded and forms in the cytoplasm; the growing inclusion recruits most of the Httex1p and depletes the cell leaving only a low concentration of monomers. The behavior of Httex1p in COS-7 and ST14A cells is compared.
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In vivo imaging of single-molecule translocation through nuclear pore complexes by pair correlation functions.
PLoS One. 2010; 5(5): e10475. PMCID: PMC2862743BACKGROUND: Nuclear pore complexes (NPCs) mediate bidirectional transport of proteins, RNAs, and ribonucleoproteins across the double-membrane nuclear envelope. Although there are many studies that look at the traffic in the nucleus and through the nuclear envelope we propose a method to detect the nucleocytoplasmic transport kinetics in an unperturbed cell, with no requirement for specific labeling of isolated molecules and, most important, in the presence of the cell milieu. METHODOLOGY: The pair correlation function method (pCF) measures the time a molecule takes to migrate from one location to another within the cell in the presence of many molecules of the same kind. The spatial and temporal correlation among two arbitrary points in the cell provides a local map of molecular transport, and also highlights the presence of barriers to diffusion with millisecond time resolution and spatial resolution limited by diffraction. We use the pair correlation method to monitor a model ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
Scanning laser image correlation for measurement of flow.
J Biomed Opt. 2010; 15(2): 026003. PMCID: PMC2869368Scanning laser image correlation (SLIC) is an optical correlation technique for measuring the fluid velocity of particles suspended in a liquid. This technique combines laser scanning of an arbitrary pattern with pair cross-correlation between any two points in the pattern. SLIC overcomes many of the limitations of other optical correlation techniques for flow measurement, such as laser speckle, spatial temporal image correlation spectroscopy, and two-foci methods. One of the main advantages of SLIC is that the concept can be applied to measurements on a range of scales through simple zooming or modifications in the instrumentation. Additionally, SLIC is relatively insensitive to instrument noise through the use of correlation analysis and is insensitive to background. SLIC can provide detailed information about the direction and pattern of flow. SLIC has potential applications ranging from microfluidics to blood flow measurements.
Jaureguiberry MS, Tricerri MA, Sánchez SA, Garda HA, Finarelli GS, Gonzalez MC, Rimoldi OJ.
Membrane organization and regulation of cellular Cholesterol homeostasis.
J Membr Biol. 2010; 234(3): 183-194. PMCID: PMC2868589An excess of intracellular free cholesterol (Chol) is cytotoxic, and its homeostasis is crucial for cell viability. Apolipoprotein A-I (apoA-I) is a highly efficient Chol acceptor because it activates complex cellular pathways that tend to mobilize and export Chol from cellular depots. We hypothesize that membrane composition and/or organization is strongly involved in Chol homeostasis. To test this hypothesis, we constructed a cell line overexpressing stearoyl coenzyme A (CoA) desaturase (SCD cells), which modifies plasma membrane (PM) composition by the enrichment of monounsaturated fatty acids, and determined this effect on membrane properties, cell viability, and Chol homeostasis. PM in SCD cells has a higher ratio of phospholipids to sphingomyelin and is slightly enriched in Chol. These cells showed an increase in the ratio of cholesteryl esters to free Chol; they were more resistant to Chol toxicity, and they exported more caveolin than control cells. The data suggest that cell functionality ... [truncated at 150 words]
Celli A, Sánchez SA, Behne MJ, Hazlett TL, Gratton E, Mauro TM.
The epidermal Ca(2+) gradient: measurement using the phasor representation of fluorescent lifetime imaging.
Biophys J. 2010; 98(5): 911-921. PMCID: PMC2830439Ionic gradients are found across a variety of tissues and organs. In this report, we apply the phasor representation of fluorescence lifetime imaging data to the quantitative study of ionic concentrations in tissues, overcoming technical problems of tissue thickness, concentration artifacts of ion-sensitive dyes, and calibration across inhomogeneous tissue. We used epidermis as a model system, as Ca(2+) gradients in this organ have been shown previously to control essential biologic processes of differentiation and formation of the epidermal permeability barrier. The approach described here allowed much better localization of Ca(2+) stores than those used in previous studies, and revealed that the bulk of free Ca(2+) measured in the epidermis comes from intracellular Ca(2+) stores such as the Golgi and the endoplasmic reticulum, with extracellular Ca(2+) making a relatively small contribution to the epidermal Ca(2+) gradient. Due to the high spatial resolution of two-photon microscopy, we were able to measure a ... [truncated at 150 words]
Yoon Y, Tong J, Lee PJ, Albanese A, Bhardwaj N, Källberg M, Digman MA, Lu H, Gratton E, Shin YK, Cho W.
Molecular basis of the potent membrane remodeling activity of the epsin1 ENTH domain.
J Biol Chem. 2010; 285(1): 531-540. PMCID: PMC2804201The mechanisms by which cytosolic proteins reversibly bind the membrane and induce the curvature for membrane trafficking and remodeling remain elusive. The epsin1 ENTH domain has potent vesicle tubulation activity despite lack of intrinsic molecular curvature. EPR revealed that the N-terminal alpha-helix penetrates the PtdIns(4,5)P(2)-containing membrane at a unique oblique angle and concomitantly interacts closely with helices from neighboring molecules in an antiparallel orientation. The quantitative fluorescence microscopy showed that the formation of highly ordered ENTH domain complexes beyond a critical size is essential for its vesicle tubulation activity. The mutations that interfere with the formation of large ENTH domain complexes abrogated the vesicle tubulation activity. Furthermore, the same mutations in the intact epsin1 abolished its endocytic activity in mammalian cells. Collectively, these results show that the ENTH domain facilitates the cellular membrane budding and fission by a novel mechanism that is distinct from that proposed for BAR domains.
Kukreti S, Cerussi AE, Tanamai W, Hsiang D, Tromberg BJ, Gratton E.
Characterization of metabolic differences between benign and malignant tumors: high-spectral-resolution diffuse optical spectroscopy.
Radiology. 2010; 254(1): 277-284. PMCID: PMC2797652Purpose: To develop a near-infrared spectroscopic method to identify breast cancer biomarkers and to retrospectively determine if benign and malignant breast lesions could be distinguished by using this method. Materials and Methods: The study was HIPAA compliant and was approved by the university institutional review board. Written informed consent was obtained. By using self-referencing differential spectroscopy (SRDS) analysis, the existence of specific spectroscopic signatures of breast lesions on images acquired by using diffuse optical spectroscopy imaging in the wavelength range (650-1000 nm) was established. The SRDS method was tested in 60 subjects (mean age, 38 years; age range, 22-74 years). There were 17 patients with benign breast tumors and 22 patients with malignant breast tumors. There were 21 control subjects. Results: Discrimination analysis helped separate malignant from benign tumors. A total of 40 lesions (22 malignant and 18 benign) were analyzed. Twenty were true-positive lesions, 17 were true-negative lesions, one ... [truncated at 150 words]
Tanner K, Ferris DR, Lanzanò L, Mandefro B, Mantulin WW, Gardiner DM, Rugg EL, Gratton E.
Coherent movement of cell layers during wound healing by image correlation spectroscopy.
Biophys J. 2009; 97(7): 2098-2106. PMCID: PMC2756390We have determined the complex sequence of events from the point of injury until reepithelialization in axolotl skin explant model and shown that cell layers move coherently driven by cell swelling after injury. We quantified three-dimensional cell migration using correlation spectroscopy and resolved complex dynamics such as the formation of dislocation points and concerted cell motion. We quantified relative behavior such as velocities and swelling of cells as a function of cell layer during healing. We propose that increased cell volume (~37% at the basal layer) is the driving impetus for the start of cell migration after injury where the enlarged cells produce a point of dislocation that foreshadows and dictates the initial direction of the migrating cells. Globally, the cells follow a concerted vortex motion that is maintained after wound closure. Our results suggest that cell volume changes the migration of the cells after injury.
Katayama Y, Burkacky O, Meyer M, Bräuchle C, Gratton E, Lamb DC.
Real-time nanomicroscopy via three-dimensional single-particle tracking.
ChemPhysChem. 2009; 10(14): 2458-2464. PMCID: PMC2857558We developed a new method for real-time, three-dimensional tracking of fluorescent particles. The instrument is based on a laser-scanning confocal microscope where the focus of the laser beam is scanned or orbited around the particle. Two confocal pinholes are used to simultaneously monitor regions immediately above and below the particle and a feedback loop is used to keep the orbit centered on the particle. For moderate count rates, this system can track particles with 15 nm spatial resolution in the lateral dimensions and 50 nm in the axial dimension at a temporal resolution of 32 ms. To investigate the interaction of the tracked particles with cellular components, we have combined our orbital tracking microscope with a dual-color, wide-field setup. Dual-color fluorescence wide-field images are recorded simultaneously in the same image plane as the particle being tracked. The functionality of the system was demonstrated by tracking fluorescent-labeled artificial viruses in tubulin-eGFP ... [truncated at 150 words]
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Fluorescence correlation spectroscopy and fluorescence cross-correlation spectroscopy, applications in systems biology.
WIREs Syst Biol Med. 2009; 1(2): 273-282. PMCID: PMC3086279This article focuses on methods based on fluctuation correlation spectroscopy to determine the formation of protein complexes in living cells. We present the principles of the fluctuation method applied to cells. We discuss the novelty and the promises of this approach. The emphasis is in the discussion of the underlying statistical assumptions of the image correlation spectroscopy analysis rather than in reviewing applications of the method. Although one example of the application of the fluctuation method is given, this article also contains simulations that are better suited to illustrate and support the basic assumptions of the method.
Portolés MJL, Nieto FR, Soria DB, Amalvy JI, Peruzzo PJ, Mártire DO, Kotler M, Holub O, Gonzalez MC.
Photophysical properties of blue-emitting silicon nanoparticles.
J Phys Chem C. 2009; 113(31): 13694-13702. PMCID: PMC3410643Silicon nanoparticles with strong blue photoluminescence were synthesized by electrochemical etching of silicon wafers and ultrasonically removed under N2 atmosphere in organic solvents to produce colloids. Thermal treatment leads to the formation of colloidal Si particles of 3 ± 1 nm diameter, which upon excitation with 340−380 nm light exhibited room temperature luminescence in the range from 400 to 500 nm. The emission and the one- and two-photon excitation spectra of the particles are not sensitive to surface functionalization with methyl 2-methylprop-2-enoate. However, the derivatized particles show higher emission quantum yields in air-saturated suspensions (44%) than the underivatized particles (27%), as well as higher stability of its dispersions. FTIR and XPS spectra indicate a significant surface oxidation of the particles. The Si:O:C ratio at the surface of the derivatized particles estimated from XPS is Si3O6(C5O2Hy)1, with y = 7−8. Vibronic spacing is observed in both the emission and excitation spectra. ... [truncated at 150 words]
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Imaging barriers to diffusion by pair correlation functions.
Biophys J. 2009; 97(2): 665-673. PMCID: PMC2711318Molecular diffusion and transport are fundamental processes in physical, chemical, biochemical, and biological systems. However, current approaches to measure molecular transport in cells and tissues based on perturbation methods such as 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 farther apart 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 by 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 ... [truncated at 150 words]
Krishnan K, Holub O, Gratton E, Clayton AHA, Cody S, Moens PDJ.
Profilin interaction with phosphatidylinositol (4,5)-bisphosphate destabilizes the membrane of giant unilamellar vesicles.
Biophys J. 2009; 96(12): 5112-5121. PMCID: PMC2712054Profilin, a small cytoskeletal protein, and phosphatidylinositol (4,5)-bisphosphate [PI(4,5)P2] have been implicated in cellular events that alter the cell morphology, such as endocytosis, cell motility, and formation of the cleavage furrow during cytokinesis. Profilin has been shown to interact with PI(4,5)P2, but the role of this interaction is still poorly understood. Using giant unilamellar vesicles (GUVs) as a simple model of the cell membrane, we investigated the interaction between profilin and PI(4,5)P2. A number and brightness analysis demonstrated that in the absence of profilin, molar ratios of PI(4,5)P2 above 4% result in lipid demixing and cluster formations. Furthermore, adding profilin to GUVs made with 1% PI(4,5)P2 leads to the formation of clusters of both profilin and PI(4,5)P2. However, due to the self-quenching of the dipyrrometheneboron difluoride-labeled PI(4,5)P2, we were unable to determine the size of these clusters. Finally, we show that the formation of these clusters results in the destabilization ... [truncated at 150 words]
Gielen E, Smisdom N, vandeVen MJ, de Clercq B, Gratton E, Digman MA, Rigo JM, Hofkens J, Engelborghs Y, Ameloot M.
Measuring diffusion of lipid-like probes in artificial and natural membranes by raster image correlation spectroscopy (RICS): use of a commercial laser-scanning microscope with analog detection.
Langmuir. 2009; 25(9): 5209-5218. PMCID: PMC2728053The heterogeneity in composition and interaction within the cellular membrane translates into a wide range of diffusion coefficients of its constituents. Therefore, several complementary microfluorimetric techniques such as fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP) and single-particle tracking (SPT) have to be applied to explore the dynamics of membrane components. The recently introduced raster image correlation spectroscopy (RICS) offers a much wider dynamic range than each of these methods separately and allows for spatial mapping of the dynamic properties. RICS is implemented on a confocal laser-scanning microscope (CLSM), and the wide dynamic range is achieved by exploiting the inherent time information carried by the scanning laser beam in the generation of the confocal images. The original introduction of RICS used two-photon excitation and photon counting detection. However, most CLSM systems are based on one-photon excitation with analog detection. Here we report on the performance of such a commercial ... [truncated at 150 words]
Henning MF, Sánchez SA, Bakás L.
Visualization and analysis of lipopolysaccharide distribution in binary phospholipid bilayers.
Biochem Biophys Res Commun. 2009; 383(1): 22-26. PMCID: PMC2865696Lipopolysaccharide (LPS) is an endotoxin released from the outer membrane of Gram-negative bacteria during infections. It have been reported that LPS may play a role in the outer membrane of bacteria similar to that of cholesterol in eukaryotic plasma membranes.
In this article we compare the effect of introducing LPS or cholesterol in liposomes made of dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine on the solubilization process by Triton X-100. The results show that liposomes containing LPS or cholesterol are more resistant to solubilization by Triton X-100 than the binary phospholipid mixtures at 4 °C.
The LPS distribution was analyzed on GUVs of DPPC:DOPC using FITC-LPS. Solid and liquid-crystalline domains were visualized labeling the GUVs with LAURDAN and GP images were acquired using a two-photon microscope. The images show a selective distribution of LPS in gel domains.
Our results support the hypothesis that LPS could aggregate and concentrate selectively in biological membranes providing a mechanism to bring together several ... [truncated at 150 words]
Caracciolo G, Digman MA, Gratton E, Sánchez SA, Caminiti R.
Efficient escape from endosomes determines the superior efficiency of multicomponent lipoplexes.
J Phys Chem B. 2009; 113(15): 4995-4997. PMCID: PMC2673460Designer 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.
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Analysis of diffusion and binding in cells using the RICS approach.
Microsc Res Tech. 2009; 72(4): 323-332. PMCID: PMC4364519The 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 article, 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]
Tyler KM, Fridberg A, Toriello KM, Olson CL, Cieslak JA, Hazlett TL, Engman DM.
Flagellar membrane localization via association with lipid rafts.
J Cell Sci. 2009; 122(Pt 6): 859-866. PMCID: PMC2714428The eukaryotic flagellar membrane has a distinct composition from other domains of the plasmalemma. Our work shows that the specialized composition of the trypanosome flagellar membrane reflects increased concentrations of sterols and saturated fatty acids, correlating with direct observation of high liquid order by laurdan fluorescence microscopy. These findings indicate that the trypanosome flagellar membrane possesses high concentrations of lipid rafts: discrete regions of lateral heterogeneity in plasma membranes that serve to sequester and organize specialized protein complexes. Consistent with this, a dually acylated Ca2+ sensor that is concentrated in the flagellum is found in detergent-resistant membranes and mislocalizes if the lipid rafts are disrupted. Detergent-extracted cells have discrete membrane patches localized on the surface of the flagellar axoneme, suggestive of intraflagellar transport particles. Together, these results provide biophysical and biochemical evidence to indicate that lipid rafts are enriched in the trypanosome flagellar membrane, providing a unique mechanism for flagellar ... [truncated at 150 words]
Rossow MJ, Mantulin WW, Gratton E.
Spatiotemporal image correlation spectroscopy measurements of flow demonstrated in microfluidic channels.
J Biomed Opt. 2009; 14(2): 024014. PMCID: PMC2702990Accurate blood flow measurements during surgery can improve an operation's chance of success. We developed near-infrared spatio-temporal image spectroscopy (NIR-STICS), which has the potential to make blood flow measurements that are difficult to accomplish with existing methods. Specifically, we propose the technique and we show feasibility on phantom measurements. NIR-STICS has the potential of measuring the fluid velocity in small blood vessels (less than 1 mm in diameter) and of creating a map of blood flow rates over an area of approximately 1 cm2. NIR-STICS employs near-infrared spectroscopy to probe inside blood vessel walls and spatiotemporal image correlation spectroscopy to directly—without the use of a model—extract fluid velocity from the fluctuations within an image. We present computer simulations and experiments on a phantom system that demonstrate the effectiveness of NIR-STICS.
Digman MA, Wiseman PW, Choi CK, Horwitz AR, Gratton E.
Stoichiometry of molecular complexes at adhesions in living cells.
Proc Natl Acad Sci USA. 2009; 106(7): 2170-2175. PMCID: PMC2630200We describe a method to detect molecular complexes and measure their stoichiometry in living cells from simultaneous fluctuations of the fluorescence intensity in two image channels, each detecting a different kind of protein. The number and brightness (N&B) analysis, namely, the use of the ratio between the variance and the average intensity to obtain the brightness of molecules, is extended to the cross-variance of the intensity fluctuations in two channels. We apply the cross-variance method to determine the stoichiometry of complexes containing paxillin and vinculin or focal adhesion kinase (FAK) in disassembling adhesions in mouse embryo fibroblasts expressing FAK, vinculin, and paxillin-tagged with EGFP and mCherry. We found no complexes of these proteins in the cytoplasm away from the adhesions. However, at the adhesions, large aggregates leave, forming a hole, during their disassembly. This hole shows cross-correlation between FAK and paxillin and vinculin and paxillin. From the amplitude of the ... [truncated at 150 words]
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One photon up, one photon down.
Nat Biotechnol. 2009; 27(2): 147-148.A microscopy technique based on stimulated Raman scattering achieves label-free imaging with very high sensitivity.
Toro CA, Sánchez SA, Zanocco A, Lemp E, Gratton E, Gunther G.
Solubilization of lipid bilayers by myristyl sucrose ester: effect of cholesterol and phospholipid head group size.
Chem Phys Lipids. 2009; 157(2): 104-112. PMCID: PMC2868593The solubilization of biological membranes by detergents has been used as a major method for the isolation and purification of membrane proteins and other constituents. Considerable interest in this field has resulted from the finding that different components can be solubilized selectively. Certain membrane constituents are incorporated into small micelles, whereas others remain in the so-called detergent-resistant membrane domains that are large enough to be separated by centrifugation. The detergent resistant fractions contain an elevated percentage of cholesterol, and thus its interaction with specific lipids and proteins may be key for membrane organization and regulation of cellular signaling events.
This report focuses on the solubilization process induced by the sucrose monoester of myristic acid, b-D-Fructofuranosyl-6-O-myristyl-a-D-glucopyranoside (MMS), a nonionic detergent. We studied the effect of the head group and the cholesterol content on the process. 1-Palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and Dioctadecyl-dimethyl-ammonium chloride (DODAC) vesicles were used, and the solubilization process was followed using Laurdan ... [truncated at 150 words]
Digman MA, Wiseman PW, Horwitz AR, Gratton E.
Detecting protein complexes in living cells from laser scanning confocal image sequences by the cross correlation raster image spectroscopy method.
Biophys J. 2009; 96(2): 707-716. PMCID: PMC2716688We describe a general method for detecting molecular complexes based on the analysis of single molecule fluorescence fluctuations from laser scanning confocal images. The method detects and quantifies complexes of two different fluorescent proteins noninvasively in living cells. Because 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 method, specific locations in the cell can be visualized where dynamics of binding and unbinding of fluorescent protein complexes occur. This ... [truncated at 150 words]
Kukreti S, Cerussi AE, Tromberg BJ, Gratton E.
Intrinsic near-infrared spectroscopic markers of breast tumors.
Dis Markers. 2008; 25(6): 281-290. PMCID: PMC2732199We have discovered quantitative optical biomarkers unique to cancer by developing a double-differential spectroscopic analysis method for near-infrared (NIR, 650–1000 nm) spectra acquired non-invasively from breast tumors. These biomarkers are characterized by specific NIR absorption bands. The double-differential method removes patient specific variations in molecular composition which are not related to cancer, and reveals these specific cancer biomarkers. Based on the spectral regions of absorption, we identify these biomarkers with lipids that are present in tumors either in different abundance than in the normal breast or new lipid components that are generated by tumor metabolism. Furthermore, the O-H overtone regions (980–1000 nm) show distinct variations in the tumor as compared to the normal breast. To quantify spectral variation in the absorption bands, we constructed the Specific Tumor Component (STC) index. In a pilot study of 12 cancer patients we found 100% sensitivity and 100% specificity for lesion identification. The STC ... [truncated at 150 words]
Sanabria H, Digman MA, Gratton E, Waxham MN.
Spatial diffusivity and availability of intracellular Calmodulin.
Biophys J. 2008; 95(12): 6002-6015. PMCID: PMC2599858Calmodulin (CaM) is the major pathway that transduces intracellular Ca2+ increases to the activation of a wide variety of downstream signaling enzymes. CaM and its target proteins form an integrated signaling network believed to be tuned spatially and temporally to control CaM's ability to appropriately pass signaling events downstream. Here, we report the spatial diffusivity and availability of CaM labeled with enhanced green fluorescent protein (eGFP)-CaM, at basal and elevated Ca2+, quantified by the novel fluorescent techniques of raster image scanning spectroscopy and number and brightness analysis. Our results show that in basal Ca2+ conditions cytoplasmic eGFP-CaM diffuses at a rate of 10 µm2/s, twofold slower than the noninteracting tracer, eGFP, indicating that a significant fraction of CaM is diffusing bound to other partners. The diffusion rate of eGFP-CaM is reduced to 7 µm2/s when a large (646 kDa) target protein Ca2+/CaM-dependent protein kinase II is coexpressed in the cells. ... [truncated at 150 words]
Watson NB, Nelson E, Digman MA, Thornburg JA, Alphenaar BW, McGregor WG.
RAD18 and associated proteins are immobilized in nuclear foci in human cells entering S-phase with ultraviolet light-induced damage.
Mutat Res. 2008; 648(1-2): 23-31. PMCID: PMC2610409Proteins required for translesion DNA synthesis localize in nuclear foci of cells with replication-blocking lesions. The dynamics of this process were examined in human cells with fluorescence-based biophysical techniques. Photobleaching recovery and raster image correlation spectroscopy experiments indicated that involvement in the nuclear foci reduced the movement of RAD18 from diffusion-controlled to virtual immobility. Examination of the mobility of REV1 indicated that it is similarly immobilized when it is observed in nuclear foci. Reducing the level of RAD18 greatly reduced the focal accumulation of REV1 and reduced UV mutagenesis to background frequencies. Fluorescence lifetime measurements indicated that RAD18 and RAD6A or polη only transferred resonance energy when these proteins colocalized in damage-induced nuclear foci, indicating a close physical association only within such foci. Our data support a model in which RAD18 within damage-induced nuclear foci is immobilized and is required for recruitment of Y-family DNA polymerases and subsequent mutagenesis. In ... [truncated at 150 words]
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Analysis of molecular concentration and brightness from fluorescence fluctuation data with an electron multiplied CCD camera.
Biophys J. 2008; 95(11): 5385-5398. PMCID: PMC2586556We demonstrate the calculation of particle brightness and concentration from fluorescence-fluctuation photon-counting statistics using an electron multiplied CCD (EMCCD) camera. This technique provides a concentration independent measure of particle brightness in dynamic systems. The high sensitivity and highly parallel detection of EMCCD cameras allow for imaging of dynamic particle brightness, providing the capability to follow aggregation reactions in real time. A critical factor of the EMCCD camera is the presence of nonlinearity at high intensities. These nonlinearities arise due to limited capacity of the CCD well and to the analog-to-digital converter maximum range. However we show that the specific camera we used 16 bit ADC cameras has sufficient dynamic range for most microscopy applications. In addition we explore the importance of camera timing behavior as it is affected by the vertical frame transfer speed of the camera. While the camera has microsecond exposure time for illumination of a few pixels, ... [truncated at 150 words]
Barreiro O, Zamai M, Yáñez-Mó M, Tejera E, López-Romero P, Monk PN, Gratton E, Caiolfa VR, Sánchez-Madrid F.
Endothelial adhesion receptors are recruited to adherent leukocytes by inclusion in preformed tetraspanin nanoplatforms.
J Cell Biol. 2008; 183(3): 527-542. PMCID: PMC2575792VCAM-1 and ICAM-1, receptors for leukocyte integrins, are recruited to cell-cell contact sites on the apical membrane of activated endothelial cells. In this study, we show that this recruitment is independent of ligand engagement, actin cytoskeleton anchorage, and heterodimer formation. Instead, VCAM-1 and ICAM-1 are recruited by inclusion within specialized preformed tetraspanin-enriched microdomains, which act as endothelial adhesive platforms (EAPs). Using advanced analytical fluorescence techniques, we have characterized the diffusion properties at the single-molecule level, nanoscale organization, and specific intradomain molecular interactions of EAPs in living primary endothelial cells. This study provides compelling evidence for the existence of EAPs as physical entities at the plasma membrane, distinct from lipid rafts. Scanning electron microscopy of immunogold-labeled samples treated with a specific tetraspanin-blocking peptide identify nanoclustering of VCAM-1 and ICAM-1 within EAPs as a novel mechanism for supramolecular organization that regulates the leukocyte integrin-binding capacity of both endothelial receptors during extravasation.
Fratoddi I, Gohlke C, Cametti C, Diociaiuti M, Russo MV.
Self-assembly of nanostructured polymetallaynes.
Polymer. 2008; 49(15): 3211-3216.Organometallic conjugated polymers containing transition metal centers in the main chain (polymetallynes) , with general formula -[M-(PBu3)2-C≡C-X-C≡C-]n- with M = Pt(II) and Pd(II), X= organic conjugated spacer, namely poly[1,1'-bis(ethynyl)-4,4'-biphenyl-(bis-tributylphosphine)Pd(II)] (Pd-DEBP), poly[1,1'-bis(ethynyl)-4,4'-biphenyl-(bis-tributylphosphine)Pt(II)] (Pt-DEBP) and poly-[1,4-bis(ethynyl)-2,5-dihexadecyloxy)benzene-bis(triphenylphosphine)platinum(II)] (Pt-BOB) were synthesized and fully characterized. The polymeric compounds were cast deposited onto glass substrates and their morphologies studied by means of SEM (Scanning Electron Microscopy) and EF-TEM (Energy Filtered-Transmission Electron Microscopy). The formation of nanostructured fibrils with diameters in the range 100-300 nm was revealed by SEM. EF-TEM images showed that the fibers are made of hollow nanotubes, randomly oriented, with external diameter of about 6-7 nm.
Malengo G, Andolfo A, Sidenius N, Gratton E, Zamai M, Caiolfa VR.
Fluorescence correlation spectroscopy and photon counting histogram on membrane proteins: functional dynamics of the glycosylphosphatidylinositol-anchored urokinase plasminogen activator receptor.
J Biomed Opt. 2008; 13(3): 031215. PMCID: PMC2718687The oligomerization of glycosylphosphatidylinositol-anchored proteins is thought to regulate their association with membrane microdomains, subcellular sorting, and activity. However, these mechanisms need to be comprehensively explored in living, unperturbed cells, without artificial clustering agents, and using fluorescent protein-tagged chimeras that are fully biologically active. We expressed in human embryo kidnay 293 (HEK293) cells a biologically active chimera of the urokinase plasminogen activator receptor (uPAR), the uPAR-mEGFP-GPI. We also produced HEK293/D2D3-mEGFP-GPI cells expressing the truncated form of the receptor, lacking biological activity. We studied the dynamics and oligomerization of the two proteins, combining fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analyses, and using subclones with homogenously low expression levels. Overall, the mobile fractions of the two proteins, constituted by monomers and dimers, had comparable diffusion coefficients. However, the diffusion coefficient decreased in monomer-enriched fractions only for the active receptor, suggesting that uPAR monomers might be preferentially engaged in multiprotein ... [truncated at 150 words]
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Chromatin dynamics during interphase explored by single-particle tracking.
Chromosome Res. 2008; 16(3): 439-449. PMCID: PMC2701671Our view of the structure and function of the interphase nucleus has changed drastically in recent years. It is now widely accepted that the nucleus is a well organized and highly compartmentalized organelle and that this organization is intimately related to nuclear function. In this context, chromatin-initially considered a randomly entangled polymer-has also been shown to be structurally organized in interphase and its organization was found to be very important to gene regulation. Relevant and not completely answered questions are how chromatin organization is achieved and what mechanisms are responsible for changes in the positions of chromatin loci in the nucleus. A significant advance in the field resulted from tagging chromosome sites with bacterial operator sequences, and visualizing these tags using green fluorescent protein fused with the appropriate repressor protein. Simultaneously, fluorescence imaging techniques evolved significantly during recent years, allowing observation of the time evolution of processes in living specimens. ... [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.
Biochem Biophys Res Comm. 2008; 369(2): 519-525. PMCID: PMC2396457Ca(2+)/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. Here, based on our previous work, we attempted to generate an optical probe for fluorescence lifetime imaging (FLIM) of CaMKII activity by fusing the protein with donor and acceptor fluorescent proteins at its amino- and carboxyl-termini. We first optimized the combinations of fluorescent proteins by taking advantage of expansion of fluorescent proteins towards longer wavelength in fluorospectrometric assay. Then using digital frequency domain FLIM (DFD-FLIM), we demonstrated that the resultant protein can indeed detect CaMKII activation in living cells. These FLIM versions of Camui could be useful for elucidating the function of CaMKII both in vitro and in vivo.
Digman MA, Brown CM, Horwitz AR, Mantulin WW, Gratton E.
Paxillin dynamics measured during adhesion assembly and disassembly by correlation spectroscopy.
Biophys J. 2008; 94(7): 2819-2831. PMCID: PMC2267137Paxillin is an adaptor molecule involved in the assembly of focal adhesions. Using different fluorescence fluctuation approaches, we established that paxillin-EGFP is dynamic on many timescales within the cell, ranging from milliseconds to seconds. In the cytoplasmic regions, far from adhesions, paxillin is uniformly distributed and freely diffusing as a monomer, as determined by single-point fluctuation correlation spectroscopy and photon-counting histogram analysis. Near adhesions, paxillin dynamics are reduced drastically, presumably due to binding to protein partners within the adhesions. The photon-counting histogram analysis of the fluctuation amplitudes reveals that this binding equilibrium in new or assembling adhesions is due to paxillin monomers binding to quasi-immobile structures, whereas in disassembling adhesions or regions of adhesions, the equilibrium is due to exchange of large aggregates. Scanning fluctuation correlation spectroscopy and raster-scan image correlation spectroscopy analysis of laser confocal images show that the environments within adhesions are heterogeneous. Relatively large adhesions appear to ... [truncated at 150 words]
Garcia-Marcos A, Sánchez SA, Parada P, Eid JS, Jameson DM, Remacha M, Gratton E, Ballesta JPG.
Yeast ribosomal stalk heterogeneity in vivo shown by two-photon FCS and molecular brightness analysis.
Biophys J. 2008; 94(7): 2884-2890. PMCID: PMC2267128The stalk of Saccharomyces cerevisiae ribosomes contains, on average, five distinct proteins, namely P0 and four acidic proteins, P1, P1β, P2, and P2β. Each ribosome contains only one copy of P0, but the distribution of the acidic proteins among the ribosome population in vivo has not been determined. Using two-photon fluorescence correlation spectroscopy and scanning FCS, on cells expressing EGFP-tagged P0, P1, and P2 proteins, we show, with brightness analysis, that individual yeast ribosomes in vivo are compositionally heterogeneous in regard to P1, P1β, P2, and P2β. These results are relevant to the hypothesis, based on in vitro studies, that the overall cellular pattern of expressed proteins can be determined by the distribution of the stalk proteins among the ribosome population.
Digman MA, Dalal RB, Horwitz AR, Gratton E.
Mapping the number of molecules and brightness in the laser scanning microscope.
Biophys J. 2008; 94(6): 2320-2332. PMCID: PMC2257897We describe a technique based on the moment analysis for the measurement of the average number of molecules and brightness in each pixel in fluorescence microscopy images. The average brightness of the particle is obtained from the ratio of the variance to the average intensity at each pixel. To obtain the average number of fluctuating particles, we divide the average intensity at one pixel by the brightness. This analysis can be used in a wide range of concentrations. In cells the intensity at any given pixel may be due to bright immobile structures, dim fast diffusing particles and to autofluorescence or scattering. The total variance is given by the variance of each of the above components in addition to the variance due to detector noise. Assuming that all sources of variance are independent, the total variance is the sum of the variances of the individual components. The variance due to ... [truncated at 150 words]
Raub CB, Unruh JR, Suresh V, Krasieva TB, Lindmo T, Gratton E, Tromberg BJ, George SC.
Image correlation spectroscopy of multiphoton images correlates with collagen mechanical properties.
Biophys J. 2008; 94(6): 2361-2373. PMCID: PMC2257909Multiphoton microscopy (MPM) holds promise as a non-invasive imaging technique for characterizing collagen structure, and thus mechanical properties, through imaging second harmonic generation (SHG) and two-photon fluorescence in engineered and real connective tissues. Controlling polymerization pH to manipulate collagen gel microstructure, we quantifed pore and fiber dimensions using both standard methods and image correlation spectroscopy (ICS) on MPM, scanning electron, and darkfield microscopy images. The latter two techniques are used to confirm microstructural measurements made from MPM images. As polymerization pH increased from 5.5 to 8.5, mean fiber diameter decreased from 3.7 ± 0.7 µm to 1.6 ± 0.3 µm, the average pore size decreased from 81.7±3.7 µm2 to 7.8±0.4 µm2, and pore area fraction decreased from 56.8±0.8% to 18.0±1.3% (measured from SHG images), while the storage modulus G' and the loss modulus G'', components of the shear modulus, increased ~33-fold and ~16-fold, respectively. A characteristic length scale measured using ... [truncated at 150 words]
Colyer RA, Lee CY, Gratton E.
A novel fluorescence lifetime imaging system that optimizes photon efficiency.
Microsc Res Tech. 2008; 71(3): 201-213.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. On the basis of the mathematical model, we present an error analysis that 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.
Sánchez SA, Tricerri MA, Gratton E.
Detecting cholesterol changes in lipid bilayers.
BioWorld Europe. 2008; 1: 8-11.Developing controlled methods for cholesterol manipulation in biological and artificial systems is an exciting goal and attracts the attention of bio-scientists. A technique is needed sensitive to cholesterol content and with good spatial resolution to look for changes in membrane cholesterol content in intact cells. This article describes the detection of cholesterol changes in lipid bilayers by laurdan generalized polarization and two-photon excitation fluorescence microscopy.
Digman MA, Caiolfa VR, Zamai M, Gratton E.
The phasor approach to fluorescence lifetime imaging analysis.
Biophys J. 2008; 94(2): L14-16. PMCID: PMC2157251Changing 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 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 simplify the way data are analyzed in FLIM, paving the way for the analysis of large data sets and, in general, making the FLIM technique accessible to the non expert in spectroscopy and data analysis.
Fiorini R, Ragni L, Ambrosi S, Littarru GP, Gratton E, Hazlett TL.
Fluorescence studies of the interactions of ubiquinol-10 with liposomes.
Photochem Photobiol. 2008; 84(1): 209-214.Ubiquinone-10 plays a central role in energy production and its reduced form, ubiquinol-10 is also capable of acting as a potent radical scavenging antioxidant against membrane lipid peroxidation. Efficiency of this protection depends mostly on its localization in lipid bilayer. The intrinsic fluorescence of ubiquinol-10 and of the exogenous probe, Laurdan, has been used to determine the location of ubiquinol-10 in unilamellar liposomes of egg phosphatidylcholine (EggPC) and dimyristoyl phosphatidylcholine. Laurdan fluorescence moiety is positioned at the hydrophilic-hydrophobic interface of the phospholipid bilayer and its parameters reflect the membrane polarity and microheterogeneity, which we have used to explore the coexistence of microdomains with distinct physical properties. In liquid-crystalline bilayers ubiquinol has a short fluorescence lifetime (0.4 ns) and a high steady-state anisotropy. In a concentration-dependent manner, ubiquinol-10 influences the Laurdan excitation, emission and generalized polarization measurements. In EggPC liposomes ubiquinol-10 induces a decrease in membrane water mobility near the probe, ... [truncated at 150 words]
Dalal RB, Digman MA, Horwitz AR, Vetri V, Gratton E.
Determination of particle number and brightness using a laser scanning confocal microscope operating in the analog mode.
Microsc Res Tech. 2008; 71(1): 69-81.We describe a method to obtain the brightness and number of molecules at each pixel of an image stack obtained with a laser scanning microscope. The method is based on intensity fluctuations due to the diffusion of molecules in a pixel. For a detector operating in the analog mode, the variance must be proportional to the intensity. Once this constant has been calibrated, we use the ratio between the variance and the intensity to derive the particle brightness. Then, from the ratio of the intensity to the brightness we obtain the average number of particles in the pixel. We show that the method works with molecules in solution and that the results are comparable to those obtained with fluctuation correlation spectroscopy. We compare the results obtained with the detector operating in the analog and photon counting mode. Although the dynamic range of the detector operating in the photon counting mode ... [truncated at 150 words]
Celli A, Beretta S, Gratton E.
Phase fluctuations on the micron-submicron scale in GUVs composed of a binary lipid mixture.
Biophys J. 2008; 94(1): 104-116. PMCID: PMC2134868We used a combination of imaging and fluctuation techniques to investigate the temporal evolution of gel phase domains at the onset of phase separation, as well as the correlation between domain topology and local lipid ordering in GUVs composed of a binary mixture of DPPC:DLPC 1:1. The data acquired at temperatures immediately above the transition temperature of the two lipids suggest fluctuations in the lipid organization with a lifetime shorter than 0.1 second and a characteristic length of 1.2 µm. As the temperature is decreased below the transition temperature of one of the lipids, coupling between the two leaflets of the bilayer is observed to begin within the first five minutes after the onset of phase separation. However, domains confined to only one leaflet can be found during the first 45-50 minutes after the onset of phase separation. Our analysis using a two-state model (liquid and gel) indicates that for ... [truncated at 150 words]
Brown CM, Dalal RB, Hebert B, Digman MA, Horwitz AR, Gratton E.
Raster image correlation spectroscopy (RICS) for measuring fast protein dynamics and concentrations with a commercial laser scanning confocal microscope.
J Microsc. 2008; 229(1): 78-91. PMCID: PMC3690660Raster image correlation spectroscopy (RICS) is a new and novel technique for measuring molecular dynamics and concentrations from fluorescence confocal images. The RICS technique extracts information about molecular dynamics and concentrations from images of living cells taken on commercial confocal systems. Here we develop guidelines for performing the RICS analysis on an analogue commercial laser scanning confocal microscope. Guidelines for typical instrument settings, image acquisition settings and analogue detector characterization are presented. Using appropriate instrument/acquisition parameters, diffusion coefficients and concentrations can be determined, even for highly dynamic dye molecules in solution. Standard curves presented herein demonstrate the ability to detect protein concentrations as low as ~ 2 nM. Additionally, cellular measurements give accurate values for the diffusion of paxillin-enhanced-green fluorescent protein (EGFP), an adhesion adaptor molecule, in the cytosol of the cell and also show slower paxillin dynamics near adhesions where paxillin interacts with immobile adhesion components. Methods are presented ... [truncated at 150 words]
Caiolfa VR, Zamai M, Malengo G, Andolfo A, Madsen CD, Sutin JDB, Digman MA, Gratton E, Blasi F, Sidenius N.
Monomer–dimer dynamics and distribution of GPI-anchored uPAR are determined by cell surface
protein assemblies.
J Cell Biol. 2007; 179(5): 1067-1082. PMCID: PMC2099195To search for functional links between glycosylphosphatidylinositol (GPI) protein monomer–oligomer exchange and membrane dynamics and confinement, we studied urokinase plasminogen activator (uPA) receptor (uPAR), a GPI receptor involved in the regulation of cell adhesion, migration, and proliferation. Using a functionally active fluorescent protein–uPAR in live cells, we analyzed the effect that extracellular matrix proteins and uPAR ligands have on uPAR dynamics and dimerization at the cell membrane. Vitronectin directs the recruitment of dimers and slows down the diffusion of the receptors at the basal membrane. The commitment to uPA–plasminogen activator inhibitor type 1–mediated endocytosis and recycling modifies uPAR diffusion and induces an exchange between uPAR monomers and dimers. This exchange is fully reversible. The data demonstrate that cell surface protein assemblies are important in regulating the dynamics and localization of uPAR at the cell membrane and the exchange of monomers and dimers. These results also provide a strong rationale for ... [truncated at 150 words]
Olopade CO, Mensah E, Gupta R, Huo D, Picchiett DL, Gratton E, Michalos A.
Noninvasive determination of brain tissue oxygenation during sleep in obstructive sleep apnea: a near-infrared spectroscopic approach.
Sleep. 2007; 30(12): 1747-1755. PMCID: PMC2276122Study Objectives: Recurrent apneas and hypoxemia during sleep in obstructive sleep apnea (OSA) are associated with profound changes in cerebral blood flow to the extent that cerebral autoregulation may be insufficient to protect the brain. Since the brain is sensitive to hypoxia, the cerebrovascular morbidity seen in OSA could be due to chronic, cumulative effects of intermittent hypoxia. Near-infrared spectroscopy (NIRS) has the potential to noninvasively monitor brain tissue oxygen saturation (SO2), and changes in concentration of oxyhemoglobin [O2Hb], deoxyhemoglobin [HHb] and total hemoglobin [tHb] with real-time resolution. We hypothesized that brain tissue oxygenation would be worse during sleep in OSA relative to controls and sought to determine the practical use of NIRS in the sleep laboratory.
Design: We evaluated changes in brain tissue oxygenation using NIRS during overnight polysomnography.
Setting: Studies were conducted at University of Illinois, Chicago and Carle Hospital, Urbana, Illinois.
Patients: Nineteen subjects with OSA and 14 healthy controls ... [truncated at 150 words]
Wolf U, Wolf M, Choi JH, Paunescu LA, Michalos A, Gratton E.
Regional differences of hemodynamics and oxygenation in the human calf muscle detected with near-infrared spectrophotometry.
J Vasc Interv Radiol. 2007; 18(9): 1094-1101.Purpose: Measurements on muscle tissue are often performed at a selected single location over the muscle of interest. The hypothesis is that the values obtained reflect the status within the entire muscle or muscle group. However, this may not be the case. The aim of our study was to investigate whether this hypothesis is true for hemodynamics and oxygenation in the healthy human calf muscle at rest.
Methods: Hemoglobin flow, blood flow, oxygen consumption and venous hemoglobin oxygen saturation were mapped at 22 locations simultaneously by frequency-domain nearinfrared spectrophotometry with a specially designed probe during venous occlusion in 30 legs of 15 healthy subjects (9 female, 6 male, age range 26-37 years).
Results: For all parameters we found spatial heterogeneity between subjects and also within individual legs. All parameters were highly significantly different when comparing proximal and distal regions. Differences were also found between medial and lateral regions. The global mean values ... [truncated at 150 words]
Gatto R, Hoffman W, Paisansathan C, Mantulin WW, Gratton E, Charbel FT.
Effect of age on brain oxygenation regulation during changes in position.
J Neurosci Methods. 2007; 164(2): 308-311.Introduction: Reports indicate that brain regulation of oxygenation is inhibited in patients with low baseline oxyhemoglobin concentrations and that brain oxyhemoglobin concentrations are decreased with aging. The purpose of this study was to determine if regulation of brain oxygenation to changes in blood pressure is inhibited by normal aging.
Methods: Brain oxyhemoglobin (OHb) and deoxyhemoglobin (HHb) concentrations were determined from the forehead using a frequency domain near infrared spectroscopy in 27 healthy volunteers. Subjects were separated into two groups by age (20–39, n = 16; 40–60, n = 11). Brain hemoglobin and non-invasive blood pressure were measured in (1) supine, (2) sitting, (3) supine and (4) sitting positions with 10-min equilibration intervals between each determination. Statistical differences were determined by two way repeated measures analysis of variance.
Results: Young subjects were 28±5 years (mean±S.D.) and older subjects were 48±6 years. In supine position, OHb and HHb were 28.4±8.3 and 15.4±2.4 µmol/L, respectively, ... [truncated at 150 words]
Sánchez SA, Tricerri MA, Gratton E.
Interaction of high density lipoprotein particles with membranes containing cholesterol.
J Lipid Res. 2007; 48(8): 1689-1700.In this study, cholesterol (FC) efflux mediated by human high-density lipoproteins (HDL) was investigated using fluorescence methodologies. The accessibility of FC to HDL may depend on whether it is located in regions rich in unsaturated phospholipids or in domains containing high levels of FC and sphingomyelin, known as "lipid-rafts". LAURDAN Generalized Polarization and two-photon microscopy was used to quantify FC removal from different pools in the bilayer of Giant Unilamellar Vesicles (GUVs). GUVs made of POPC and FC were observed after incubation with reconstituted particles containing apolipoprotein A-I and POPC (78A diameter rHDL). Fluorescence Correlation Spectroscopy (FCS) data show an increase in the rHDL size during the incubation period. GUVs made of two "raft-like" mixtures (DOPC:DPPC:FC 1:1:1 and POPC:SPM:FC 6:1:1) were used to model liquidordered/ liquiddisordered phase coexistence. Through these experiments we concluded that rHDL preferentially removes cholesterol from the more fluid phases. These data, and their extrapolation to in ... [truncated at 150 words]
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Exploring dynamics in living cells by tracking single particles.
Cell Biochem Biophys. 2007; 48(1): 1-15.In the last years, significant advances in microscopy techniques and the introduction of a novel technology to label living cells with genetically encoded fluorescent proteins revolutionized the field of Cell Biology. Our understanding on cell dynamics built from snapshots on fixed specimens has evolved thanks to our actual capability to monitor in real time the evolution of processes in living cells. Among these new tools, single particle tracking techniques were developed to observe and follow individual particles. Hence, we are starting to unravel the mechanisms driving the motion of a wide variety of cellular components ranging from organelles to protein molecules by following their way through the cell. In this review, we introduce the single particle tracking technology to new users. We briefly describe the instrumentation and explain some of the algorithms commonly used to locate and track particles. Also, we present some common tools used to analyze trajectories and ... [truncated at 150 words]
Ermolenko DN, Spiegel PC, Majumdar ZK, Hickerson RP, Clegg RM, Noller HF.
The antibiotic viomycin traps the ribosome in an intermediate state of translocation.
Nat Struct Mol Biol. 2007; 14: 493-497.During protein synthesis, transfer RNA and messenger RNA undergo coupled translocation through the ribosome's A, P and E sites, a process catalyzed by elongation factor EF-G. Viomycin blocks translocation on bacterial ribosomes and is believed to bind at the subunit interface. Using fluorescent resonance energy transfer and chemical footprinting, we show that viomycin traps the ribosome in an intermediate state of translocation. Changes in FRET efficiency show that viomycin causes relative movement of the two ribosomal subunits indistinguishable from that induced by binding of EF-G with GDPNP. Chemical probing experiments indicate that viomycin induces formation of a hybrid-state translocation intermediate. Thus, viomycin inhibits translation through a unique mechanism, locking ribosomes in the hybrid state; the EF-G-induced 'ratcheted' state observed by cryo-EM is identical to the hybrid state; and, since translation is viomycin sensitive, the hybrid state may be present in vivo.
Holub O, Seufferheld MJ, Gohlke C, Govindjee, Heiss GJ, Clegg RM.
Fluorescence lifetime imaging microscopy of chlamydomonas reinhardtii: non-photochemical quenching mutants and the effect of photosynthetic inhibitors on the slow chlorophyll fluorescence transient.
J Microsc. 2007; 226(2): 90-120.Fluorescence lifetime-resolved images of chlorophyll fluorescence were acquired at the maximum P-level and during the slower transient (up to 250 s, including P-S-M-T) in the green photosynthetic alga Chlamydomonas reinhardtii. At the P-level, wild type and the violaxanthin-accumulating mutant npq1 show similar fluorescence intensity and fluorescence lifetime-resolved images. The zeaxanthin-accumulating mutant npq2 displays reduced fluorescence intensity at the P-level (about 25–35% less) and corresponding lifetime-resolved frequency domain phase and modulation values compared to wild type/npq1. A two-component analysis of possible lifetime compositions shows that the reduction of the fluorescence intensity can be interpreted as an increase in the fraction of a short lifetime component. This supports the important photoprotection function of zeaxanthin in photosynthetic samples, and is consistent with the notion of a ‘dimmer switch’. Similar, but quantitatively different, behaviour was observed in the intensity and fluorescence lifetime-resolved imaging measurements for cells that were treated with the electron transport inhibitor ... [truncated at 150 words]
Ermolenko DN, Majumdar ZK, Hickerson RP, Spiegel PC, Clegg RM, Noller HF.
Observation of intersubunit movement of the ribosome in solution using FRET.
J Mol Biol. 2007; 370(3): 530-540.Protein synthesis is believed to be a dynamic process, involving structural rearrangements of the ribosome. Cryo-EM reconstructions of certain elongation factor G (EF-G)-containing complexes have led to the proposal that translocation of tRNA and mRNA through the ribosome, from the A to P to E sites, is accompanied by a rotational movement between the two ribosomal subunits. Here, we have used Forster resonance energy transfer (FRET) to monitor changes in the relative orientation of the ribosomal subunits in different complexes trapped at intermediate stages of translocation in solution. Binding of EF-G to the ribosome in the presence of the non-hydrolyzable GTP analogue GDPNP or GTP plus fusidic acid causes an increase in the efficiency of energy transfer between fluorophores introduced into proteins S11 in the 30 S subunit and L9 in the 50 S subunit, and a decrease in energy transfer between S6 and L9. Similar anti-correlated changes in energy ... [truncated at 150 words]
Kukreti S, Cerussi AE, Tromberg BJ, Gratton E.
Intrinsic tumor biomarkers revealed by novel double-differential spectroscopic analysis of near-infrared spectra.
J Biomed Opt. 2007; 12(2): 020509.We develop a double-differential spectroscopic analysis method for broadband near-infrared (NIR, 650 to 1000 nm) absorption spectra. Application of this method to spectra of tumor-containing breast tissue reveals specific cancer biomarkers. In this method, patient-specific variations in molecular composition are removed by using the normal tissue as an internal control. The effects of concentration differences of the four major tissue absorbers (oxyhemoglobin, deoxyhemoglobin, water, and bulk lipid) between the tumor and normal tissue are accounted for to reveal small spectral components unique to cancer. From a pilot study of 15 cancer patients, we find these spectral components to be characterized by specific NIR absorption bands. Based on the spectral regions of absorption at about 760, 930, and 980 nm, we identify these biomarkers with changes in state or addition of lipid and/or water. To quantify spectral variation in the absorption bands, we construct the specific tumor component (STC) index. ... [truncated at 150 words]
Boens N, Qin W, Basari N, Hofkens J, Ameloot M, Pouget J, Lefèvre JP, Valeur B, Gratton E, vandeVen MJ, Silva N Jr, Engelborghs Y, Willaert K, Sillen A, Rumbles G, Phillips D, Visser AJWG, van Hoek A, Lakowicz JR, Malak H, Gryczynski I, Szabo AG, Krajcarski DT, Tamai N, Miura A.
Fluorescence lifetime standards for time and frequency domain fluorescence spectroscopy.
Anal Chem. 2007; 79(5): 2137-49.A series of fluorophores with single-exponential fluorescence decays in liquid solution at 20 degrees C were measured independently by nine laboratories using single-photon timing and multifrequency phase and modulation fluorometry instruments with lasers as excitation source. The dyes that can serve as fluorescence lifetime standards for time-domain and frequency-domain measurements are all commercially available, are photostable under the conditions of the measurements, and are soluble in solvents of spectroscopic quality (methanol, cyclohexane, water). These lifetime standards are anthracene, 9-cyanoanthracene, 9,10-diphenylanthracene, N-methylcarbazole, coumarin 153, erythrosin B, N-acetyl-l-tryptophanamide, 1,4-bis(5-phenyloxazol-2-yl)benzene, 2,5-diphenyloxazole, rhodamine B, rubrene, N-(3-sulfopropyl)acridinium, and 1,4-diphenylbenzene. At 20 degrees C, the fluorescence lifetimes vary from 89 ps to 31.2 ns, depending on fluorescent dye and solvent, which is a useful range for modern pico- and nanosecond time-domain or mega- to gigahertz frequency-domain instrumentation. The decay times are independent of the excitation and emission wavelengths. Dependent on the structure of the dye and ... [truncated at 150 words]
Calderon-Arnulphi M, Alaraj AM, Amin-Hanjani S, Mantulin WW, Polzonetti CM, Gratton E, Charbel FT.
Detection of cerebral ischemia in neurovascular surgery using quantitative frequency-domain near-infrared spectroscopy.
J Neurosurg. 2007; 106(2): 283-290.OBJECT. There is great value in monitoring for signs of ischemia during neurovascular procedures. Current intraoperative monitoring techniques provide real-time feedback with limited accuracy. Quantitative frequency-domain near-infrared spectroscopy (Q-NIRS) allows measurement of tissue oxyhemoglobin (HbO2), deoxyhemoglobin (HHb), and total hemoglobin (tHb) concentrations and brain tissue oxygen saturation (SO2), which could be useful when monitoring for evidence of intraoperative ischemia.
METHODS. Using Q-NIRS, the authors monitored 25 neurovascular procedures including aneurysm clip placement, arteriovenous malformation resection, carotid endarterectomy, superficial temporal artery–middle cerebral artery (MCA) bypass surgery, external carotid artery–MCA bypass surgery, encephaloduromyosynangiosis, and balloon occlusion testing. The Q-NIRS technology provides measurable cerebral oxygenation values independent from those of the scalp tissue. Thus, alterations in the variables measured with Q-NIRS quantitatively reflect cerebral tissue perfusion. Bilateral monitoring was performed in all cases. Five of the patients exhibited evidence of clinical ischemic events during the procedures. One patient suffered blood loss with systemic hypotension ... [truncated at 150 words]
Tanner K, Beitel E, D'Amico E, Mantulin WW, Gratton E.
Effects of vasodilation on intrinsic optical signals in the mammalian brain: a phantom study.
J Biomed Opt. 2007; 11(6): 064020.Using a broadband spectral technique, we recently showed [J. Biomed. Opt. 10, 064009 (2005)] that during visual stimulation of the cat brain there were not only changes in oxy- and deoxyhemoglobin levels, reminiscent of the optical blood oxygenation level dependence (BOLD) effect reported in humans, but also the apparent water content of the tissue and the optical scattering contribution decreased during stimulation. These relatively fast changes (in seconds) in water tissue content are difficult to explain in physiological terms. We developed a simple model to explain how local vasodilation, which occurs as a result of the stimulation, could cause this apparent change in water content. We show that in a phantom model we can obtain spectral effects similar to those observed in the cat brain such as the apparent decrease of the water spectral component without changing the water content of the bath in which the phantom measurements were performed. ... [truncated at 150 words]
Hanson KM, Gratton E, Bardeen CJ.
Sunscreen enhancement of UV-induced reactive oxygen species in the skin.
Free Radic Biol Med. 2006; 41(8): 1205-12.The number of UV-induced (20 mJ cm(-2)) reactive oxygen species (ROS) generated in nucleated epidermis is dependent upon the length of time the UV filter octocrylene, octylmethoxycinnamate, or benzophenone-3 remains on the skin surface. Two-photon fluorescence images acquired immediately after application of each formulation (2 mg cm(-2)) to the skin surface show that the number of ROS produced is dramatically reduced relative to the skin-UV filter control. After each UV filter remains on the skin surface for t=20 min, the number of ROS generated increases, although it remains below the number generated in the control. By t=60 min, the filters generate ROS above the control. The data show that when all three of the UV filters penetrate into the nucleated layers, the level of ROS increases above that produced naturally by epidermal chromophores under UV illumination.
Nicolini C, Celli A, Gratton E, Winter R.
Pressure tuning of the morphology of heterogeneous lipid vesicles: a two-photon-excitation fluorescence microscopy study.
Biophys J. 2006; 91(8): 2936-42. PMCID: PMC1578477We used a technique that allows us to visualize local and morphological changes of the membrane of more component giant unilamellar vesicles due to high pressure perturbation. Under these conditions, thermally induced processes are largely suppressed, and the bending rigidity and line tension are influenced by pressure-induced changes in lipid molecular packing and shape only. We studied the effect of pressure on the lateral organization and morphology of the model raft system DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)/sphingomyelin/cholesterol as well as of the fluid mixture POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine)/DLPC (1,2-dilauroyl-sn-glycero-3-phosphocholine) by two-photon excitation fluorescence microscopy. The pressure-dependent experiments were carried out using a sample cell made from a thin fused silica capillary. The use of Laurdan as fluorescence label allowed us to also follow the lipid phase state by calculating the generalized polarization (GP) values of the vesicles and extracting their average value. During the compression cycle, a reduction in the volume of the vesicles is ... [truncated at 150 words]
Eckhoff DA, Stuart JN, Sutin JDB, Sweedler JV, Gratton E.
Capillary electrophoresis of ultrasmall carboxylate functionalized silicon nanoparticles.
J Chem Phys. 2006; 125(8): 081103.Capillary electrophoresis is used to separate ultrasmall ( approximately 1 nm) carboxylate functionalized Si nanoparticles (Si-np-COO(-)) prepared via hydrosilylation with an omega-ester 1-alkene. The electropherograms show a monodisperse Si core size with one or two carboxylate groups added to the surface. On-column detection of their laser-induced fluorescence demonstrates that the individual Si-np-COO(-) have narrow emissions (full width at half maximum = 30-40 nm) with a nearly symmetric lineshape. Preparative scale electrophoresis should be a viable route for purification of the Si-np-COO(-) for further study and future applications.
Ragan TM, Huang H, So PTC, Gratton E.
3D particle tracking on a two-photon microscope.
J Fluoresc. 2006; 16(3): 325-36.A 3D single-particle-tracking (SPT) system was developed based on two-photon excitation fluorescence microscopy that can track the motion of particles in three dimensions over a range of 100 mum and with a bandwidth up to 30 Hz. We have implemented two different techniques employing feedback control. The first technique scans a small volume around a particle to build up a volumetric image that is then used to determine the particle's position. The second technique scans only a single plane but utilizes optical aberrations that have been introduced into the optical system that break the axial symmetry of the point spread function and serve as an indicator of the particle's axial position. We verified the performance of the instrument by tracking particles in well-characterized models systems. We then studied the 3D viscoelastic mechanical response of 293 kidney cells using the techniques. Force was applied to the cells, by using a magnetic ... [truncated at 150 words]
Lee NY, Hazlett TL, Koland JG.
Structure and dynamics of the epidermal growth factor receptor C-terminal phosphorylation domain.
Protein Sci. 2006; 15(5): 1142-52. PMCID: PMC2242510The C-terminal phosphorylation domain of the epidermal growth factor receptor is believed to regulate protein kinase activity as well as mediate the assembly of signal transduction complexes. The structure and dynamics of this proposed autoregulatory domain were examined by labeling the extreme C terminus of the EGFR intracellular domain (ICD) with an extrinsic fluorophore. Fluorescence anisotropy decay analysis of the nonphosphorylated EGFR-ICD yielded two rotational correlation times: a longer time, consistent with the global rotational motion of a 60- to 70-kDa protein with an elongated globular conformation, and a shorter time, presumably contributed by segmental motion near the fluorophore. A C-terminally truncated form of EGFR-ICD yielded a slow component consistent with the rotational motion of the 38-kDa kinase core. These findings suggested a structural arrangement of the EGFR-ICD in which the C-terminal phosphorylation domain interacts with the kinase core to move as an extended structure. A marked reduction in the ... [truncated at 150 words]
Rogozhina EV, Eckhoff DA, Gratton E, Braun PV.
Carboxyl functionalization of ultrasmall luminescent silicon nanoparticles through thermal hydrosilylation.
J Mater Chem. 2006; 16: 1421-1430.Functionalization of ultrasmall semiconductor nanoparticles to develop new luminescent probes that are optically bright, stable in aqueous environments, and sized comparably to small organic fluorophores would be of considerable utility for myriad applications in biology. Here, we report one of the first examples of thermal hydrosilylation between a bi-functional alkene and ultrasmall (~1 nm) H-passivated silicon nanoparticles (Si-np-H) to prepare strongly luminescent, water stable, carboxyl functionalized nanoparticles (Si-np-COOH). Nuclear magnetic resonance, infrared absorption spectroscopy (FTIR), size exclusion chromatography (SEC), and photoluminescence spectroscopy are used to characterize the Si-np dispersions. Based on the SEC and FTIR data, a reaction scheme is proposed to account for side products formed through a free radical cross-linking mechanism. The Si-np-COOH may find use in applications such as biomolecular labeling and biological imaging.
Nicolini C, Baranski J, Schlummer S, Palomo J, Lumbierres-Burgues M, Kahms M, Kuhlmann J, Sánchez SA, Gratton E, Waldmann H, Winter R.
Visualizing association of N-ras in lipid microdomains: influence of domain structure and interfacial adsorption.
J Am Chem Soc. 2006; 128(1): 192-201.In this study, two-photon fluorescence microscopy on giant unilamellar vesicles and tapping-mode atomic force microscopy (AFM) are applied to follow the insertion of a fluorescently (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene, BODIPY) labeled and completely lipidated (hexadecylated and farnesylated) N-Ras protein into heterogeneous lipid bilayer systems. The bilayers consist of the canonical raft mixture 1-palmitoyl-2-oleoylphosphatidylcholine (POPC), sphingomyelin, and cholesterol, which-depending on the concentration of the constituents-separates into liquid-disordered (l(d)), liquid-ordered (l(o)), and solid-ordered (s(o)) phases. The results provide direct evidence that partitioning of N-Ras occurs preferentially into liquid-disordered lipid domains, which is also reflected in a faster kinetics of incorporation into the fluid lipid bilayers. The phase sequence of preferential binding of N-Ras to mixed-domain lipid vesicles is l(d) > l(o) >> s(o). Intriguingly, we detect, using the better spatial resolution of AFM, also a large proportion of the lipidated protein located at the l(d)/l(o) phase boundary, thus leading to a favorable decrease in line ... [truncated at 150 words]
Simonson PD, D'Amico E, Gratton E.
Modulation of an optical needle's reflectivity alters the average photon path through scattering media.
J Biomed Opt. 2006; 11(1): 014023.We introduce the concept of deliberate placement of absorbers to alter the average path of photons through tissue for a biomedical optical device. By changing the reflectivity of a needle that separates a source and detector, the average photon path through a turbid medium can be changed. Totally reflective needles have photon scattering density functions similar to a point source and detector in an infinite medium. An absorbing needle moves the average photon path of photons that reach the detector away from the needle. Thus, by modulating the reflectivity of the needle, it is possible to modify the sensitive volume, and simple tomography data should be possible. These results are confirmed by Monte Carlo simulations and experiment. Experiments include moving a black target relative to an optical "needle" and measuring the resulting intensity and phase lag of light reaching a detector at the distal end of the needle.
Levi V, Gelfand VI, Serpinskaya AS, Gratton E.
Melanosomes transported by myosin-V in Xenopus melanophores perform slow 35 nm steps.
Biophys J. 2006; 90(1): L07-9. PMCID: PMC1367043We studied the motion of pigment organelles driven by myosin-V in Xenopus melanophores using a tracking technique with precision of 2 nm. The organelle trajectories showed occasional steps with a distribution centered at 35 nm and a standard deviation of 13 nm, in agreement with the step size of myosin-V determined in vitro. In contrast, trajectories of melanosomes in cells expressing a dominant negative form of myosin-V did not show steps. The step duration was in the range 20-80 ms, slower than what it would be expected from in vitro results. We speculate that the cytoplasm high viscosity may affect significantly the melanosomes' motion.
Levi V, Serpinskaya AS, Gratton E, Gelfand VI.
Organelle transport along microtubules in Xenopus melanophores: evidence for cooperation between multiple motors.
Biophys J. 2006; 90(1): 318-27. PMCID: PMC1367030Xenopus melanophores have pigment organelles or melanosomes which, in response to hormones, disperse in the cytoplasm or aggregate in the perinuclear region. Melanosomes are transported by microtubule motors, kinesin-2 and cytoplasmic dynein, and an actin motor, myosin-V. We explored the regulation of melanosome transport along microtubules in vivo by using a new fast-tracking routine, which determines the melanosome position every 10 ms with 2-nm precision. The velocity distribution of melanosomes transported by cytoplasmic dynein or kinesin-2 under conditions of aggregation and dispersion presented several peaks and could not be fit with a single Gaussian function. We postulated that the melanosome velocity depends linearly on the number of active motors. According to this model, one to three dynein molecules transport each melanosome in the minus-end direction. The transport in the plus-end direction is mainly driven by one to two copies of kinesin-2. The number of dyneins transporting a melanosome increases during ... [truncated at 150 words]
Gokhale NA, Abraham A, Digman MA, Gratton E, Cho W.
Phosphoinositide specificity of and mechanism of lipid domain formation by annexin A2-p11 heterotetramer.
J Biol Chem. 2005; 280(52): 42831-40.Annexin A2 is a phospholipid-binding protein that forms a heterotetramer (annexin II-p11 heterotetramer; A2t) with p11 (S100A10). It has been reported that annexin A2 is involved in binding to phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) and in inducing membrane microdomain formation. To understand the mechanisms underlying these findings, we determined the membrane binding properties of annexin A2 wild type and mutants both as monomer and as A2t. Our results from surface plasmon resonance analysis showed that A2t and annexin A2 has modest selectivity for PtdIns(4,5)P2 over other phosphoinositides, which is conferred by conserved basic residues, including Lys279 and Lys281, on the convex surface of annexin A2. Fluorescence microscopy measurements using giant unilamellar vesicles showed that A2t of wild type, but not (K279A)2-(p11)2 or (K281A)2-(p11)2, specifically induced the formation of 1-microm-sized PtdIns(4,5)P2 clusters, which were stabilized by cholesterol. Collectively, these studies elucidate the structural determinant of the PtdIns(4,5)P2 selectivity of A2t and suggest that ... [truncated at 150 words]
Redford GI, Majumdar ZK, Sutin JDB, Clegg RM.
Properties of microfluidic turbulent mixing revealed by fluorescence lifetime imaging.
J Chem Phys. 2005; 123(22): 224504.We present a new method of measurement based on fluorescence lifetime imaging that reveals molecular-scale details of the mixing process in a continuous-flow turbulent microfluidic reactor. Our data provide a glimpse of the cascade to the minimal eddy size, followed by rapid diffusion involving the smallest eddies for final mixing.
Levi V, Ruan Q, Plutz M, Belmont AS, Gratton E.
Chromatin dynamics in interphase cells revealed by tracking in a two-photon excitation microscope.
Biophys J. 2005; 89(6): 4275-85. PMCID: PMC1366992Increasing 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 approximately 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 approximately 150 nm lasting 0.3-2 s. During these jumps, the particle moved an average of four 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 mechanism driving ... [truncated at 150 words]
Majumdar ZK, Sutin JDB, Clegg RM.
Microfabricated continuous-flow, turbulent, microsecond mixer.
Rev Sci Instrum. 2005; 76(12): 125103.We present a microfabricated, continuous-flow, turbulent mixing device that can mix two or more fluids to complete homogeneity on the molecular scale in the microsecond range. The current design is compact, portable, relatively simple to fabricate, adaptable for various measurement techniques, and consumes small sample volumes. The entire mixing process is observable and we use this feature to characterize the dependence of the progress of mixing on the flow velocity. We present details of the mixer's construction and optical data acquisition using fluorescence. Because the mixer is constructed using microfabrication technology, it is inexpensive and alterations are easy to explore. We show that the dependence of mixing times and pressure drop on the flow velocity agree well with theoretical expectations for turbulent pipe flow.
Hickerson RP, Majumdar ZK, Baucom A, Clegg RM, Noller HF.
Measurement of internal movements within the 30 S ribosomal subunit using Förster resonance energy transfer.
J Mol Biol. 2005; 354(2): 459-72.We have used Förster resonance energy transfer (FRET) to study specific conformational changes in the Escherichia coli 30 S ribosomal subunit that occur upon association with the 50 S subunit. By measuring energy transfer between 13 different pairs of fluorescent probes attached to specific positions on 30 S subunit proteins, we have monitored changes in distance between different locations within the 30 S subunit in its free and 50 S-bound states. The measured distance changes provide restraints for modeling the movement that occurs within the 30 S subunit upon formation of the 70 S ribosome in solution. Treating the head, body, and platform domains of the 30 S subunit as simple rigid bodies, the lowest-energy solution converges on a model that satisfies each of the individual FRET restraints. In this model, the 30 S subunit head tilts towards the 50 S subunit, similar to the movement found in comparing 30 ... [truncated at 150 words]
Tanner K, D'Amico E, Kaczmarowski A, Kukreti S, Malpeli JG, Mantulin WW, Gratton E.
Spectrally resolved neurophotonics: a case report of hemodynamics and vascular components in the mammalian brain.
J Biomed Opt. 2005; 10(6): 064009.We developed a spectral technique that is independent of the light transport modality (diffusive or nondiffusive) to separate optical changes in scattering and absorption in the cat's brain due to the hemodynamic signal following visual stimulation. We observe changes in oxyhemoglobin and deoxyhemoglobin concentration signals during visual stimulation reminiscent of the functional magnetic resonance imaging (fMRI) blood oxygenation level dependence (BOLD) effect. Repeated measurements at different locations show that the observed changes are local rather than global. We also determine that there is an apparent large decrease in the water concentration and scattering coefficient during stimulation. We model the apparent change in water concentration on the separation of the optical signal from two tissue compartments. One opaque compartment is featureless (black), due to relatively large blood vessels. The other compartment is the rest of the tissue. When blood flow increases due to stimulation, the opaque compartment increases in volume, resulting ... [truncated at 150 words]
Eckhoff DA, Sutin JDB, Clegg RM, Gratton E, Rogozhina EV, Braun PV.
Optical characterization of ultrasmall Si nanoparticles prepared through electrochemical dispersion of bulk Si.
J Phys Chem B. 2005; 109(42): 19786-97.Studying the properties and stability of silicon nanoparticles (Si-np) in aqueous environments may lead to novel applications in biological systems. In this work, we use absorption and photoluminescence (PL) spectroscopy to characterize ultrasmall Si-np prepared through anodic etching and ultrasonic fractionation of a crystalline Si wafer. Their behavior is studied over time in 2-propanol and during treatments with water, NaOH, HCl, and H(2)O(2). The observed population is divided into two types of material: bright species consisting of well-etched Si-np, approximately 1 nm in diameter, and dark species derived from partially etched or aggregated Si structures. The dark material is seen by its scattering in the 2-propanol and water solutions and is largely removed via precipitation with the NaOH or HCl treatment. The bright material includes three distinct species with their respective emissions in the UV-B, UV-A, and hard-blue regions of the spectrum. The hard-blue PL is shown to have a ... [truncated at 150 words]
Majumdar ZK, Hickerson RP, Noller HF, Clegg RM.
Measurements of internal distance changes of the 30S ribosome using FRET with multiple donor-acceptor pairs: quantitative spectroscopic methods.
J Mol Biol. 2005; 351(5): 1123-45.We present analytical and experimental procedures for determining distance changes within the 30 S subunit of the Escherichia coli ribosome using Förster resonance energy transfer (FRET). We discuss ways to contend with complexities when using FRET to measure distance changes within large multi-subunit macromolecular complexes, such as the ribosome. 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. We also discuss 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. Thirteen dye-pair locations with different distances using 18 independent FRET pairs conjugated to ... [truncated at 150 words]
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Polar plot representation for frequency-domain analysis of fluorescence lifetimes.
J Fluoresc. 2005; 15(5): 805-15.We present applications of polar plots for analyzing fluorescence lifetime data acquired in the frequency domain. This graphical, analytical method is especially useful for rapid FLIM measurements. The usual method for sorting out and determining the underlying lifetime components from a complex fluorescence signal is to carry out the measurement at multiple frequencies. When it is not possible to measure at more than one frequency, such as rapid lifetime imaging, specific features of the polar plot analysis yield valuable information, and provide a diagnostic visualization of the participating fluorescent species underlying a complex lifetime distributions. Data are presented where this polar plot presentation is useful to derive valuable, unique information about the underlying component distributions. We also discuss artifacts of photolysis and how this method can also be applied to samples where each fluorescence species shows a continuous distribution of lifetimes. Polar plots of frequency-domain data are commonly used for ... [truncated at 150 words]
Digman MA, Brown CM, Sengupta P, Wiseman PW, Horwitz AR, Gratton E.
Measuring fast dynamics in solutions and cells with a laser scanning microscope.
Biophys J. 2005; 89(2): 1317-27. PMCID: PMC1366616Single-point fluorescence correlation spectroscopy (FCS) allows measurements of fast diffusion and dynamic processes in the microsecond-to-millisecond time range. For measurements on living cells, image correlation spectroscopy (ICS) and temporal ICS extend the FCS approach to diffusion times as long as seconds to minutes and simultaneously provide spatially resolved dynamic information. However, ICS is limited to very slow dynamics due to the frame acquisition rate. Here we develop novel extensions to ICS that probe spatial correlations in previously inaccessible temporal windows. We show that using standard laser confocal imaging techniques (raster-scan mode) not only can we reach the temporal scales of single-point FCS, but also have the advantages of ICS in providing spatial information. This novel method, called raster image correlation spectroscopy (RICS), rapidly measures during the scan many focal points within the cell providing the same concentration and dynamic information of FCS as well as information on the spatial correlation ... [truncated at 150 words]
Arnulphi C, Sánchez SA, Tricerri MA, Gratton E, Jonas A.
Interaction of human apolipoprotein A-I with model membranes exhibiting lipid domains.
Biophys J. 2005; 89(1): 285-95. PMCID: PMC1366526Several mechanisms for cell cholesterol efflux have been proposed, including membrane microsolubilization, suggesting that the existence of specific domains could enhance the transfer of lipids to apolipoproteins. In this work isothermal titration calorimetry, circular dichroism spectroscopy, and two-photon microscopy are used to study the interaction of lipid-free apolipoprotein A-I (apoA-I) with small unilamellar vesicles (SUVs) of 1-palmitoyl, 2-oleoyl phosphatidylcholine (POPC) and sphingomyelin (SM), with and without cholesterol. Below 30 degrees C the calorimetric results show that apoA-I interaction with POPC/SM SUVs produces an exothermic reaction, characterized as nonclassical hydrophobic binding. The heat capacity change (DeltaCp degrees ) is small and positive, whereas it was larger and negative for pure POPC bilayers, in the absence of SM. Inclusion of cholesterol in the membranes induces changes in the observed thermodynamic pattern of binding and counteracts the formation of alpha-helices in the protein. Above 30 degrees C the reactions are endothermic. Giant unilamellar ... [truncated at 150 words]
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Lipid-protein interactions revealed by two-photon microscopy and fluorescence correlation spectroscopy.
Acc Chem Res. 2005; 38(6): 469-77.Cellular processes involve a multitude of chemical reactions that must be kept in delicate equilibrium to maintain cellular homeostasis. Powerful biophysical techniques are needed to measure the localization and concentration of target molecules as well as to quantify complex molecular processes in model and in vivo systems. Two-photon microscopy and fluorescence correlation spectroscopy (FCS) can measure association and dynamics of appropriate molecules under equilibrium conditions. FCS provides information on motility (diffusion coefficients), concentration (number of particles), association (molecular brightness), and localization (image) of the target molecules. All of this information, in conjunction with computational modeling techniques, can help us to better understand the network of complex molecular interactions, which are at the basis of cellular processes. Fluorescence imaging techniques add the beauty of visualization to the scientific information. Photons emitted by a fluorescent dye are digitized, and the associated spatial information and intensity can be translated into different colors and ... [truncated at 150 words]
Stahelin RV, Digman MA, Medkova M, Ananthanarayanan B, Melowic HR, Rafter JD, Cho W.
Diacylglycerol-induced membrane targeting and activation of protein kinase Cε. Mechanistic differences between protein kinases Cδ and Cε.
J Biol Chem. 2005; 280(20): 19784-19793.Two novel protein kinases C (PKC), PKCδ and PKCε, have been reported to have opposing functions in some mammalian cells. To understand the basis of their distinct cellular functions and regulation, we investigated the mechanism of in vitro and cellular sn-1,2-diacylglycerol (DAG)-mediated membrane binding of PKCε and compared it with that of PKCδ. The regulatory domains of novel PKC contain a C2 domain and a tandem repeat of C1 domains (C1A and C1B), which have been identified as the interaction site for DAG and phorbol ester. Isothermal titration calorimetry and surface plasmon resonance measurements showed that isolated C1A and C1B domains of PKCε have comparably high affinities for DAG and phorbol ester. Furthermore, in vitro activity and membrane binding analyses of PKCε mutants showed that both the C1A and C1B domains play a role in the DAG-induced membrane binding and activation of PKCε. The C1 domains of PKCε are not ... [truncated at 150 words]
Digman MA, Sengupta P, Wiseman PW, Brown CM, Horwitz AR, Gratton E.
Fluctuation correlation spectroscopy with a laser-scanning microscope: exploiting the hidden time structure.
Biophys J. 2005; 88(5): L33-6. PMCID: PMC1305524Images obtained with a laser-scanning microscope contain a time structure that can be exploited to measure fast dynamics of molecules in solution and in cells. The spatial correlation approach provides a simple algorithm to extract this information. We describe the analysis used to process laser-scanning images of solutions and cells to obtain molecular diffusion constant in the microsecond to second timescale.
Tricerri MA, Toledo JD, Sánchez SA, Hazlett TL, Gratton E, Jonas A, Garda HA.
Visualization and analysis of apolipoprotein A-I interaction with binary phospholipid bilayers.
J Lipid Res. 2005; 46(4): 669-78.Apolipoprotein A-I (apoA-I) interaction with specific cell lipid domains was suggested to trigger cholesterol and phospholipid efflux. We analyzed here apoA-I interaction with dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) bilayers at a temperature showing phase coexistence. Solid and liquid-crystalline domains were visualized by two-photon fluorescence microscopy on giant unilamellar vesicles (GUVs) labeled with 6-dodecanoyl-2-dimethyl-amino-naphthalene (Laurdan). A decrease of vesicle size was detected as long as they were incubated with lipid-free apoA-I, together with a shape deformation and a relative enrichment in DSPC. Selective lipid removal mediated by apoA-I from different domains was followed in real time by changes in the Laurdan generalized polarization. The data show a selective interaction of apoA-I with liquid-crystalline domains, from which it removes lipids, at a molar ratio similar to the domain compositions. Next, apoA-I was incubated with DMPC/DSPC small unilamellar vesicles, and products were isolated and quantified. Protein solubilized both lipids but formed complexes relatively enriched in the ... [truncated at 150 words]
Levi V, Ruan Q, Gratton E.
3-D particle tracking in a two-photon microscope: application to the study of molecular dynamics in cells.
Biophys J. 2005; 88(4): 2919-28. PMCID: PMC1305386We developed a method for tracking particles in three dimensions designed for a two-photon microscope, which holds great promise to study cellular processes because of low photodamage, efficient background rejection, and improved depth discrimination. During a standard cycle of the tracking routine (32 ms), the laser beam traces four circular orbits surrounding the particle in two z planes above and below the particle. The radius of the orbits is half of the x,y-width of the point spread function, and the distance between the z planes is the z-width of the point spread function. The z-position is adjusted by moving the objective with a piezoelectric-nanopositioner. The particle position is calculated on the fly from the intensity profile obtained during the cycle, and these coordinates are used to set the scanning center for the next cycle. Applying this method, we were able to follow the motion of 500-nm diameter fluorescent polystyrene microspheres ... [truncated at 150 words]
Gratton E, Toronov VY, Wolf U, Wolf M, Webb A.
Measurement of brain activity by near-infrared light.
J Biomed Opt. 2005; 10(1): 11008.We review our most recent results on near-IR studies of human brain activity, which have been evolving in two directions: detection of neuronal signals and measurements of functional hemodynamics. We discuss results obtained so far, describing in detail the techniques we developed for detecting neuronal activity, and presenting results of a study that, as we believe, confirms the feasibility of neuronal signal detection. We review our results on near-IR measurements of cerebral hemodynamics, which are performed simultaneously with functional magnetic resonance imaging (MRI) These results confirm the cerebral origin of hemodynamic signals measured by optical techniques on the surface of the head. We also show how near-IR methods can be used to study the underlying physiology of functional MRI signals.
Inoue M, Digman MA, 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.
J Biol Chem. 2004; 279(47): 49160-71.In dietary potassium deficiency there is a decrease in the transport activity of the type IIa sodium/phosphate cotransporter protein (NaPi) despite 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 activity have not been determined. We determined if in potassium deficiency NaPi is increasingly partitioned in cholesterol-, sphingomyelin-, and glycosphingolipid-enriched microdomains of the apical membrane and if the increased presence of NaPi in these microdomains modulates its activity. By using a detergent-free density gradient flotation technique, we found that 80% of the apical membrane NaPi partitions into the low density cholesterol-, sphingomyelin-, and GM1-enriched fractions characterized as "lipid raft" fractions. In potassium deficiency, a higher proportion of NaPi was localized in the lipid raft fractions. By combining fluorescence correlation spectroscopy and photon counting histogram ... [truncated at 150 words]
Barcellona ML, Gammon S, Hazlett TL, Digman MA, Gratton E.
Polarized fluorescence correlation spectroscopy of DNA-DAPI complexes.
Microsc Res Tech. 2004; 65(4-5): 205-17.We discuss the use of fluorescence correlation spectroscopy for the measurement of relatively slow rotations of large macromolecules in solution or attached to other macromolecular structures. We present simulations and experimental results to illustrate the range of rotational correlation times and diffusion times that the technique can analyze. In particular, we examine various methods to analyze the polarization fluctuation data. We have found that by first constructing the polarization function and then calculating the autocorrelation function, we can obtain the rotational motion of the molecule with very little interference from the lateral diffusion of the macromolecule, as long as the rotational diffusion is significantly faster than the lateral diffusion. Surprisingly, for common 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 are present at any instant of ... [truncated at 150 words]
Masters BR, So PTC, Buehler C, Barry NP, Sutin JDB, Mantulin WW, Gratton E.
Mitigating thermal mechanical damage potential during two-photon dermal imaging.
J Biomed Opt. 2004; 9(6): 1265-70.Two-photon excitation fluorescence microscopy allows in vivo high-resolution imaging of human skin structure and biochemistry with a penetration depth over 100 microm. The major damage mechanism during two-photon skin imaging is associated with the formation of cavitation at the epidermal-dermal junction, which results in thermal mechanical damage of the tissue. In this report, we verify that this damage mechanism is of thermal origin and is associated with one-photon absorption of infrared excitation light by melanin granules present in the epidermal-dermal junction. The thermal mechanical damage threshold for selected Caucasian skin specimens from a skin bank as a function of laser pulse energy and repetition rate has been determined. The experimentally established thermal mechanical damage threshold is consistent with a simple heat diffusion model for skin under femtosecond pulse laser illumination. Minimizing thermal mechanical damage is vital for the potential use of two-photon imaging in noninvasive optical biopsy of human skin ... [truncated at 150 words]
Safonova LP, Michalos A, Wolf U, Wolf M, Hueber DM, Choi JH, Gupta R, Polzonetti CM, Mantulin WW, Gratton E.
Age-correlated changes in cerebral hemodynamics assessed by near-infrared spectroscopy.
Arch Gerontol Geriatr. 2004; 39(3): 207-25.Cerebral hemodynamic responses due to normal aging may interfere with hormonal changes, drug therapy, diseases, life style, and other factors. Age-correlated alterations in cerebral vasculature and autoregulatory mechanisms are the subject of interest in many studies. Near-infrared spectroscopy (NIRS) is widely used for monitoring cerebral hemodynamics and oxygenation changes at the level of small vessels. We believe that the compensatory ability of cerebral arterioles under hypoxic conditions and the dilatatory ability of cerebral vessels due to vasomotion may decline with normal aging. To test this hypothesis we used frequency-domain NIRS to measure changes in cerebral tissue oxygenation and oxy- and deoxy-hemoglobin concentrations caused by hypoxia during breath holding. We also assessed cerebral vasomotion during profound relaxation. Thirty seven healthy volunteers, 12 females and 25 males, ranging from 22 to 56 years of age (mean age 35 ± 11 years) participated in the study. We observed age-correlated changes in the cerebral ... [truncated at 150 words]
Hart AJ, Slocum AH, Sutin JDB.
Segmented and shielded structures for reduction of thermal expansion-induced tilt errors.
Precision Eng. 2004; 28(4): 443-458.The design and prototype tests of a segmented and shielded instrumentation structure, based on tube modules connected by canoe ball type kinematic couplings, are presented with application to a high-precision microscope for biological science experiments. The segmented tube design is shown to be significantly less sensitive to deformation from uneven thermal disturbances than a single tube structure. The gaps between the tube segments relieve thermal strains and restrict axial heat conduction, and the large-radius kinematic couplings provide high thermal resistance interconnects without significantly degrading structural stiffness. Simulation results, based on two- and three-dimensional models of heat conduction, are verified with experimental measurements of prototype structures and are used to predict the dependence of the tilt error on the geometry, boundary conditions, and material properties of the tubular segments. Adding layers of low-cost foam insulation and thin metal shielding to the walls of the structure further improves performance. Kinematic couplings could ... [truncated at 150 words]
Czeslik C, Jackler G, Hazlett TL, Gratton E, Steitz R, Wittemann A, Ballauff M.
Salt-induced protein resistance of polyelectrolyte brushes studied using fluorescence correlation spectroscopy and neutron reflectometry.
Phys Chem Chem Phys. 2004; 6: 5557-5563.We used two-photon excitation fluorescence correlation spectroscopy (FCS) and neutron reflectometry to study in situ the effect of salt concentration on the degree of protein binding to 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. Spherical PAA brush particles were used in the FCS experiments, whereas a planar PAA brush served as protein substrate in the neutron reflectometry experiments. It has been found that BSA binds strongly to both the spherical and the planar PAA 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 the PAA brush. However, the analysis of the FCS data reveals that the protein affinity of the spherical PAA brush particles decreases drastically when increasing ... [truncated at 150 words]
Ruan Q, Cheng MA, Levi M, Gratton E, Mantulin WW.
Spatial-temporal studies of membrane dynamics: scanning fluorescence correlation spectroscopy (SFCS).
Biophys J. 2004; 87(2): 1260-7. PMCID: PMC1304464Giant unilamellar vesicles (GUVs) have been widely used as a model membrane system to study membrane organization, dynamics, and protein-membrane interactions. Most recent studies have relied on imaging methods, which require good contrast for image resolution. Multiple sequential image processing only detects slow components of membrane dynamics. We have developed a new fluorescence correlation spectroscopy (FCS) technique, termed scanning FCS (i.e., SFCS), which 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. The scan rate is fast compared to the diffusion of the membrane proteins and even small molecules in the GUVs. Scanning FCS outputs a "carpet" of timed fluorescence intensity fluctuations at specific points along the scan. In this study, GUVs were assembled from rat kidney brush border membranes, which included the integral membrane proteins. Scanning FCS measurements on GUVs allowed ... [truncated at 150 words]
Janosch S, Nicolini C, Ludolph B, Peters C, Völkert M, Hazlett TL, Gratton E, Waldmann H, Winter R.
Partitioning of dual-lipidated peptides into membrane microdomains: lipid sorting vs peptide aggregation.
J Am Chem Soc. 2004; 126(24): 7496-503.The lateral membrane organization and phase behavior of the lipid mixture DMPC(di-C(14))/DSPC(di-C(18))/cholesterol (0-33 mol %) with and without an incorporated fluorescence-labeled palmitoyl/farnesyl dual-lipidated peptide, BODIPY-Gly-Cys(Pal)-Met-Gly-Leu-Pro-Cys(Far)-OMe, which represents a membrane recognition model system for Ras proteins, was studied by two-photon excitation fluorescence microscopy. Measurements were performed on giant unilamellar vesicles (GUVs) over a large temperature range, ranging from 30 to 80 degrees C to cover different lipid phase states (all-gel, fluid/gel, liquid-ordered, all-fluid). At temperatures where the fluid-gel coexistence region of the pure binary phospholipid system occurs, large-scale concentration fluctuations appear. Incorporation of cholesterol levels up to 33 mol % leads to a significant increase of conformational order in the membrane system and a reduction of large domain structures. Adding the peptide leads to dramatic changes in the lateral organization of the membrane. With cholesterol present, a phase separation is induced by a lipid sorting mechanism owing to the high affinity ... [truncated at 150 words]
Borovikov YS, Dedova IV, Remedios CGd, Vikhoreva NN, Vikhorev PG, Avrova SV, Hazlett TL, van der Meer BW.
Fluorescence depolarization of actin filaments in reconstructed myofibers: the effect of S1 or pPDM-S1 on movements of distinct areas of actin.
Biophys J. 2004; 86(5): 3020-9. PMCID: PMC1304168Fluorescence polarization measurements were used to study changes in the orientation and order of different sites on actin monomers within muscle thin filaments during weak or strong binding states with myosin subfragment-1. Ghost muscle fibers were supplemented with actin monomers specifically labeled with different fluorescent probes at Cys-10, Gln-41, Lys-61, Lys-373, Cys-374, and the nucleotide binding site. We also used fluorescent phalloidin as a probe near the filament axis. Changes in the orientation of the fluorophores depend not only on the state of acto-myosin binding but also on the location of the fluorescent probes. We observed changes in polarization (i.e., orientation) for those fluorophores attached at the sites directly involved in myosin binding (and located at high radii from the filament axis) that were contrary to the fluorophores located at the sites close to the axis of thin filament. These altered probe orientations suggest that myosin binding alters the conformation ... [truncated at 150 words]
Rao S, Sutin JDB, Clegg RM, Gratton E, Nayfeh MH, Habbal S, Tsolakidis A, Martin RM.
Excited states of tetrahedral single-core Si29 nanoparticles.
Phys Rev B. 2004; 69(20): 205319.We dispersed bulk crystalline Si into identical hydrogenated nanoparticles with negligible impurities and defects, which provide the opportunity for detailed comparison between measurement and theory. The UV photoluminescence of a dispersion of 1 nm silicon particles was studied. Distinct bands appear in the emission spectra with the lowest peaks in wavelength identified to be at 400, 360, and 310 nm with optimal excitation at 3.7, 4.0, and 4.6 eV, respectively. The multiple photoluminescence bands are analyzed in terms of the molecularlike energy levels of one bulklike and two nonbulklike reconstruction configurations of the filled fullerene single-core Si29H24, calculated by quantum Monte Carlo calculations and by time-dependent density functional theory. The measured bands are in close agreement with the excited states of the ideal bulklike configuration. However, there is a possibility that some of the observed bands might originate from the nonbulklike reconstructions. The Stokes shifts are discussed in terms of ... [truncated at 150 words]
Hohng S, Wilson TJ, Tan E, Clegg RM, Lilley DMJ, Ha T.
Conformational flexibility of four-way junctions in RNA.
J Mol Biol. 2004; 336(1): 69-79.Helical junctions are common architectural features in RNA. They are particularly important in autonomously folding molecules, as exemplified by the hairpin ribozyme. We have used single-molecule fluorescence spectroscopy to study the dynamic properties of the perfect (4H) four-way helical junction derived from the hairpin ribozyme. In the presence of Mg(2+), the junction samples parallel and antiparallel conformations and both stacking conformers, with a bias towards one antiparallel stacking conformer. There is continual interconversion between the forms, such that there are several transitions per second under physiological conditions. Our data suggest that interconversion proceeds via an open intermediate with reduced cation binding in which coaxial stacking between helices is disrupted. The rate of interconversion becomes slower at higher Mg(2+) concentrations, yet the activation barrier decreases under these conditions, indicating that entropic effects are important. Transitions also occur in the presence of Na(+) only; however, the coaxial stacking appears incomplete under these ... [truncated at 150 words]
Czeslik C, Jansen RP, Ballauff M, Wittemann A, Royer CA, Gratton E, Hazlett TL.
Mechanism of protein binding to spherical polyelectrolyte brushes studied in situ using two-photon excitation fluorescence fluctuation spectroscopy.
Phys Rev E. 2004; 69(2): 021401.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. Whereas fluorescence fluctuations are traditionally evaluated by calculating the autocorrelation function (fluorescence correlation spectroscopy), a complementary PCH analysis has been performed in this study which is advantageous when particle concentrations of a multicomponent system are of interest and the particles can be distinguished through particle brightness differences. The binding of two proteins, staphylococcal nuclease (SNase) and bovine serum albumin (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 regardless of the protein charge. When the ionic strength of the solution is raised to 100 mM, ... [truncated at 150 words]
Lee EH, Faulhaber D, Hanson KM, Ding W, Peters S, Kodali S, Granstein RD.
Dietary Lutein reduces ultraviolet radiation-induced inflammation and immunosuppression.
J Invest Dermatol. 2004; 122(2): 510-517.Ultraviolet radiation (UVR) promotes skin cancer development by mutagenic, immunosuppressive, and oxidative-stress-inducing mechanisms; however, certain antioxidants may counteract and prevent UVR-induced photodamage. Lutein is a xanthophyll carotenoid with potent antioxidant activity. Because reactive oxygen species (ROS) are believed to have a role in UVR-induced skin damage, we investigated whether lutein can modify UVR effects including the tissue swelling response to midrange UVR (280–320 nm, ultraviolet B (UVB) radiation) and UVB suppression of contact hypersensitivity (CHS) in both the local and the systemic models of UV-induced immunosuppression. We found that compared to mice fed the standard laboratory diet, mice fed dietary lutein demonstrated significant inhibition of ear swelling owing to UVB radiation. Mice exposed to 1700 J per m2 UVB radiation four times at daily intervals and then sensitized to dinitrofluorobenzene at the site of irradiation showed a decreased CHS response upon challenge. This suppression by UVB radiation was significantly inhibited ... [truncated at 150 words]
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The vital contributions of Perrin and Förster.
Biophotonics Int. 2004; 11(9): 42-45.The theories of the pioneers of energy transfer, which provides useful insight into the mechanism and leads to a better appreciation of the broad applicability of the technique, are discussed. Förster resonance energy transfer (FRET) is used extensively to monitor macromolecule interactions, to determine molecular distances and to follow conformational changes. Perrin demonstrated that if the molecules were separated within the nonradiating near field of the donor dipole, energy could be transferred to the acceptor molecule. He assumed that identical molecules have exactly the same fundamental oscillation frequency, that is, the two Hertzian oscillators are in exact resonance.
Choi JH, Wolf M, Toronov VY, Wolf U, Polzonetti CM, Hueber DM, Safonova LP, Gupta R, Michalos A, Mantulin WW, Gratton E.
Noninvasive determination of the optical properties of adult brain: near-infrared spectroscopy approach.
J Biomed Opt. 2004; 9(1): 221-9.The basic parameters for physiological measurements provided by near-infrared spectroscopy are the local absorption and scattering coefficients. For the adult human head, they have been difficult to measure noninvasively because of the layered structure of the head. The results of measurements of absorption and reduced scattering coefficients through the forehead on 30 adult volunteers using a multidistance frequency domain method are reported. The optode separation distance ranged from 10 to 80 mm and measurements were recorded at 758 and 830 nm. The measured absorption and reduced scattering coefficients of the forehead were used to evaluate the hemoglobin content in the scalp and brain as well as cerebral oxygen saturation. We found that cerebral oxygenation was relatively narrowly distributed within the subject group (the standard deviation was about 3% for scalp and 6% for brain, respectively), whereas hemoglobin concentrations had a relatively broader distribution. We found that as the optode distance ... [truncated at 150 words]
Kis-Petiková K, Gratton E.
Distance measurement by circular scanning of the excitation beam in the two-photon microscope.
Microsc Res Tech. 2004; 63(1): 34-49.We developed a method to measure relative distances with nanometer accuracy of fluorescent particles of different color in a two-photon scanning fluorescence microscope, with two-channel photon counting detection. The method can be used in the 10-500 nm range, for distances below the resolution limit of standard far field microscopy. The proposed technique is more efficient than the methods using raster scanning. To achieve maximum sensitivity in the radial direction, the excitation beam is moved periodically in a circular orbit with a radius of the size of the point spread function. The phase and the modulation of the periodic fluorescence signal, calculated by fast Fourier transform, gives the phase and the radial distance of the particle from the center of scanning. The coordinates of particles are recovered simultaneously in the two channels and the relative distance is calculated in real time. Particles can be tracked by moving the center of scanning ... [truncated at 150 words]
Dedova IV, Avrova SV, Vikhoreva NN, Vikhorev PG, Hazlett TL, van der Meer BW, Remedios CGd, Borovikov YS.
Conformational changes of actin induced by strong or weak myosin subfragment-1 binding.
Tsitologiia. 2004; 46(8): 719-34.Movements of different areas of polypeptide chains within F-actin monomers induced by S1 or pPDM-S1 binding were studied by polarized fluorimetry. Thin filaments of ghost muscle were reconstructed by adding G-actin labeled with fluorescent probes attached alternatively to different sites of actin molecule. These sites were: Cys-374 labeled with 1,5-IAEDANS, TMRIA or 5-IAF; Lys-373 labeled with NBD-Cl; Lys-113 labeled with Alexa-488; Lys-61 labeled with FITC; Gln-41 labeled with DED and Cys-10 labeled with 1,5-IAEDANS, 5-IAF or fluorescein-maleimid. In addition, we used TRITC-, FITC-falloidin and e-ADP that were located, respectively, in filament groove and interdomain cleft. The data were analysed by model-dependent and model-independent methods (see appendixes). The orientation and mobility of fluorescent probes were significantly changed when actin and myosin interacted, depending on fluorophore location and binding site of actomyosin. Strong binding of S with actin leads to 1) a decrease in the orientation of oscillators of derivatives of falloidin ... [truncated at 150 words]
Sánchez SA, Brunet JE, Jameson DM, Lagos R, Monasterio O.
Tubulin equilibrium unfolding followed by time-resolved fluorescence and fluorescence correlation spectroscopy.
Protein Sci. 2004; 13(1): 81-88. PMCID: PMC2286518The pathway for the in vitro equilibrium unfolding of the tubulin heterodimer by guanidinium chloride (GdmCl) has been studied using several spectroscopic techniques, specifically circular dichroism (CD), two-photon Fluorescence Correlation Spectroscopy (FCS), and time-resolved fluorescence, including lifetime and dynamic polarization. The results show that tubulin unfolding is characterized by distinct processes that occur in different GdmCl concentration ranges. From 0 to 0.5 M GdmCl, a slight alteration of the tubulin heterodimer occurs, as evidenced by a small, but reproducible increase in the rotational correlation time of the protein and a sharp decrease in the secondary structure monitored by CD. In the range 0.5-1.5 M GdmCl, significant decreases in the steady-state anisotropy and average lifetime of the intrinsic tryptophan fluorescence occur, as well as a decrease in the rotational correlation time, from 48 to 26 nsec. In the same GdmCl range, the number of protein molecules (labeled with Alexa 488), as ... [truncated at 150 words]
Morren G, Wolf U, Lemmerling P, Wolf M, Choi JH, Gratton E, de Lathauwer L, van Huffel S.
Detection of fast neuronal signals in the motor cortex from functional near infrared spectroscopy measurements using independent component analysis.
Med Biol Eng Comput. 2004; 42(1): 92-99.Fast changes, in the range of milliseconds, in the optical properties of cerebral tissue are associated with brain activity and can be detected using non-invasive near-infrared spectroscopy (NIRS). These changes are assumed to be caused by changes in the light scattering properties of the neuronal tissue. The aim of this study was to develop highly sensitive data analysis algorithms to detect this fast signal, which is small compared with other physiological signals. A frequency-domain tissue oximeter, whose laser diodes were intensity modulated at 110MHz, was used. The amplitude, mean intensity and phase of the modulated optical signal were measured at a sample rate of 96 Hz. The probe, consisting of four crossed source detector pairs was placed above the motor cortex, contralateral to the hand performing a tapping exercise consisting of alternating rest and tapping periods of 20s each. An adaptive filter was used to remove the arterial pulsatility from ... [truncated at 150 words]
Gaus K, Gratton E, Kable EPW, Jones AS, Gelissen I, Kritharides L, Jessup W.
Visualizing lipid structure and raft domains in living cells with two-photon microscopy.
Proc Natl Acad Sci USA. 2003; 100(26): 15554-9. PMCID: PMC307606The lateral organization of cellular membranes is formed by the clustering of specific lipids, such as cholesterol and sphingolipids, into highly condensed domains (termed lipid rafts). Hence such domains are distinct from the remaining membrane by their lipid structure (liquid-ordered vs. -disordered domains). Here, we directly visualize membrane lipid structure of living cells by using two-photon microscopy. In macrophages, liquid-ordered domains are particularly enriched on membrane protrusions (filopodia), adhesion points and cell-cell contacts and cover 10-15% of the cell surface at 37 degrees C. By deconvoluting the images, we demonstrate the existence of phase separation in vivo. We compare the properties of microscopically visible domains (<1 microm2), with those of isolated detergent-resistant membranes and provide evidence that membrane coverage by lipid rafts and their fluidity are principally governed by cholesterol content, thereby providing strong support for the lipid raft hypothesis.
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Bioconvertible vitamin antioxidants improve sunscreen photoprotection against UV-induced reactive oxygen species.
J Cosmet Sci. 2003; 54(6): 589-598.The ability of sunscreens and antioxidants to deactivate highly destructive reactive oxygen species in human skin has remained inconclusive. Two-photon fluorescence imaging microscopy was used to determine the effect of sunscreen/antioxidant combinations upon UV-induced ROS generation in ex vivo human skin. A sunscreen combination containing octylmethoxycinnamate (Parsol MCX) and avobenzone (Parsol 1789) at SPF 8 and SPF 15 was tested for its ability to prevent UV radiation from generating ROS in the viable epidermal strata of ex vivo human skin. A UV dose equivalent to two hours of North American solar UV was used to irradiate the skin. Each sunscreen reduced the amount of ROS induced in the viable strata by a value consistent with the SPF level. UV photons that were not absorbed/scattered by the sunscreen formulations generated ROS within the viable epidermal layers. The addition of the bioconvertible antioxidants vitamin E acetate and sodium ascorbyl phosphate (STAY-C 50) ... [truncated at 150 words]
Toronov VY, D'Amico E, Hueber DM, Gratton E, Barbieri BB, Webb A.
Optimization of the signal-to-noise ratio of frequency-domain instrumentation for near-infrared spectro-imaging of the human brain.
Opt Express. 2003; 11(21): 2717-2729.Frequency-domain near-infrared spectro-imaging offers significant advantages over the continuous-wave method in human brain applications. However, the drawback of existing instruments is a low signal-to-noise ratio for measured phase and modulation depth changes caused by cerebral activation. In this paper we show that in the case of the geometry specific for the activated area in the human brain, the SNR can be significantly improved by increasing the modulation frequency. We present the results of two studies: one performed experimentally using a subnanosecond pulsed light source and a spherical absorbing inhomogeneity immersed in a highly scattering solution, and the other performed numerically using Monte Carlo simulations of light transport in an MRI based digital phantom of the adult human head. We show that changes caused by the absorbing inhomogeneity in both phase and modulation depth increase with frequency and reach maximum values at frequencies between 400 and 1400 MHz, depending on the ... [truncated at 150 words]
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High-pressure fluorescence correlation spectroscopy.
Biophys J. 2003; 85(4): 2711-9. PMCID: PMC1303495We demonstrate that a novel high-pressure cell is suitable for fluorescence correlation spectroscopy (FCS). The pressure cell consists of a single fused silica microcapillary. The cylindrical shape of the capillary leads to refraction of the excitation light, which affects the point spread function of the system. We characterize the influence of these beam distortions by FCS and photon-counting histogram (PCH) analysis and identify the optimal position for fluorescence fluctuation experiments in the capillary. At this position within the capillary, FCS and photon-counting histogram experiments are described by the same equations as used in standard FCS experiments. We report the first experimental realization of fluorescence fluctuation spectroscopy under high pressure. A fluorescent dye was used as a model system for evaluating the properties of the capillary under pressure. The autocorrelation function and the photon count distribution were measured in the pressure range from 0 to 300 MPa. The fluctuation amplitude and ... [truncated at 150 words]
Campanini B, Raboni S, Vaccari S, Zhang L, Cook PF, Hazlett TL, Mozzarelli A, Bettati S.
Surface-exposed tryptophan residues are essential for O-acetylserine sulfhydrylase structure, function, and stability.
J Biol Chem. 2003; 278(39): 37511-9.O-Acetylserine sulfhydrylase is a homodimeric enzyme catalyzing the last step of cysteine biosynthesis via a Bi Bi ping-pong mechanism. The subunit is composed of two domains, each containing one tryptophan residue, Trp50 in the N-terminal domain and Trp161 in the C-terminal domain. Only Trp161 is highly conserved in eucaryotes and bacteria. The coenzyme pyridoxal 5'-phosphate is bound in a cleft between the two domains. The enzyme undergoes an open to closed conformational transition upon substrate binding. The effect of single Trp to Tyr mutations on O-acetylserine sulfhydrylase structure, function, and stability was investigated with a variety of spectroscopic techniques. The mutations do not significantly alter the enzyme secondary structure but affect the catalysis, with a predominant influence on the second half reaction. The W50Y mutation strongly affects the unfolding pathway due to the destabilization of the intersubunit interface. The W161Y mutation, occurring in the C-terminal domain, produces a reduction of ... [truncated at 150 words]
Tan E, Wilson TJ, Nahas MK, Clegg RM, Lilley DMJ, Ha T.
A four-way junction accelerates hairpin ribozyme folding via a discrete intermediate.
Proc Natl Acad Sci USA. 2003; 100(16): 9308-13. PMCID: PMC170914The natural form of the hairpin ribozyme comprises two major structural elements: a four-way RNA junction and two internal loops carried by adjacent arms of the junction. The ribozyme folds into its active conformation by an intimate association between the loops, and the efficiency of this process is greatly enhanced by the presence of the junction. We have used single-molecule spectroscopy to show that the natural form fluctuates among three distinct states: the folded state and two additional, rapidly interconverting states (proximal and distal) that are inherited from the junction. The proximal state juxtaposes the two loop elements, thereby increasing the probability of their interaction and thus accelerating folding by nearly three orders of magnitude and allowing the ribozyme to fold rapidly in physiological conditions. Therefore, the hairpin ribozyme exploits the dynamics of the junction to facilitate the formation of the active site from its other elements. Dynamic interplay between ... [truncated at 150 words]
Toronov VY, Walker SA, Gupta R, Choi JH, Gratton E, Hueber DM, Webb A.
The roles of changes in deoxyhemoglobin concentration and regional cerebral blood volume in the fMRI BOLD signal.
NeuroImage. 2003; 19(4): 1521-31.To study the behavior of cerebral physiological parameters and to further the understanding of the functional magnetic resonance imaging (fMRI) blood-oxygen-level-dependent (BOLD) effect, multisource frequency-domain near-infrared and BOLD fMRI signals were recorded simultaneously during motor functional activation in humans. From the near-infrared data information was obtained on the changes in cerebral blood volume and oxygenation. To relate our observations to changes in cerebral blood flow the well-known "balloon" model was employed. Our data showed that the deoxyhemoglobin concentration is the major factor determining the time course of the BOLD signal. The increase in cerebral blood oxygenation during functional activation is due to an increase in the velocity of blood flow, and occurs without significant swelling of the blood vessels.
Wolf M, Wolf U, Choi JH, Toronov VY, Paunescu LA, Michalos A, Gratton E.
Fast cerebral functional signal in the 100-ms range detected in the visual cortex by frequency-domain near-infrared spectrophotometry.
Psychophysiology. 2003; 40(4): 521-8.Brain activity is associated with physiological changes, which alter the optical properties of the tissue in the near-infrared part of the spectrum. Two major types of optical signals following functional brain activation can be distinguished: a slow signal due to hemodynamic changes and a fast signal, which is directly related to neuronal activity. The fast signal is small and therefore difficult to detect. We used a specially noise-optimized frequency-domain near-infrared spectrometer with a pi-sensor, which was expected to be particularly sensitive to deeper tissue layers, to investigate the human visual cortex during visual stimulation generated by a checkerboard. We were able to detect significant fast signals in single light bundles, but not in pi-signals. The fast signals were mostly collocated with strong slow hemodynamic signals, but showed a higher degree of localization than the latter. The latencies of 40 ± 16 ms of the fast signals were similar between locations. ... [truncated at 150 words]
Gratton E, Breusegem SY, Sutin JDB, Ruan Q, Barry NP.
Fluorescence lifetime imaging for the two-photon microscope: time-domain and frequency-domain methods.
J Biomed Opt. 2003; 8(3): 381-90.Fluorescence lifetime images are obtained with the laser scanning microscope using two methods: the time-correlated single-photon counting method and the frequency-domain method. In the same microscope system, we implement both methods. We perform a comparison of the performance of the two approaches in terms of signal-to-noise ratio (SNR) and the speed of data acquisition. While in our practical implementation the time-correlated single-photon counting technique provides a better SNR for low-intensity images, the frequency-domain method is faster and provides less distortion for bright samples.
Behne MJ, Barry NP, Hanson KM, Aronchik I, Clegg RM, Gratton E, Feingold K, Holleran WM, Elias PM, Mauro TM.
Neonatal development of the stratum corneum pH gradient: localization and mechanisms leading to emergence of optimal barrier function.
J Invest Dermatol. 2003; 120(6): 998-1006.Although basal permeability barrier function is established at birth, the higher risk for infections, dermatitis, and percutaneous absorption of toxic agents may indicate incomplete permeability barrier maturation in the early neonatal period. Since stratum corneum (SC) acidification in adults is required for normal permeability barrier homeostasis, and lipid processing occurs via acidic pH dependent enzymes, we hypothesized that, in parallel with the less acidic surface pH, newborn SC would exhibit signs of incomplete barrier formation. Fluorescence lifetime imaging reveals that neonatal rat SC acidification first becomes evident by postnatal day 3, in extracellular "microdomains" at the SC- stratum granulosum (SG) interface, where pH-sensitive lipid processing is known to occur. This localized acidification correlated temporally with efficient processing of secreted lamellar body contents to mature extracellular lamellar bilayers. Since expression of the key acidifying mechanism NHE1 is maximal just prior to birth, and gradually declines over the first postnatal week, suboptimal ... [truncated at 150 words]
Wolf U, Wolf M, Choi JH, Levi M, Choudhury D, Hull S, Coussirat D, Paunescu LA, Safonova LP, Michalos A, Mantulin WW, Gratton E.
Localized irregularities in hemoglobin flow and oxygenation in calf muscle in patients with peripheral vascular disease detected with near-infrared spectrophotometry.
J Vasc Surg. 2003; 37(5): 1017-26.PURPOSE: Near-infrared spectrophotometry is used to measure flow, concentration, and oxygenation of hemoglobin in arterioles, capillaries, and venules several centimeters deep in tissue. The purpose of this study was to investigate the distribution of flow, concentration, and oxygenation of hemoglobin in calf muscle in patients with documented peripheral arterial occlusive disease (PVD), patients with risk factors for PVD,and healthy younger subjects at rest. METHOD: With a frequency-domain near-infrared spectrophotometer and a specially designed probe, we generated maps at 22 locations simultaneously of hemoglobin flow, concentration, and oxygenation, with the venous occlusion method. Eight legs of 7 patients with diagnosed PVD (PVD group), 10 legs of 8 patients with normal ankle-brachial index but with risk factors for PVD (RF group), and 16 legs of 8 healthy subjects (H group) were studied. RESULTS: Global mean values were significantly (P <.05) different between the three groups for oxygen consumption (PVD group, 0.027 ± ... [truncated at 150 words]
Czeslik C, Royer CA, Hazlett TL, Mantulin WW.
Reorientational dynamics of enzymes adsorbed on quartz: a temperature-dependent time-resolved TIRF anisotropy study.
Biophys J. 2003; 84(4): 2533-41. PMCID: PMC1302819The preservation of enzyme activity and protein binding capacity upon protein adsorption at solid interfaces is important for biotechnological and medical applications. Because these properties are partly related to the protein flexibility and mobility, we have studied the internal dynamics and the whole-body reorientational rates of two enzymes, staphylococcal nuclease (SNase) and hen egg white lysozyme, over the temperature range of 20-80 degrees C when the proteins are adsorbed at the silica/water interface and, for comparison, when they are dissolved in buffer. The data were obtained using a combination of two experimental techniques, total internal reflection fluorescence spectroscopy and time-resolved fluorescence anisotropy measurements in the frequency domain, with the protein Trp residues as intrinsic fluorescence probes. It has been found that the internal dynamics and the whole-body rotation of SNase and lysozyme are markedly reduced upon adsorption over large temperature ranges. At elevated temperatures, both protein molecules appear completely immobilized ... [truncated at 150 words]
Wurzburger RJ, Gupta R, Parnassa AP, Jain S, Wexler JA, Chu JL, Elkon KB, Blank RD.
Use of GC clamps in DHPLC mutation scanning.
Clin Med Res. 2003; 1(2): 111-118. PMCID: PMC1069033OBJECTIVE: Development of a systematic mutation detection assay strategy for denaturing high performance liquid chromatography (DHPLC). DESIGN: Adaptation of Guanine and Cytosine (GC)-clamping from denaturing gradient gel electrophoresis (DGGE) to DHPLC. METHODS: Three target sequences harboring known allelic variants were studied to develop a general DHPLC assay design strategy. These were exon 10 of the human RET (REarranged during Transfection) gene, exon 52 of the mouse Col1a2 gene, and exon 9 of the human FAS (APO-1, CD-95) gene. Available software was used to analyze melting curves and determine assay conditions. GC clamps of 20 bp or 36 bp were added to polymerase chain reaction (PCR) primers to introduce a high melting temperature (T(m)) domain to each of the target molecules. DHPLC was performed under partially denaturing conditions. RESULTS: DHPLC assays of PCR-amplified sequences can be developed using a personal computer. The following three steps allowed for mutation detection in all ... [truncated at 150 words]
Zamai M, vandeVen MJ, Farao M, Gratton E, Ghiglieri A, Castelli MG, Fontana E, D'Argy R, Fiorino A, Pesenti E, Suarato A, Caiolfa VR.
Camptothecin poly[n-(2-hydroxypropyl) methacrylamide] copolymers in antitopoisomerase-I tumor therapy: intratumor release and antitumor efficacy.
Mol Cancer Ther. 2003; 2(1): 29-40.Soluble copolymers of camptothecin (CPT), based on poly[N-(2-hydroxypropyl) methacrylamide] (pHPMA), were obtained by conjugation through the degradable spacers -Gly-Phe-Leu-Gly- or -Gly-6-aminohexanoyl-Gly-. We investigated to what extent passive accumulation and retention of hydroxypropyl methacrylamide copolymer of CPT (pHPMA-CPT) in tumors and modulation of the drug release influence efficacy. Release of CPT in vivo was detected by time-resolved phase-shift fluorescence imaging on tumor specimens, based on the evidence that free and bound drug had different fluorescence lifetimes in solution. HT-29 murine specimens, obtained at several times after treatment with (3)H-labeled free CPT, pHPMA-Gly-Phe-Leu-Gly-CPT, or pHPMA-Gly-6-aminohexanoyl-Gly-CPT, were either imaged for time-resolved phase-shift fluorescence or subjected to autoradiography. Phase shifts of CPT conjugates were equal or longer than those of free CPT, indicating the presence of both free and polymer-bound drug in the tumor, in agreement with autoradiograms. pHPMA-Gly-Phe-Leu-Gly-CPT underwent relevant intratumor hydrolysis during the first 24 h, whereas the hydrolysis of pHPMA-Gly-6-aminohexanoyl-Gly-CPT was ... [truncated at 150 words]
Safonova LP, Michalos A, Wolf U, Choi JH, Wolf M, Mantulin WW, Hueber DM, Gratton E.
Diminished cerebral circulatory autoregulation in obstructive sleep apnea investigated by near-infrared spectroscopy.
Sleep Research Online. 2003; 5(4): 123-132.We applied near-infrared spectroscopy (NIRS) to assess cerebral tissue oxygenation and hemodynamics in obstructive sleep apnea (OSA) sufferers and control volunteers. We designed NIRS sensors and applied measurement schemes that included certain polysomnography parameters, such as arterial hemoglobin oxygen saturation (SaO2), heart rate (HR), and respiratory signal (RS), together with NIRS parameters, such as oxy- ([O2Hb]), deoxy- ([HHb]), total hemoglobin ([tHb]) concentrations, and tissue hemoglobin oxygen saturation (SO2). Twenty-one volunteers (8 females, 13 males, age 22-74 years) participated in the study. Eight were OSA sufferers, while 13 constituted the control group. Measurements were conducted during breath holding exercises and 30-45 minute daytime naps. In comparing OSA subjects with controls during breath holding, a smaller increase or even a decrease in SO2, [O2Hb], and [tHb] and a simultaneous larger increase in [HHb], confirmed insufficiency of the circulatory compensatory mechanism that prevents brain tissue hypoxia. Changes in cerebral oxygenation and hemodynamics due ... [truncated at 150 words]
Behne MJ, Meyer JW, Hanson KM, Barry NP, Murata S, Crumrine D, Clegg RM, Gratton E, Holleran WM, Elias PM, Mauro TM.
NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging.
J Biol Chem. 2002; 277(49): 47399-406.The outermost epidermal layer, the stratum corneum (SC), exhibits an acidic surface pH, whereas the pH at its base approaches neutrality. NHE1 is the only Na(+)/H(+) antiporter isoform in keratinocytes and epidermis, and has been shown to regulate intracellular pH. We now demonstrate a novel function for NHE1, as we find that it also controls acidification of extracellular "microdomains" in the SC that are essential for activation of pH-sensitive enzymes and the formation of the epidermal permeability barrier. NHE1 expression in epidermis is most pronounced in granular cell layers, and although the surface pH of NHE1 knockout mice is only slightly more alkaline than normal using conventional pH measurements, a more sensitive method, fluorescence lifetime imaging, demonstrates that the acidic intercellular domains at the surface and of the lower SC disappear in NHE1 -/- animals. Fluorescence lifetime imaging studies also reveal that SC acidification does not occur through a uniform ... [truncated at 150 words]
Wolf M, Wolf U, Choi JH, Gupta R, Safonova LP, Paunescu LA, Michalos A, Gratton E.
Functional frequency-domain near-infrared spectroscopy detects fast neuronal signal in the motor cortex.
NeuroImage. 2002; 17(4): 1868-75.Millisecond changes in the optical properties of the human brain during stimulation were detected in five volunteers using noninvasive frequency-domain near-infrared spectroscopy. During a motor stimulation task we found highly significant signals, which were directly related to neuronal activity and exhibited much more localized patterns than the slow hemodynamic signals that are also detected by the near-infrared method. We considerably reduced the noise in the instrumental system and improved data analysis algorithms. With the achieved signal-to-noise ratio, single subject measurements were feasible without the requirement of particularly strong stimuli and within a reasonable period of measurement of 5 min at a mean signal-to-noise ratio of 3.6. The advantage of this noninvasive technique with respect to electrical recording is that it is able to detect neuronal activity with the relatively high spatial resolution of 8 mm.
Ruan Q, Chen Y, Gratton E, Glaser M, Mantulin WW.
Cellular characterization of adenylate kinase and its isoform: two-photon excitation fluorescence imaging and fluorescence correlation spectroscopy.
Biophys J. 2002; 83(6): 3177-87. PMCID: PMC1302395Adenylate kinase (AK) is a ubiquitous enzyme that regulates the homeostasis of adenine nucleotides in the cell. AK1beta (long form) from murine cells shares the same protein sequence as AK1 (short form) except for the addition of 18 amino acid residues at its N-terminus. It is hypothesized that these residues serve as a signal for protein lipid modification and targeting of the protein to the plasma membrane. To better understand the cellular function of these AK isoforms, we have used several modern fluorescence techniques to characterize these two isoforms of AK enzyme. We fused cytosolic adenylate kinase (AK1) and its isoform (AK1beta) with enhanced green fluorescence protein (EGFP) and expressed the chimera proteins in HeLa cells. Using two-photon excitation scanning fluorescence imaging, we were able to directly visualize the localization of AK1-EGFP and AK1beta-EGFP in live cells. AK1beta-EGFP mainly localized on the plasma membrane, whereas AK1-EGFP distributed throughout the cell ... [truncated at 150 words]
Best KB, Ohran AJ, Hawes AC, Hazlett TL, Gratton E, Judd AM, Bell JD.
Relationship between erythrocyte membrane phase properties and susceptibility to secretory phospholipase A2.
Biochemistry. 2002; 41(47): 13982-8.Normally, cell membranes resist hydrolysis by secretory phospholipase A(2). However, upon elevation of intracellular calcium, the cells become susceptible. Previous investigations demonstrated a possible relationship between changes in lipid order caused by increased calcium and susceptibility to phospholipase A(2). To further explore this relationship, we used temperature as an experimental means of manipulating membrane physical properties. We then compared the response of human erythrocytes to calcium ionophore at various temperatures in the range of 20-50 degrees C using fluorescence spectroscopy and two-photon fluorescence microscopy. The steady state fluorescence emission of the environment-sensitive probe, laurdan, revealed that erythrocyte membrane order decreases systematically with temperature throughout this range, especially between 28 and 45 degrees C. Furthermore, the ability of calcium ionophore to induce increased membrane order and susceptibility to phospholipase A(2) depended similarly on temperature. Both responses to calcium influx were enhanced as membrane fluidity increased. Analysis of the spatial distribution of ... [truncated at 150 words]
Hanson KM, Behne MJ, Barry NP, Mauro TM, Gratton E, Clegg RM.
Two-photon fluorescence lifetime imaging of the skin stratum corneum pH gradient.
Biophys J. 2002; 83(3): 1682-90. PMCID: PMC1302264Two-photon fluorescence lifetime imaging is used to identify microdomains (1-25 microm) of two distinct pH values within the uppermost layer of the epidermis (stratum corneum). The fluorophore used is 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF), whose lifetime tau (pH 4.5, tau = 2.75 ns; pH 8.5, tau = 3.90 ns) is pH dependent over the pH range of the stratum corneum (pH 4.5 to pH 7.2). Hairless mice (SKH1-hrBR) are used as a model for human skin. Images (< or =50 microm x 50 microm) are acquired every 1.7 microm from the stratum corneum surface to the first viable layer (stratum granulosum). Acidic microdomains (average pH 6.0) of variable size (~1 microm in diameter with variable length) are detected within the extracellular matrix of the stratum corneum, whereas the intracellular space of the corneocytes in mid-stratum corneum (25 microm diameter) approaches neutrality (average pH 7.0). The surface is acidic. The average pH of the ... [truncated at 150 words]
Wolf M, Wolf U, Toronov VY, Michalos A, Paunescu LA, Choi JH, Gratton E.
Different time evolution of oxyhemoglobin and deoxyhemoglobin concentration changes in the visual and motor cortices during functional stimulation: a near-infrared spectroscopy study.
NeuroImage. 2002; 16(3 Pt 1): 704-12.Neurovascular coupling is the generic term for changes in cerebral metabolic rate of oxygen (CMRO(2)), cerebral blood flow, and cerebral blood volume related to brain activity. The goal of this paper is to better understand the effects of neurovascular coupling in the visual and motor cortices using frequency-domain near-infrared spectroscopy. Maps of concentration changes in oxyhemoglobin [O(2)Hb], deoxyhemoglobin [HHb], and total hemoglobin of the visual and motor cortices were generated during stimulation using a reversing checkerboard screen and palm-squeezing, respectively. Seven healthy volunteers of 18-37 years of age were included. In the visual cortex the patterns of [O(2)Hb] and [HHb] were strongly linearly correlated (r(2) > 0.8 in 13 of a total of 24 locations). In 20 locations the change in [O(2)Hb] was larger than 0.25 microM. The mean slope of the linear regression between [O(2)Hb] and [HHb] was -3.93 ± 0.31 (SE). The patterns of the [O(2)Hb] and [HHb] ... [truncated at 150 words]
Fahsel S, Pospiech EM, Zein M, Hazlett TL, Gratton E, Winter R.
Modulation of concentration fluctuations in phase-separated lipid membranes by polypeptide insertion.
Biophys J. 2002; 83(1): 334-44. PMCID: PMC1302151The lateral membrane organization and phase behavior of the binary lipid mixture DMPC (1,2-dimyristoyl-sn-glycero-3-phosphatidylcholine) - DSPC (1,2-distearoyl-sn-glycero-3-phosphatidylcholine) without and with incorporated gramicidin D (GD) as a model biomembrane polypeptide was studied by small-angle neutron scattering, Fourier-transform infrared spectroscopy, and by two-photon excitation fluorescence microscopy on giant unilamellar vesicles. The small-angle neutron scattering method allows the detection of concentration fluctuations in the range from 1 to 200 nm. Fluorescence microscopy was used for direct visualization of the lateral lipid organization and domain shapes on a micrometer length scale including information of the lipid phase state. In the fluid-gel coexistence region of the pure binary lipid system, large-scale concentration fluctuations appear. Infrared spectral parameters were used to determine the peptide conformation adopted in the different lipid phases. The data show that the structure of the temperature-dependent lipid phases is significantly altered by the insertion of 2 to 5 mol% GD. At temperatures ... [truncated at 150 words]
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Observation and quantification of ultraviolet-induced reactive oxygen species in ex vivo human skin.
Photochem Photobiol. 2002; 76(1): 57-63.Two-photon fluorescence imaging is used to detect UV-induced reactive oxygen species (ROS) in ex vivo human skin in this study. ROS (potentially H202, singlet oxygen or peroxynitrite [or all]) are detected after reaction with nonfluorescent dihydrorhodamine-123 (DHR) and the consequent formation of fluorescent rhodamine-123 (R123). The cellular regions at each epidermal stratum that generate ROS are identified. R-123 fluorescence is detected predominately in the lipid matrix of the stratum corneum. In contrast, the strongest R123 fluorescence signal is detected in the intracellular cytoplasm of the viable epidermal keratinocytes. A simple bimolecular one-step kinetic model is used for estimating the upper bound of the number of ROS that are generated in the skin and that react with DHR. After ultraviolet-B radiation (280-320 nm) (UVB) equivalent to 2 h of noonday summer North American solar exposure (1600 J m(-2) UVB), the model finds that 14.70 × 10(-3) mol of ROS that react ... [truncated at 150 words]
Bettati S, Campanini B, Vaccari S, Mozzarelli A, Schianchi G, Hazlett TL, Gratton E, Benci S.
Unfolding of pyridoxal 5'-phosphate-dependent O-acetylserine sulfhydrylase probed by time-resolved tryptophan fluorescence.
Biochim Biophys Acta. 2002; 1596(1): 47-54.Proteins utilizing pyridoxal 5'-phosphate as a coenzyme constitute a large superfamily and are currently classified into three functional groups and five structural fold types. Despite the variability of sequences and catalyzed reactions, they share relevant structural, dynamic and functional properties. Therefore, they constitute an optimal system to investigate the relative influence of primary sequence and coenzyme interactions on folding pathways, structural stability and enzymatic function. O-Acetylserine sulfhydrylase is a dimeric pyridoxal 5'-phosphate dependent enzyme that catalyzes the synthesis of L-cysteine from O-acetylserine and sulfide. The time-resolved fluorescence study of O-acetylserine sulfhydrylase unfolding, here reported, indicates that the coenzyme stabilizes the protein structure. The dependence on denaturant concentration of tryptophan lifetimes in the holo- and apo-enzyme demonstrates that the interactions with the coenzyme stabilize the C-terminal domain to a higher extent with respect to the N-terminal domain. This result is discussed in terms of a linkage between the differential stabilization brought ... [truncated at 150 words]
Sánchez SA, Bagatolli LA, Gratton E, Hazlett TL.
A two-photon view of an enzyme at work: Crotalus atrox venom PLA2 interaction with single-lipid and mixed-lipid giant unilamellar vesicles.
Biophys J. 2002; 82(4): 2232-43. PMCID: PMC1302016We describe the interaction of Crotalus atrox-secreted phospholipase A2 (sPLA2) with giant unilamellar vesicles (GUVs) composed of single and binary phospholipid mixtures visualized through two-photon excitation fluorescent microscopy. The GUV lipid compositions that we examined included 1-palmitoyl-2-oleoyl-phosphatidylcholine, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC), and 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) (above their gel-liquid crystal transition temperatures) and two well characterized lipid mixtures, 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine (DMPE):DMPC (7:3) and 1,2-dilauroyl-sn-glycero-3-phosphocholine (DLPC)/1,2-diarachidoyl-sn-glycero-3-phosphocholine (DAPC) (1:1) equilibrated at their phase-coexistence temperature regime. The membrane fluorescence probes, 6-lauroyl-2-(dimethylamino) napthalene, 6-propionyl-2-(dimethylamino) naphthalene, and rhodamine-phosphatidylethanolamine, were used to assess the state of the membrane and specifically mark the phospholipid domains. Independent of their lipid composition, all GUVs were reduced in size as sPLA2-dependent lipid hydrolysis proceeded. The binding of sPLA2 was monitored using a fluorescein-sPLA2 conjugate. The sPLA2 was observed to associate with the entire surface of the liquid phase in the single phospholipid GUVs. In the mixed-lipid GUV's, at temperatures promoting domain coexistence, a preferential binding ... [truncated at 150 words]
Huang H, Dong CY, Kwon HS, Sutin JDB, Kamm RD, So PTC.
Three-dimensional cellular deformation analysis with a two-photon magnetic manipulator workstation.
Biophys J. 2002; 82(4): 2211-23. PMCID: PMC1302014The ability to apply quantifiable mechanical stresses at the microscopic scale is critical for studying cellular responses to mechanical forces. This necessitates the use of force transducers that can apply precisely controlled forces to cells while monitoring the responses noninvasively. This paper describes the development of a micromanipulation workstation integrating two-photon, three-dimensional imaging with a high-force, uniform-gradient magnetic manipulator. The uniform-gradient magnetic field applies nearly uniform forces to a large cell population, permitting statistical quantification of select molecular responses to mechanical stresses. The magnetic transducer design is capable of exerting over 200 pN of force on 4.5-microm-diameter paramagnetic particles and over 800 pN on 5.0-microm ferromagnetic particles. These forces vary within ±10% over an area 500 × 500 microm2. The compatibility with the use of high numerical aperture (approximately 1.0) objectives is an integral part of the workstation design allowing submicron-resolution, three-dimensional, two-photon imaging. Three-dimensional analyses of cellular deformation under ... [truncated at 150 words]
Patel RC, Kumar U, Lamb DC, Eid JS, Rocheville M, Grant M, Rani A, Hazlett TL, Patel SC, Gratton E, Patel YC.
Ligand binding to somatostatin receptors induces receptor-specific oligomer formation in live cells.
Proc Natl Acad Sci USA. 2002; 99(5): 3294-9. PMCID: PMC122512Heptahelical receptors (HHRs) are generally thought to function as monomeric entities. Several HHRs such as somatostatin receptors (SSTRs), however, form homo- and heterooligomers when activated by ligand binding. By using dual fluorescent ligands simultaneously applied to live cells monotransfected with SSTR5 (R5) or SSTR1 (R1), or cotransfected with R5 and R1, we have analyzed the ligand receptor stoichiometry and aggregation states for the three receptor systems by fluorescence resonance energy transfer and fluorescence correlation spectroscopy. Both homo- and heterooligomeric receptors are occupied by two ligand molecules. We find that monomeric, homooligomeric, and heterooligomeric receptor species occur in the same cell cotransfected with two SSTRs, and that oligomerization of SSTRs is regulated by ligand binding by a selective process that is restricted to some (R5) but not other (R1) SSTR subtypes. We propose that induction by ligand of different oligomeric states of SSTRs represents a unique mechanism for generating signaling specificity ... [truncated at 150 words]
Tricerri MA, Sánchez SA, Arnulphi C, Durbin DM, Gratton E, Jonas A.
Interaction of apolipoprotein A-I in three different conformations with palmitoyl oleoyl phosphatidylcholine vesicles.
J Lipid Res. 2002; 43(2): 187-97.Interactions of apolipoprotein A-I (apoA-I) with cell membranes appear to be important in the initial steps of reverse cholesterol transport. The objective of this work was to examine the effect of three distinct conformations of apoA-I (lipid-free and in 78 A or 96 A reconstituted high density lipoproteins, rHDL) on its ability to bind to, and abstract lipids from, palmitoyl oleoyl phosphatidylcholine membrane vesicles (small unilamellar vesicles, SUV, and giant unilamellar vesicles, GUV). The molecular interactions were observed by two-photon fluorescence microscopy, and the binding parameters were quantified by gel-permeation chromatography or isothermal titration microcalorimetry. Rearrangement of apoA-I-containing particles after exposure to SUVs was examined by native gel electrophoresis. The results indicate that lipid-free apoA-I binds reversibly, with high affinity, to the vesicles but does not abstract a significant amount of lipid nor perturb the vesicle structure. The 96 A rHDL, where all the amphipathic helices of apoA-I are saturated ... [truncated at 150 words]
Breusegem SY, Clegg RM, Loontiens FG.
Base-sequence specificity of Hoechst 33258 and DAPI binding to five (A/T)4 DNA sites with kinetic evidence for more than one high-affinity Hoechst 33258-AATT complex.
J Mol Biol. 2002; 315(5): 1049-61.The binding of Hoechst 33258 and DAPI to five different (A/T)4 sequences in a stable DNA hairpin was studied exploiting the substantial increase in dye fluorescence upon binding. The two dyes have comparable affinities for the AATT site (e.g. association constant K(a)=5.5 x 10(8) M(-1) for DAPI), and their affinities decrease in the series AATT >> TAAT approximately equal to ATAT > TATA approximately equal to TTAA. The extreme values of K(a) differ by a factor of 200 for Hoechst 33258 but only 30 for DAPI. The binding kinetics of Hoechst 33258 were measured by stopped-flow under pseudo-first order conditions with an (A/T)4 site in excess. The lower-resolution experiments can be well represented by single exponential processes, corresponding to a single-step binding mechanism. The calculated association-rate parameters for the five (A/T)4 sites are similar (2.46 x 10(8) M(-1) s(-1) to 0.86 x 10(8) M(-1) s(-1)) and nearly diffusion-controlled, while the ... [truncated at 150 words]
Sukhishvili SA, Chen Y, Müller JD, Gratton E, Schweizer KS, Granick S.
Surface diffusion of Poly(ethylene glycol).
Macromolecules. 2002; 35(5): 1776-1784.We report direct measurement of the center-of-mass diffusion coefficient, D, of uncharged flexible linear chains adsorbed at the solid-liquid interface at dilute surface coverage. We find D ~ N^3/2 (N is degree of polymerization) when N was varied by more than an order of magnitude (N = 48, 113, 244, 456, and 693) and the scatter of the data was low. The experimental system was poly(ethylene glycol), PEG, adsorbed from dilute aqueous solution onto a self-assembled hydrophobic monolayer, condensed octadecyltriethoxysilane. The method of measurement was fluorescence correlation spectroscopy of a rhodamine green derivative dye that was end-attached to one sole end of the adsorbed PEG chains. The observed scaling implies the diffusion time ~ N^3 if Rg ~ N^3/4 as expected for a chain in good solvent in two dimensions (Rg is the radius of gyration), but a variety of other theoretical approaches to describe the dynamical scaling are ... [truncated at 150 words]
Chen Y, Müller JD, Ruan Q, Gratton E.
Molecular brightness characterization of EGFP in vivo by fluorescence fluctuation spectroscopy.
Biophys J. 2002; 82(1): 133-44. PMCID: PMC1302455We characterize the molecular properties of autofluorescence and transiently expressed EGFP in the nucleus and in the cytoplasm of HeLa cells by fluorescence correlation spectroscopy (FCS) and by photon counting histogram (PCH) analysis. PCH has been characterized and applied in vitro, but its potential for in vivo studies needs to be explored. Thus, this study mainly focuses on the characterization of PCH analysis in vivo. The strength of PCH lies in its ability to distinguish biomolecules by their molecular brightness value. Because the concept of molecular brightness is crucial for PCH analysis, we study the molecular brightness of EGFP and determine the statistical accuracy of its measurement under in vivo conditions. We started by characterizing the influence of autofluorescence on EGFP measurements. We found a molecular brightness of EGFP that is a factor of 10 higher than the brightness of the autofluorescence. Moment analysis demonstrates that the contribution of autofluorescence ... [truncated at 150 words]
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FRET tells us about proximities, distances, orientations and dynamic properties.
J Biotechnol. 2002; 82(3): 177-9.Excerpt: A major challenge in biological science is to determine the spatial juxtapositions and distributions of molecular and supramolecular components that constitute biological structures. A great deal, if not the major part, of biology happens at the interface between interacting molecules. Techniques that inform us about these molecular interactions are of crucial importance...
Jasuja R, Hazlett TL, Helms MK, Lee SH, Jameson DM, Larsen RW.
Temperature dependence of photoinduced electron transfer within self-associated porphyrin: guanine monophosphate complexes.
Chem Phys Lett. 2001; 350(5-6): 515-521.In the current report, the temperature dependence of photoinduced electron transfer between tetrakis-(4-tetramethylpyridyl)porphine (T4MPyP) and guanine monophosphate (GMP) has been examined. In the presence of GMP the fluorescence lifetime analysis reveals a Lorentzian distribution of lifetimes centered at 0.7 ns with a width of 0.9 ns displaying significant temperature dependence. Fitting temperature dependent data to the Marcus equation gives a reorganizational energy (λ) for the electron transfer reaction of 0.6 eV and an electronic coupling factor (HAB) of 3×10−3 eV. These results suggest conformational regulation of electron transfer within the non-covalent porphyrin:nucleotide complex.
Ruan Q, Ruan K, Balny C, Glaser M, Mantulin WW.
Protein folding pathways of adenylate kinase from E. coli: hydrostatic pressure and stopped-flow studies.
Biochemistry. 2001; 40(48): 14706-14.Adenylate kinase (AKe) from E. coli is a small, single-chain, monomeric enzyme with no tryptophan and a single cysteine residue. We have constructed six single-Trp mutants of AKe to facilitate optical studies of these proteins and to specifically examine the interrelationship between their structure, function, dynamics, and folding reactions. In this study, the effects of hydrostatic pressure on the folding reactions of AKe were studied. The native structure of AKe was transformed to a non-native, yet pressure stable, conformation by hydrostatic pressure of about 300 MPa. This pressure lability of AKe is rather low for a monomeric protein and presumably may be attributed to substantial conformational flexibility and a correspondingly large volume change. The refolding of AKe after pressure-induced denaturation was reversible under ambient conditions. At low temperature (near 0 degrees C), the refolding process of pressure-exposed AKe mutants displayed a significant hysteresis. The observation of a slow refolding rate ... [truncated at 150 words]
Bismuto E, Gratton E, Lamb DC.
Dynamics of ANS binding to tuna apomyoglobin measured with fluorescence correlation spectroscopy.
Biophys J. 2001; 81(6): 3510-21. PMCID: PMC1301806The dynamics of the binding reaction of ANS to native and partly folded (molten globule) tuna and horse apomyoglobins has been investigated by fluorescence correlation spectroscopy and frequency domain fluorometry. The reaction rate has been measured as a function of apomyoglobin and ANS concentrations, pH, and temperature. Examination of the autocorrelation functions shows that the reaction rate is fast enough to be observed in tuna apomyoglobin, whereas the reaction rate in horse apomyoglobin is on the same time scale as diffusion through the volume or longer. Specifically, for tuna apomyoglobin at pH 7 and room temperature the on rate is 2200 microM(-1) s(-1) and the off rate is 5900 s(-1), in comparison with k(on) = 640 microM(-1) s(-1) and k(off) = 560 s(-1) for horse myoglobin as measured previously. The independence of the reaction rate from the ANS concentration indicates that the reaction rate is dominated by the off rate. ... [truncated at 150 words]
Toronov VY, Webb A, Choi JH, Wolf M, Safonova LP, Wolf U, Gratton E.
Study of local cerebral hemodynamics by frequency-domain near-infrared spectroscopy and correlation with simultaneously acquired functional magnetic resonance imaging.
Opt Express. 2001; 9(8): 417-427.The aim of our study was to explore the possibility of detecting hemodynamic changes in the brain using the phase of the intensity modulated optical signal. To obtain optical signals with the highest possible signal-to-noise ratio, we performed a series of simultaneous NIRS-fMRI measurements, with subsequent correlation of the time courses of both measurements. The cognitive paradigm used arithmetic calculations, with optical signals acquired with sensors placed on the forehead. Measurements were done on seven healthy subjects. In five subjects we demonstrated correlation between the hemodynamic signals obtained using NIRS and BOLD fMRI. In four subjects correlation was found for the hemodynamic signal obtained using the phase of the intensity modulated signal.
Gratton E, Barry NP, Beretta S, Celli A.
Multiphoton fluorescence microscopy.
Methods. 2001; 25(1): 103-10.Multiphoton fluorescence microscopy has now become a relatively common tool among biophysicists and biologists. The intrinsic sectioning achievable by multiphoton excitation provides a simple means to excite a small volume inside cells and tissues. Multiphoton microscopes have a simplified optical path in the emission side due to the lack of an emission pinhole, which is necessary with normal confocal microscopes. This article illustrates examples in which this advantage in the simplified optics is exploited to achieve a new type of measurements. First, dual-emission wavelength measurements are used to identify regions of different phase domains in giant vesicles and to perform fluctuation experiments at specific locations in the membrane. Second, we show how dual-wavelength measurements are used in conjunction with scanning fluctuation analysis to measure the changes in the geometry of the domains and the incipient formation of gel domains when the temperature of the giant vesicles is gradually lowered.
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Direct observation of lipid domains in free-standing bilayers using two-photon excitation fluorescence microscopy.
J Fluoresc. 2001; 11(3): 141-160.The direct observation of temperature-dependent lipid phase equilibria, using two-photon excitation fluorescence microscopy on giant unilamellar vesicles (GUVs) composed of different lipid mixtures, provides novel information about the physical characteristics of lipid domain coexistence. Physical characteristics such as the shape, size, and time evolution of different lipid domains are not directly accessible from the traditional experimental approaches that employ either small and large unilamellar vesicles or multilamellar vesicles. In this review article, we address the most relevant findings reported from our laboratory regarding the direct observation of lipid domain coexistence at the level of single vesicles in artificial and natural lipid mixtures. In addition, key points concerning our experimental approach will be discussed. The unique advantages of the fluorescent probe 6-dodecanoyl-2-dimethylaminonaphthalene (LAURDAN) under two-photon excitation fluorescence microscopy is particularly addressed, especially, the possibility of obtaining information on the phase state of different lipid domains directly from the fluorescent images.
Smith SK, Farnbach AR, Harris FM, Hawes AC, Jackson LR, Judd AM, Vest RS, Sánchez SA, Bell JD.
Mechanisms by which intracellular calcium induces susceptibility to secretory phospholipase A2 in human erythrocytes.
J Biol Chem. 2001; 276(25): 22732-22741.Exposure of human erythrocytes to the calcium ionophore ionomycin rendered them susceptible to the action of secretory phospholipase A2 (sPLA2). Analysis of erythrocyte phospholipid metabolism by thin-layer chromatography revealed significant hydrolysis of both phosphatidylcholine and phosphatidylethanolamine during incubation with ionomycin and sPLA2. Several possible mechanisms for the effect of ionomycin were considered. Involvement of intracellular phospholipases A2 was excluded since inhibitors of these enzymes had no effect. Assessment of membrane oxidation by cis-parinaric acid fluorescence and comparison to the oxidants diamide and phenylhydrazine revealed that oxidation does not participate in the effect of ionomycin. Incubation with ionomycin caused classical physical changes to the erythrocyte membrane such as morphological alterations (spherocytosis), translocation of aminophospholipids to the outer leaflet of the membrane, and release of microvesicles. Experiments with phenylhydrazine, KCl, quinine, merocyanine 540, the calpain inhibitor E-64d, and the scramblase inhibitor R5421 revealed that neither phospholipid translocation nor vesicle release was required ... [truncated at 150 words]
Sánchez SA, Chen Y, Müller JD, Gratton E, Hazlett TL.
Solution and interface aggregation states of Crotalus atrox venom phospholipase A2 by two-photon excitation fluorescence correlation spectroscopy.
Biochemistry. 2001; 40(23): 6903-11.The dimeric Crotalus atrox venom PLA2 is part of the secreted phospholipase A2 (PLA2) enzyme family that interacts at the lipid-solution interface to hydrolyze the sn-2 acyl ester bond of phospholipids. We have employed fluorescence correlation spectroscopy (FCS) to study the monomer-dimer equilibrium of the C. atrox venom PLA2 in solution, in the presence of urea, and in the presence of monomeric and micellar n-dodecylphosphocholine (C12-PN), a phosphatidylcholine analogue. Dilution experiments show that PLA2 is an extremely tight dimer, Kd < or = 0.01 nM, in solution. Urea was introduced to weaken the subunit's association, and an estimate for the PLA(2) dimer dissociation constant in buffer was obtained by linear extrapolation. The derived dissociation constant was at least several orders of magnitude greater than that suggested from the dilution experiments, indicating a complex interaction between urea and the PLA2 dimer. FCS data indicate that the PLA2 dimer begins to dissociate ... [truncated at 150 words]
Breusegem SY, Sadat-Ebrahimi SE, Douglas KT, Clegg RM, Loontiens FG.
Increased stability and lifetime of the complex formed between DNA and meta-phenyl-substituted Hoechst dyes as studied by fluorescence titrations and stopped-flow kinetics.
J Mol Biol. 2001; 308(4): 649-63.The large increase in fluorescence upon binding of five para- and meta-phenyl substituted hydroxy and methoxy derivatives of the Hoechst dye with poly[d(A-T)], d(CGCGAATTCGCG)2, and its corresponding T4-looped 28-mer hairpin was used to monitor the binding by equilibrium titrations and by stopped-flow kinetics. The affinity increases in the same order for the three DNAs: p-OH<m-OCH3, p-OH<m-OH<m-OH, p-OCH3<bis-m-OH. The association constants K(a) are three to 11 times larger for the AATT site than for poly[d(A-T)]. The AATT site binds m-OH Hoechst with K(a)=3.8 x 10(9 )M(-1) and bis-m-OH Hoechst with K(a)=1.9 x 10(10 )M(-1), which are seven and 37 times higher than p-OH Hoechst (Hoechst 33258), respectively. The high K(a )values determined at equilibrium agree with the kinetically defined association constants K(kin)=k(on)/k(off). The association-rate parameters k(on) were obtained by stopped-flow kinetics and the dissociation-rate parameters k(off) by dissociation kinetics using poly[d(A-5BrU)]. For binding to the AATT site, k(on) values are similar ... [truncated at 150 words]
Tricerri MA, Agree AKB, Sánchez SA, Bronski J, Jonas A.
Arrangement of apolipoprotein A-I in reconstituted high-density lipoprotein disks: an alternative model based on fluorescence resonance energy transfer experiments.
Biochemistry. 2001; 40(16): 5065-74.The folding and organization of apolipoprotein A-I (apoA-I) in discoidal, high-density lipoprotein (HDL) complexes with phospholipids are not yet completely resolved. For about 20 years, it was generally accepted that the amphipathic helices of apoA-I lie parallel to the acyl chains of the phospholipids ("picket fence" model). However, based on the X-ray crystal structure of a large, lipid-free fragment of apoA-I, a "belt model" was recently proposed. In this model, the helices of two antiparallel apoA-I molecules are extended in a circular arrangement and lie perpendicular to the phospholipid acyl chains. To obtain conclusive information on the spatial organization of apoA-I in discoidal HDL, we engineered three separate cysteine mutants of apoA-I (D9C, A124C, A232C) for specific labeling with the fluorescence probes ALEXA-488 or ALEXA-546 (fluorescein and rhodamine derivatives). The labeled apoA-I was reconstituted into well-defined HDL complexes containing two molecules of protein and dipalmitoylphosphatidylcholine, and the complexes were used ... [truncated at 150 words]
Krasnowska EK, Bagatolli LA, Gratton E, Parasassi T.
Surface properties of cholesterol-containing membranes detected by Prodan fluorescence.
Biochim Biophys Acta. 2001; 1511(2): 330-40.The fluorescent membrane probe 6-propionyl-2-dimethylaminonaphthalene (Prodan) displays a high sensitivity to the polarity and packing properties of lipid membrane. Contrary to 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), Prodan can also monitor the properties of the membrane surface, i.e., the polar-head pretransition. In bilayers composed of coexisting gel and liquid-crystalline phases, Prodan shows a preferential partitioning in the latter, so that the detected membrane properties mainly belong to fluid domains. In the presence of cholesterol, the packing properties of the gel phase phospholipids are modified in such a way that Prodan can penetrate and label the membrane. Although Prodan labeling of the gel phase is a function of cholesterol concentration, 3 mol percent cholesterol is sufficient for a 60% Prodan labeling with respect to the maximum labeling reached at 15 mol percent cholesterol. We present steady-state and dynamical fluorescence measurements of Prodan in bilayers in the presence of cholesterol. Our results fit the liquid-ordered/liquid-disordered phase ... [truncated at 150 words]
Toronov VY, Webb A, Choi JH, Wolf M, Michalos A, Gratton E, Hueber DM.
Investigation of human brain hemodynamics by simultaneous near-infrared spectroscopy and functional magnetic resonance imaging.
Med Phys. 2001; 28(4): 521-7.The aim of this study was to compare functional cerebral hemodynamic signals obtained simultaneously by near infrared spectroscopy (NIRS) and by functional magnetic resonance imaging (fMRI). The contribution of superficial layers (skin and skull) to the NIRS signal was also assessed. Both methods were used to generate functional maps of the motor cortex area during a periodic sequence of stimulation by finger motion and rest. In all subjects we found a good collocation of the brain activity centers revealed by both methods. We also found a high temporal correlation between the BOLD signal (fMRI) and the deoxy-hemoglobin concentration (NIRS) in the subjects who exhibited low fluctuations in superficial head tissues.
Chirico G, Bettati S, Mozzarelli A, Chen Y, Müller JD, Gratton E.
Molecular heterogeneity of O-acetylserine sulfhydrylase by two-photon excited fluorescence fluctuation spectroscopy.
Biophys J. 2001; 80(4): 1973-85. PMCID: PMC1301386O-acetylserine sulfhydrylase, a homo-dimeric enzyme from Salmonella typhimurium, covalently binds one pyridoxal 5'-phosphate molecule per subunit as a fluorescent coenzyme. Different tautomers of the Schiff base between the coenzyme and lysine 41 generate structured absorption and fluorescence spectra upon one-photon excitation. We investigated the protein population heterogeneity by fluorescence correlation spectroscopy and lifetime techniques upon two-photon excitation. We sampled the fluorescence intensity from a small number of molecules (approximately 10) and analyzed the distribution of photon counts to separately determine the number and the fluorescence brightness of excited protein molecules. The changes in the average number of molecules and in the fluorescence brightness with the excitation wavelength indicate the presence of at least two fluorescent species, with two-photon excitation maxima at 660 and 800 nm. These species have been identified as the enolimine and ketoenamine tautomers of the protein-coenzyme internal aldimine. Their relative abundance is estimated to be 4:1, whereas ... [truncated at 150 words]
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Hydration and protein substates: fluorescence of proteins in reverse micelles.
J Mol Liq. 2001; 45(3): 253-272.The fluorescence properties of indole derivatives, lysozyme and azurin were investigated in reverse micelles of detergent sodium bis[2-ethylhexl]sulfosuccinate (Aerosol OT)* in n-hexane. L-tryptophan, l-methyl-tryptophan and n-acetyl-l-tryptophanamide exhibited complex fluorescence decays in reverse micelles. Fluorescence decays were best described using Gaussian bimodal distributions of lifetimes. Increasing hydration levels in the micelles resulted in a decrease in decay heterogeneity, as indicated by a large decrease in lifetime distribution widths. Steady-state polarization and fluorescence emission measurements indicated both an increase in average polarity of the environment around the indole derivatives and an increase in the mobility of the probes with increasing hydration levels. The fluorescence decays of lysozyme and azurin in reverse micelles were also found to be very complex and were described with Gaussian lifetime distributions. Increasing water content in the micelles caused marked decreases in both center and width of the lifetime distributions for these two proteins. Steady-state polarization measurements as ... [truncated at 150 words]
van Rompaey E, Chen Y, Müller JD, Gratton E, van Craenenbroeck E, Engelborghs Y, de Smedt S, Demeester J.
Fluorescence fluctuation analysis for the study of interactions between oligonucleotides and polycationic polymers.
Biol Chem. 2001; 382(3): 379-386.The interactions between a cationic polymer, poly(2-dimethylamino)ethyl methacrylate (pDMAEMA), and negatively charged rhodamine-labeled 25-mer phosphodiester oligonucleotides (Rh-ONs) were studied by fluorescence fluctuation spectroscopy and other techniques. The composition of the pDMAEMA/Rh-ON complexes was investigated as a function of the charge ratio (±) by increasing the pDMAEMA concentration and keeping the Rh-ON concentration constant. We applied two different methods for analyzing the fluorescence fluctuation profiles of the pDMAEMA/Rh-ON complexes, which depended on their composition. First, we analyzed the data with the photon counting histogram (PCH) technique, which determines the molecular brightness and the concentration of fluorophores (Chen et al, 1999). A particular challenge for the data analysis is the occurrence of sudden fluorescence bursts in the fluorescence fluctuation profiles, which are linked to the appearance of multimolecular complexes (i. e. when several Rh-ONs were present in one complex). A quantitative interpretation of the analysis for the complexes remains challenging and is ... [truncated at 150 words]
Dietrich C, Bagatolli LA, Volovyk ZN, Thompson NL, Levi M, Jacobson KA, Gratton E.
Lipid rafts reconstituted in model membranes.
Biophys J. 2001; 80(3): 1417-28. PMCID: PMC1301333One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a ... [truncated at 150 words]
Dong CY, Buehler C, So PTC, French TE, Gratton E.
Implementation of intensity-modulated laser diodes in time-resolved, pump-probe fluorescence microscopy.
Appl Opt. 2001; 40(7): 1109-1115.We present the implementation of intensity-modulated laser diodes for applications in frequency-domain pump-probe fluorescence microscopy. Our technique, which is based on the stimulated-emission approach, uses two sinusoidally modulated laser diodes. One laser (635 nm) excites the chromophores under study, and the other laser (680 nm) is responsible for inducing stimulated emission from excited-state molecules. Both light sources are modulated in the 80-MHz range but with an offset of 5 kHz between them. The result of the interaction of the pump and the probe beams is that a cross-correlation fluorescence signal at 5 kHz is generated primarily at the focal volume. Microscope imaging at the cross-correlation signal results in images with high contrast, and time-resolved high-frequency information can be acquired without high-speed detection. A detailed experimental arrangement of our methodology is presented along with images acquired from a 4.0- m-diameter fluorescent sphere and TOTO-3 -labeled mouse STO cells. (TOTO-3 is a ... [truncated at 150 words]
Margeat E, Poujol N, Boulahtouf A, Chen Y, Müller JD, Gratton E, Cavailles V, Royer CA.
The human estrogen receptor alpha dimer binds a single SRC-1 coactivator molecule with an affinity dictated by agonist structure.
J Mol Biol. 2001; 306(3): 433-42.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 study we have quantitatively characterized the interactions of full-length baculovirus expressed human estrogen receptor alpha (ERalpha), as well as ERalpha hormone binding domain (ERHBD) with a fragment of the coactivator protein SRC-1 (amino acid residues 570 to 780). Fluorescence anisotropy and fluorescence correlation spectroscopy of fluorescently labeled SRC-1(570-780) demonstrate unambiguously that the stoichiometry of the SRC-1/ERalpha/estradiol complex is one coactivator molecule per ERalpha dimer. The affinity of the estradiol or estriol bound ERalpha/SRC-1 complexes was found to be significantly higher than that observed in the presence of estrone. No binding was observed in the absence of ligand or ... [truncated at 150 words]
Nayfeh MH, Barry NP, Therrien J, Akcakir O, Gratton E, Belomoin G.
Stimulated blue emission in reconstituted films of ultrasmall silicon nanoparticles.
Appl Phys Lett. 2001; 78(8): 1131-1133.We dispersed electrochemical etched Si into a colloid of ultrabright blue luminescent nanoparticles (1 nm in diameter) and reconstituted it into films or microcrystallites. When the film is excited by a near-infrared two-photon process at 780 nm, the emission exhibits a sharp threshold near 106 W/cm2, rising by many orders of magnitude, beyond which a low power dependence sets in. Under some conditions, spontaneous recrystallization forms crystals of smooth shape from which we observe collimated beam emission, pointing to very large gain coefficients. The results are discussed in terms of population inversion, produced by quantum tunneling or/and thermal activation, and stimulated emission in the quantum confinement-engineered Si–Si phase found only on ultrasmall Si nanoparticles. The Si–Si phase model provides gain coefficients as large as 103–105 cm–1.
Barcellona ML, Chen Y, Müller JD, Gratton E.
4',6-diamidino-2-phenylindole (DAPI) interacts with rare structures of GC polymers.
Eur Biophys J. 2001; 30(2): 98-109.The binding of 4',6-diamidino-2-phenylindole (DAPI) to double-stranded GC polymers either in the alternating or in homopolymer sequence was investigated using fluorescence techniques. We employed fluctuation correlation spectroscopy, which measures the diffusion coefficient of fluorescent particles, to demonstrate that the fluorescence was originating from relatively slowly diffusing entities. These entities display a very large heterogeneity of diffusing coefficients, indicating that molecular aggregation is extensive in our samples. We used frequency domain fluorometry to characterize the fluorescence lifetime of the species, while varying the GC polymer-dye coverage systematically. At very low coverage we observed a relatively bright fluorescent component with a lifetime value of approximately 4 ns. The stoichiometry of binding of this bright species was such that it can only arise from rare molecular structures, either unusual loops or large molecular aggregates. The amount and characteristics of this bright fluorescent component were different between the homo and the alternating polymer, indicating ... [truncated at 150 words]
Bettati S, Benci S, Campanini B, Raboni S, Chirico G, Beretta S, Schnackerz KD, Hazlett TL, Gratton E, Mozzarelli A.
Role of pyridoxal 5'-phosphate in the structural stabilization of O-acetylserine sulfhydrylase.
J Biol Chem. 2000; 275(51): 40244-51.Proteins belonging to the superfamily of pyridoxal 5'-phosphate-dependent enzymes are currently classified into three functional groups and five distinct structural fold types. The variation within this enzyme group creates an ideal system to investigate the relationships among amino acid sequences, folding pathways, and enzymatic functions. The number of known three-dimensional structures of pyridoxal 5'-phosphate-dependent enzymes is rapidly increasing, but only for relatively few have the folding mechanisms been characterized in detail. The dimeric O-acetylserine sulfhydrylase from Salmonella typhimurium belongs to the beta-family and fold type II group. Here we report the guanidine hydrochloride-induced unfolding of the apo- and holoprotein, investigated using a variety of spectroscopic techniques. Data from absorption, fluorescence, circular dichroism, (31)P nuclear magnetic resonance, time-resolved fluorescence anisotropy, and photon correlation spectroscopy indicate that the O-acetylserine sulfhydrylase undergoes extensive disruption of native secondary and tertiary structure before monomerization. Also, we have observed that the holo-O-acetylserine sulfhydrylase exhibits a greater ... [truncated at 150 words]
Nayfeh MH, Akcakir O, Belomoin G, Barry NP, Therrien J, Gratton E.
Second harmonic generation in microcrystallite films of ultrasmall Si nanoparticles.
Appl Phys Lett. 2000; 77(25): 4086-4088.We dispersed crystalline Si into a colloid of ultrasmall nano particles (~1 nm), and reconstituted it into microcrystallites films on device-quality Si. The film is excited by near-infrared femtosecond two-photon process in the range 765-835 nm, with incident average power in the range 15-70 mW, focused to ~1 μm. We have observed strong radiation at half the wavelength of the incident beam. The results are analyzed in terms of second-harmonic generation, a process that is not allowed in silicon due to the centrosymmetry. Ionic vibration of or/and excitonic self-trapping on novel radiative Si-Si dimer phase, found only in ultrasmall nanoparticles, are suggested as a basic mechanism for inducing anharmonicity that breaks the centrosymmetry.
Holub O, Seufferheld MJ, Gohlke C, Govindjee, Clegg RM.
Fluorescence lifetime imaging (FLI) in real-time - a new technique in photosynthesis research.
Photosynthetica. 2000; 8(4): 581-599.We describe an instrument that allows the rapid measurement of fluorescence lifetime-resolved images of leaves as well as sub-cellular structures of intact plants or single cells of algae. Lifetime and intensity fluorescence images can be acquired and displayed in real time (up to 55 lifetime-resolved images per s). Our imaging technique therefore allows rapid measurements that are necessary to determine the fluorescence lifetimes at the maximum (P level) fluorescence following initial illumination during the chlorophyll (Chl) a fluorescence transient (induction) in photosynthetic organisms. We demonstrate the application of this new instrument and methodology to measurements of: (1) Arabidopsis thaliana leaves showing the effect of dehydration on the fluorescence lifetime images; (2) Zea mays leaves showing differences in the fluorescence lifetimes due to differences in the bundle sheath cells (having a higher amount of low yield photosystem 1) and the mesophyll cells (having a higher amount of high yield photosystem 2); ... [truncated at 150 words]
Tricerri MA, Agree AKB, Sánchez SA, Jonas A.
Characterization of apolipoprotein A-I structure using a cysteine-specific fluorescence probe.
Biochemistry. 2000; 39(47): 14682-91.Two new Cys mutants of proapolipoprotein A-I, D9C and A232C, were created and expressed in Escherichia coli systems. Specific labeling with the thiol-reactive fluorescence probe, 6-acryloyl-2-dimethylaminonaphthalene (acrylodan), was used to study the structural organization and dynamic properties of the extreme regions of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound states. Spectroscopic approaches, including circular dichroism and various fluorescence methods, were used to examine the properties of the mutant proteins and of their covalent adducts with the fluorescence probe. The mutations themselves had no effect on the structure and stability of apoA-I in the lipid-free state and in reconstituted HDL (rHDL) complexes. Furthermore, covalent modification with acrylodan did not alter the properties of the apoA-I variants in the lipid-bound state nor in the lipid-free A232C mutant, but it affected the structure and local stability of the lipid-free protein in the D9C mutant. Fluorescence results using the acrylodan probe confirmed a ... [truncated at 150 words]
Lamb DC, Schenk A, Röcker C, Nienhaus GU.
Determining chemical rate coefficients using time-gated fluorescence correlation spectroscopy.
J Phys Org Chem. 2000; 13(10): 654 - 658.In recent years, fluorescence correlation spectroscopy (FCS) has become an important technique for studying dynamic processes of molecules in thermodynamic equilibrium. Fluorescent organic molecules are excited by laser light, and the emitted light quanta from a small number of molecules in a volume of ~1 fl are collected using a high numerical aperture microscope objective and photon counting detection. Translational and rotational diffusion, chemical reactions (including photochemistry) and conformational changes of the molecules give rise to temporal correlations in the fluorescence intensity fluctuations that can be revealed by autocorrelation analysis. A method is presented to improve the sensitivity of FCS measurements on samples containing multiple fluorescent species. Using pulsed laser excitation in conjunction with electronic gating in the detection channel, we preferentially suppress the emission from the short lifetime components by fluorescence lifetime separation. We demonstrate the usefulness of this technique by applying it to the binding reaction of the ... [truncated at 150 words]
Chen Y, Müller JD, Tetin SY, Tyner JD, Gratton E.
Probing ligand protein binding equilibria with fluorescence fluctuation spectroscopy.
Biophys J. 2000; 79(2): 1074-1084. PMCID: PMC1301003We examine the binding of fluorescent ligands to proteins by analyzing the fluctuation amplitude g(0) of fluorescence fluctuation experiments. The normalized variance g(0) depends on the molecular brightness and the concentration of each species in the sample. Thus a single g(0) measurement is not sufficient to resolve individual species. Titration of the ligand with protein establishes the link between molecular brightness and concentration by fitting g(0) to a binding model and allows the separation of species. We first apply g(0) analysis to binary dye mixtures with brightness ratios of 2 and 4 to demonstrate the feasibility of this technique. Next we consider the influence of binding on the fluctuation amplitude g(0). The dissociation coefficient, the molecular brightness ratio, and the stochiometry of binding strongly influence the fluctuation amplitude. We show that proteins with a single binding site can be clearly differentiated from proteins with two independent binding sites. The binding ... [truncated at 150 words]
Lamb DC, Schenk A, Röcker C, Scalfi-Happ C, Nienhaus GU.
Sensitivity enhancement in fluorescence correlation spectroscopy of multiple species using time-gated detection.
Biophys J. 2000; 79(2): 1129-1138. PMCID: PMC1301008Fluorescence correlation spectroscopy (FCS) is a powerful technique to measure chemical reaction rates and diffusion coefficients of molecules in thermal equilibrium. The capabilities of FCS can be enhanced by measuring the energy, polarization, or delay time between absorption and emission of the collected fluorescence photons in addition to their arrival times. This information can be used to change the relative intensities of multiple fluorescent species in FCS measurements and, thus, the amplitude of the intensity autocorrelation function. Here we demonstrate this strategy using lifetime gating in FCS experiments. Using pulsed laser excitation and laser-synchronized gating in the detection channel, we suppress photons emitted within a certain time interval after excitation. Three applications of the gating technique are presented: suppression of background fluorescence, simplification of FCS reaction studies, and investigation of lifetime heterogeneity of fluorescently labeled biomolecules. The usefulness of this technique for measuring forward and backward rates of protein fluctuations ... [truncated at 150 words]
Sukhishvili SA, Chen Y, Müller JD, Gratton E, Schweizer KS, Granick S.
Materials science: diffusion of a polymer 'pancake'.
Nature. 2000; 406(6792): 146.Thread-like chains of flexible polymers that adsorb to a solid surface assume a flat 'pancake' conformation when the surface coverage is low and are only able to diffuse in two dimensions because so many segments are adsorbed. Here we show that the centre-of-mass diffusion coefficient of the polymer chain, measured at dilute coverage to ensure minimal chain–chain interaction, has a strong power-law dependence on the degree of polymerization. This nonlinear dependence of polymer diffusion on a solid surface contrasts with the linear dependence observed on a fluid membrane.
Casavola C, Paunescu LA, Fantini S, Gratton E.
Blood flow and oxygen consumption with near-infrared spectroscopy and venous occlusion: spatial maps and the effect of time and pressure of inflation.
J Biomed Opt. 2000; 5(3): 269-76.We have measured the local blood flow (BF) and oxygen consumption (OC) in the human calf muscle using near-infrared spectroscopy during venous occlusion. Venous occlusion was achieved by inflating a pneumatic cuff around the thigh of the subject. We have investigated the influence of the inflation time and cuff pressure on the recovered values of BF and OC. We have found that if the cuff pressure is increased from a threshold pressure (approximately 30 mm Hg) to a critical pressure (approximately 45 mm Hg) in less than about 6 s, one measures the same values of BF and OC independent of the total inflation time and final cuff pressure. We also report nine-pixel spatial maps of BF and OC to show that this technique can lead to spatially resolved measurements of blood flow and oxygen consumption in tissues.
Buehler C, Dong CY, So PTC, French TE, Gratton E.
Time-resolved polarization imaging by pump-probe (stimulated emission) fluorescence microscopy.
Biophys J. 2000; 79(1): 536-49. PMCID: PMC1300957We report the application of pump-probe fluorescence microscopy in time-resolved polarization imaging. We derived the equations governing the pump-probe stimulated emission process and characterized the pump and probe laser power levels for signal saturation. Our emphasis is to use this novel methodology to image polarization properties of fluorophores across entire cells. As a feasibility study, we imaged a 15-microm orange latex sphere and found that there is depolarization that is possibly due to energy transfer among fluorescent molecules inside the sphere. We also imaged a mouse fibroblast labeled with CellTracker Orange CMTMR (5-(and-6)-(((4-chloromethyl)benzoyl)amino)tetramethyl-rhodamine). We observed that Orange CMTMR complexed with gluthathione rotates fast, indicating the relatively low fluid-phase viscosity of the cytoplasmic microenvironment as seen by Orange CMTMR. The measured rotational correlation time ranged from approximately 30 to approximately 150 ps. This work demonstrates the effectiveness of stimulated emission measurements in acquiring high-resolution, time-resolved polarization information across the entire cell.
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A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: a two-photon fluorescence microscopy study.
Biophys J. 2000; 79(1): 434-47. PMCID: PMC1300947Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixtures were studied at different temperatures, by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temperatures corresponding to the fluid phase and the fluid-solid phase coexistence. In addition, we studied the solid-solid temperature regime for the DMPC/DSPC and DMPE/DMPC mixtures. From the Laurdan intensity images the generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region for all of the ... [truncated at 150 words]
Bagatolli LA, Gratton E, Khan TK, Chong PLG.
Two-photon fluorescence microscopy studies of bipolar tetraether giant liposomes from thermoacidophilic archaebacteria Sulfolobus acidocaldarius.
Biophys J. 2000; 79(1): 416-25. PMCID: PMC1300945The effects of temperature and pH on Laurdan (6-lauroyl-2-(dimethylamino)naphthalene) fluorescence intensity images of giant unilamellar vesicles (GUVs) ( approximately 20-150 microm in diameter) composed of the polar lipid fraction E (PLFE) from the thermoacidophilic archaebacteria Sulfolobus acidocaldarius have been studied using two-photon excitation. PLFE GUVs made by the electroformation method were stable and well suited for microscopy studies. The generalized polarization (GP) of Laurdan fluorescence in the center cross section of the vesicles has been determined as a function of temperature at pH 7.23 and pH 2.68. At all of the temperatures and pHs examined, the GP values are low (below or close to 0), and the GP histograms show a broad distribution width (> 0.3). When excited with light polarized in the y direction, Laurdan fluorescence in the center cross section of the PLFE GUVs exhibits a photoselection effect showing much higher intensities in the x direction of the ... [truncated at 150 words]
Zolese G, Falcioni G, Bertoli E, Galeazzi R, Wozniak M, Wypych Z, Gratton E, Ambrosini A.
Steady-state and time resolved fluorescence of albumins interacting with N-oleylethanolamine, a component of the endogenous N-acylethanolamines.
Proteins. 2000; 40(1): 39-48.The functions of N-acylethanolamines, minor constituents of mammalian cells, are poorly understood. It was suggested that NAEs might have some pharmacological actions and might serve as a cytoprotective response, whether mediated by physical interactions with membranes or enzymes or mediated by activation of cannabinoid receptors. Albumins are identified as the major transport proteins in blood plasma for many compounds including fatty acids, hormones, bilirubin, ions, and many drugs. Moreover, albumin has been used as a model protein in many areas, because of its multifunctional binding properties. Bovine (BSA) and human (HSA) serum albumin are similar in sequence and conformation, but differ for the number of tryptophan residues. This difference can be used to monitor unlike protein domains. Our data suggest that NOEA binds with high affinity to both albumins, modifying their conformational features. In both proteins, NOEA molecules are linked with higher affinity to hydrophobic sites near Trp-214 in HSA ... [truncated at 150 words]
Parasassi T, Yu W, Durbin DM, Kuriashkina LR, Gratton E, Maeda N, Ursini F.
Two-photon microscopy of aorta fibers shows proteolysis induced by LDL hydroperoxides.
Free Radic Biol Med. 2000; 28(11): 1589-1597.Oxidatively modified LDL mimics several aspects of atherogenesis. In this disease, degradation of the matrix proteins' network also occurs. By a new morphological ex vivo approach, not requiring sample processing, we explored the relationship between the degradation of matrix protein and oxidatively modified LDL. Two-photon excitation fluorescence microscopy images of fresh cross-section rings of rat aorta, acquired while the sample was maintained in a glucose- and oxygen-supplemented buffer, showed straight, parallel, thick, long extracellular matrix proteins. Traditional microscopic examination, requiring sample fixation and staining, shows smaller and curved fibers. Instead, we observed curved and broken fibers after a 30-min incubation of aorta with either LDL containing lipid hydroperoxides, or tert-butyl-hydroperoxide. The adhesion of LDL to the endothelium and its internalization was directly visualized by using a lipid fluorophore. The damage to aorta matrix proteins induced by LDL and tert-butyl-hydroperoxide was fully prevented by antioxidants, such as ascorbate or Trolox C, ... [truncated at 150 words]
Frolov A, Petrescu A, Atshaves BP, So PTC, Gratton E, Serrero G, Schroeder F.
High density lipoprotein-mediated cholesterol uptake and targeting to lipid droplets in intact L-cell fibroblasts. A single- and multiphoton fluorescence approach.
J Biol Chem. 2000; 275(17): 12769-12780.Fluorescent sterols, dehydroergosterol and NBD-cholesterol, were used to examine high density lipoprotein-mediated cholesterol uptake and intracellular targeting in L-cell fibroblasts. The uptake, but not esterification or targeting to lipid droplets, of these sterols differed >100-fold, suggesting significant differences in uptake pathways. NBD-cholesterol uptake kinetics and lipoprotein specificity reflected high density lipoprotein-mediated sterol uptake via the scavenger receptor B1. Fluorescence energy transfer showed an average intermolecular distance of 26 A between the two fluorescent sterols in L-cells. Indirect immunofluorescence revealed that both fluorescent sterols localized to L-cell lipid droplets, the surface of which contained adipose differentiation-related protein. This lipid droplet-specific protein specifically bound NBD-cholesterol with high affinity (K(d) = 2 nM) at a single site. Thus, NBD-cholesterol and dehydroergosterol were useful fluorescent probes of sterol uptake and intracellular sterol targeting. NBD-cholesterol more selectively probed high density lipoprotein-mediated uptake and rapid intracellular targeting of sterol to lipid droplets. Targeting of sterol to ... [truncated at 150 words]
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.
Appl Phys Lett. 2000; 76(14): 1857-1859.We dispersed electrochemical etched Si into a colloid of ultrasmall blue luminescent nanoparticles, observable with the naked eye, in room light. We use two-photon near-infrared femtosecond excitation at 780 nm to record the fluctuating time series of the luminescence, and determine the number density, brightness, and size of diffusing fluorescent particles. The luminescence efficiency of particles is high enough such that we are able to detect a single particle, in a focal volume, of 1 pcm. The measurements yield a particle size of 1 nm, consistent with direct imaging by transmission electron microscopy. They also yield an excitation efficiency under two-photon excitation two to threefold larger than that of fluorescein. Detection of single particles paves the way for their use as labels in biosensing applications.
Bagatolli LA, Parasassi T, Gratton E.
Giant phospholipid vesicles: comparison among the whole lipid sample characteristics using different preparation methods: a two photon fluorescence microscopy study.
Chem Phys Lipids. 2000; 105(2): 135-47.Several methods for the preparation of giant unilamellar vesicles (GUVs) using synthetic phosphatidylcholine phospholipids were evaluated. We compared the physical characteristics–in terms of lamellarity and morphology–of the whole lipid sample for each different lipid preparation using the sectioning capability of the two-photon excitation fluorescence microscope. From the evaluation of the entire lipid sample we determined that vesicle size, internal shape and shell thickness distributions depend on the vesicle's preparation method. Our results show that the preparation of giant unilamellar vesicles by the application of external electric fields offers several advantages among the other methods tested here. Using this method a high yield (approximately 95%) of giant unilamellar vesicles with a narrow size distribution was obtained. Independently of the preparation method, some lipid structures, which are held together by lipid tethers, were identified and resolved. These particular lipid structures show shell thickness and size heterogeneity. Labeling the lipid samples with 6-lauroyl-2-(N,N-dimethylamino)naphtalene ... [truncated at 150 words]
Toronov VY, Franceschini MA, Filiaci ME, Fantini S, Wolf M, Michalos A, Gratton E.
Near-infrared study of fluctuations in cerebral hemodynamics during rest and motor stimulation: temporal analysis and spatial mapping.
Med Phys. 2000; 27(4): 801-15.We have noninvasively studied the motor cortex hemodynamics in human subjects under rest and motor stimulation conditions using a multichannel near-infrared tissue spectrometer. Our instrument measures optical maps of the cerebral cortex at two wavelengths (758 and 830 nm), with an acquisition time of 160 ms per map. We obtained optical maps of oxy- and deoxy-hemoglobin concentration changes in terms of amplitudes of folding average, power spectrum and coherence at the stimulation repetition frequency, and the phase synchronization index. Under periodic motor stimulation conditions, we observed coherence and frequency or phase synchronization of the local hemodynamic changes with stimulation. Our main findings are the following: (1) The amplitude of the hemodynamic response to the motor stimulation is comparable to the amplitude of the fluctuations at rest. (2) The spatial patterns of the oxy- and deoxy-hemoglobin responses to the stimulation are different. (3) The hemodynamic response to stimulation shows a spatial ... [truncated at 150 words]
Stankovic MR, Maulik D, Rosenfeld W, Stubblefield PG, Kofinas AD, Gratton E, Franceschini MA, Fantini S, Hueber DM.
Role of frequency domain optical spectroscopy in the detection of neonatal brain hemorrhage - a newborn piglet study.
J Mat Fet Med. 2000; 9(2): 142-9.OBJECTIVE: Inability of continuous wave (CW) optical spectroscopy to measure changes in scattering, and the use of an arbitrary rather than an actual baseline, makes the CW method highly susceptible to errors that can lead to a false-positive or false-negative diagnosis. Our objective was to assess whether, and to what extent, the use of quantitative frequency domain spectroscopy would improve our ability to detect and monitor the development of brain hemorrhage. METHODS: A dual-channel frequency-domain tissue spectrometer (Model 96208, ISS, Inc., Champaign, IL) was used to monitor the development of experimental subcortical and periventricular-intraventricular hemorrhage (IVH) in 10 newborn piglets (blood injection model). The multidistance approach was employed to calculate the absorption and reduced scattering coefficients and hemoglobin changes from the ac, dc, and phase values acquired at four different source-detector distances and at 752 nm and 830 nm. RESULTS: There were significant absorption and scattering changes in the subcortical ... [truncated at 150 words]
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Optical spectroscopy in studies of antibody-hapten interactions.
Methods. 2000; 20(3): 341-61.This article describes the use of optical spectroscopy in studying antibody-hapten interactions and in determining the equilibrium binding constants. Along with equilibrium binding data, spectroscopic tools often deliver structural information on binding-induced conformational changes of antibodies (or haptens). Structural implications of results from example antibody-hapten systems are included. Fluorescence spectroscopy has been particularly useful in the area of ligand binding, and thus steady-state fluorescence quenching and fluorescence polarization are the primary techniques under discussion. A brief description of fluorescence correlation spectroscopy is also provided. Absorption techniques, including circular dichroism, are mentioned to a lesser extent. A basic description of the mathematical models involved in the analysis of binding equilibria is provided along with references to more complete works. Simulated and experimental data are used to illustrate the various experimental protocols and the appropriate analytical methods. Typical sources of errors and experimental precautions are indicated throughout the general discussion.
Eid JS, Müller JD, Gratton E.
Data acquisition card for fluctuation correlation spectroscopy allowing full access to the detected photon sequence.
Rev Sci Instrum. 2000; 71(2): 361-368.Typically, fluctuation correlation spectroscopy (FCS) data acquisition cards measure the number of photon events per time interval (i.e., bin)—time mode. Commercial FCS cards combine the bins through hardware in order to calculate the autocorrelation function. Such a design therefore does not yield the time resolved photon sequence, but only the autocorrelation of that sequence. A different acquisition method which measures the number of time intervals between photon events has been implemented—photon mode. This method takes advantage of the fact that in FCS the rate of photon counts is much less than the frequency of the clock that is used to determine the temporal location of the photons. By using this new mode of data acquisition, the current card design allows for 25 ns time resolution. The data acquisition card can operate in both time and photon mode and yields the time resolved sequence of photon arrivals in both cases. Therefore, ... [truncated at 150 words]
Franceschini MA, Toronov VY, Filiaci ME, Gratton E, Fantini S.
On-line optical imaging of the human brain with 160-ms temporal resolution.
Opt Express. 2000; 6(3): 49-57.We have developed an instrument for non-invasive optical imaging of the human brain that produces on-line images with a temporal resolution of 160 ms. The imaged quantities are the temporal changes in cerebral oxy-hemoglobin and deoxy-hemoglobin concentrations. We report real-time videos of the arterial pulsation and motor activation recorded on a 4 × 9 cm^2 area of the cerebral cortex in a healthy human subject. This approach to optical brain imaging is a powerful tool for the investigation of the spatial and temporal features of the optical signals collected on the brain.
Arcangeli C, Yu W, Cannistraro S, Gratton E.
Two-photon autofluorescence microscopy and spectroscopy of Antarctic fungus: new approach for studying effects of UV-B irradiation.
Biopolymers. 2000; 57(4): 218-25.We combined two-photon fluorescence microscopy and spectroscopy to provide functional images of UV-B (280-315 nm) induced stress on an Antarctic fungus. Two-photon excitation microscopy was used to characterize the distribution of autofluorescence inside the spore and the hyphae of the fungus. The imaging analysis clearly shows that the autofluorescence response of spores is higher than that of hyphae. The imaging analysis at different depths shows that, strikingly enough, the spore autofluorescence originates from the cell wall and membrane fluorophores. The spectroscopic results show moreover that the fluorescence spectra of spores are redshifted upon UV-B irradiation. Tentative identification of the chromophores involved in the autofluorescence response and their biological relevance are also discussed on the basis of a previous steady-state fluorescence spectroscopic study performed on both whole spore suspension and organic-soluble extracts.
Müller JD, Chen Y, Gratton E.
Resolving heterogeneity on the single molecular level with the photon-counting histogram.
Biophys J. 2000; 78(1): 474-86. PMCID: PMC1300655The diffusion of fluorescent particles through a small, illuminated observation volume gives rise to intensity fluctuations caused by particle number fluctuations in the open observation volume and the inhomogeneous excitation-beam profile. The intensity distribution of these fluorescence fluctuations is experimentally captured by the photon-counting histogram (PCH). We recently introduced the theory of the PCH for diffusing particles (Chen et al., Biophys. J., 77:553-567), where we showed that we can uniquely describe the distribution of photon counts with only two parameters for each species: the molecular brightness of the particle and the average number of particles within the observation volume. The PCH is sensitive to the molecular brightness and thus offers the possibility to separate a mixture of fluorescent species into its constituents, based on a difference in their molecular brightness alone. This analysis is complementary to the autocorrelation function, traditionally used in fluorescence fluctuation spectroscopy, which separates a mixture of ... [truncated at 150 words]
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Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures.
Biophys J. 2000; 78(1): 290-305. PMCID: PMC1300637Images of giant unilamellar vesicles (GUVs) formed by different phospholipid mixtures (1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1, 2-dilauroyl-sn-glycero-3-phosphocholine (DPPC/DLPC) 1:1 (mol/mol), and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPE/DPPC), 7:3 and 3:7 (mol/mol) at different temperatures were obtained by exploiting the sectioning capability of a two-photon excitation fluorescence microscope. 6-Dodecanoyl-2-dimethylamino-naphthalene (LAURDAN), 6-propionyl-2-dimethylamino-naphthalene (PRODAN), and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE) were used as fluorescent probes to reveal domain coexistence in the GUVs. We report the first characterization of the morphology of lipid domains in unsupported lipid bilayers. From the LAURDAN intensity images the excitation generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domain. On the basis of the phase diagram of each lipid mixture, we found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region in all lipid mixtures. At temperatures corresponding to the phase coexistence region we observed lipid domains of different sizes and ... [truncated at 150 words]
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Fluorescence photon-density waves in optically diffusive media.
Optics Communications. 2000; 173(1): 73-79.Intensity-modulated light launches traveling photon-density waves into optically diffusive media. In the presence of a fluorophore, excitation photon-density waves generate fluorescence photon-density waves that can be quantitatively described using diffusion theory. We examine a number of limiting cases of the fluorescence photon-density wave to clarify its physical meaning and its implications in quantitative fluorescence spectroscopy of diffusive media. Our discussion may guide the development of experimental protocols for quantitative fluorescence spectroscopy in optically diffusive media.
Nayfeh MH, Akcakir O, Therrien J, Yamani Z, Barry NP, Yu W, Gratton E.
Highly nonlinear photoluminescence threshold in porous silicon.
Appl Phys Lett. 1999; 75(26): 4112.Porous silicon is excited using near-infrared femtosecond pulsed and continuous wave radiation at an average intensity of ~106 W/cm2 (8×1010 W/cm2 peak intensity in pulsed mode). Our results demonstrate the presence of micron-size regions for which the intensity of the photoluminescence has a highly nonlinear threshold, rising by several orders of magnitude near this incident intensity for both the pulsed and continuous wave cases. These results are discussed in terms of stimulated emission from quantum confinement engineered intrinsic Si-Si radiative traps in ultrasmall nanocrystallites, populated following two-photon absorption.
Davidson WS, Arnvig-McGuire K, Kennedy A, Kosman J, Hazlett TL, Jonas A.
Structural organization of the N-terminal domain of apolipoprotein A-I: studies of tryptophan mutants.
Biochemistry. 1999; 38(43): 14387-95.Site-directed mutagenesis and detailed fluorescence studies were used to study the structure and dynamics of recombinant human proapolipoprotein (proapo) A-I in the lipid free state and in reconstituted high-density lipoprotein (rHDL) particles. Five different mutants of proapoA-I, each containing a single tryptophan residue, were produced in bacteria corresponding to each of the naturally occurring Trp residues (position -3 in the pro-segment, 8, 50, 72, and 108) in the N-terminal half of the protein. Structural analyses indicated that the conservative Phe-Trp substitutions did not perturb the conformation of the mutants with respect to the wild-type protein. Steady-state fluorescence studies indicated that all of the Trp residues exist in nonpolar environments that are highly protected from solvent in both the lipid-free and lipid-bound forms. Time-resolved lifetime and anisotropy studies indicated that the shape of the monomeric form of proapoA-I is a prolate ellipsoid with an axial ratio of about 6:1. In addition, ... [truncated at 150 words]
Bagatolli LA, Parasassi T, Fidelio GD, Gratton E.
A model for the interaction of 6-lauroyl-2-(N,N-dimethylamino)naphthalene with lipid environments: implications for spectral properties.
Photochem Photobiol. 1999; 70(4): 557-64.Although 6-lauroyl-2-(N,N-dimethylamino)naphthalene (LAURDAN) is now widely used as a probe for lipid systems, most studies focus on the effect of the lipid environment on its emission properties but not on the excitation properties. The present study is intended to investigate the excitation properties of LAURDAN in diverse lipid environments. To this end, the fluorescence properties of LAURDAN were studied in synthetic ester and ether phosphatidylcholines and sphingomyelin vesicles below, at and above the corresponding lipid main phase-transition temperature. The excitation spectra of LAURDAN in these environments always show at least two well-resolved bands. In the different lipid vesicles the behavior of the red band in the LAURDAN excitation spectra is sensitive to the lipid chemical environment near the probe fluorescent moiety and to the packing of the different lipid phases (gel and liquid crystalline). We propose that the interaction between the LAURDAN dimethylamino group and the ester linkage of ester ... [truncated at 150 words]
Chen Y, Müller JD, Berland KM, Gratton E.
Fluorescence fluctuation spectroscopy.
Methods. 1999; 19(2): 234-52.The analysis of the intensity fluctuation of a fluorescence signal from a relatively small volume and from a few molecules contains information about the distribution of different species present in the solution and about kinetic parameters of the system. The same information is generally averaged out when the fluorescence experiment is performed in a much larger volume, typically a cuvette experiment. The fundamental reason for this difference is that the fluctuations of the fluorescence signal from a few molecules directly reflect the molecular nature of the matter. Only recently, with the advent of confocal microscopy and two-photon excitation, it has become practical to achieve small excitation volumes in which only a few fluorescent molecules are present. We introduce the concept of fluctuation spectroscopy and highlight some of the technical aspects. We discuss different analysis methods used in fluctuation spectroscopy and evaluate their use for studying protein-protein interactions.
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Two-photon fluorescence microscopy observation of shape changes at the phase transition in phospholipid giant unilamellar vesicles.
Biophys J. 1999; 77(4): 2090-101. PMCID: PMC1300490Using the sectioning effect of the two-photon fluorescence microscope, we studied the behavior of phospholipid giant unilamellar vesicles (GUVs) composed of pure diacylphosphatidylcholine phospholipids during the gel-to-liquid crystalline phase transition. We used the well-characterized excitation generalized polarization function (GP(ex)) of 6-dodecanoyl-2-dimethylamine-naphthalene (LAURDAN), which is sensitive to the changes in water content in the lipid vesicles, to monitor the phase transition in the GUVs. Even though the vesicles do not show temperature hysteresis at the main phase transition, we observed different behaviors of the vesicle shape, depending on how the GUV sample reaches the main phase transition. During the cooling cycles, we observed an increase in the vesicle diameter at the phase transition ( approximately 0.5-1%), followed by a decrease in the diameter when the vesicle reached the gel phase. During the heating cycles and close to the phase transition temperature, a surprising behavior is observed, showing a sequence of different ... [truncated at 150 words]
Parasassi T, Gratton E, Zajicek HK, Levi M, Yu W.
Detecting membrane lipid microdomains by two-photon fluorescence microscopy.
IEEE Eng Med Biol Mag. 1999; 18(5): 92-9.Two photon excitation fluorescence microscopy is an emerging imaging tool for the study of biological samples [1]. The unique characteristics of two-photon excitation, such as the reduced sample photodamage and probe photobleaching, and a better background rejection compared to one-photon excitation, allow the prolonged observation and the study of samples that are difficult to measure using one-photon fluorescence microscopy [2,3], The spectroscopic properties of the emitting fluorophores can be characterized using two-photon microscopy [4].
This article discusses our ability to obtain, using two-photon excitation, microscopy images of the generalized polarization (GP) of the lipid probe 2-dimethylamino-6-lauroylnaphthalene (LAURDAN) in phospholipid vesicles and in natural membranes. The images show different distribution of the GP value depending on membrane composition. The use of linearly polarized excitation allowed the attribution of the GP heterogeneity to coexisting membrane domains of different dynamic properties. Based on the photoselection operated by the excitation polarization, we propose a model ... [truncated at 150 words]
Stankovic MR, Maulik D, Rosenfeld W, Stubblefield PG, Kofinas AD, Drexler S, Nair R, Franceschini MA, Hueber DM, Gratton E, Fantini S.
Real-time optical imaging of experimental brain ischemia and hemorrhage in neonatal piglets.
J Perinat Med. 1999; 27(4): 279-86.Our objective was to study the development of experimental brain ischemia and hemorrhage by real-time optical imaging. Optical imaging is based on the ability of near infrared light to non-invasively penetrate through the intact scalp and skull and measure brain concentrations of oxy- and deoxyhemoglobin, dominant brain absorbers. Optical imaging was performed in 7 anesthetized, instrumented, and ventilated newborn piglets subjected to the injection of 0.3 cc of saline followed by 2 cc of blood into the left frontal subcortical brain region via a needle inserted through the skull with stereotactic guidance. The image-acquisition rate of 5.26 images per sec allowed for real-time imaging. The detection threshold of the imager at the estimated depth of 1-1.5 cm was approximately 70 microL for saline and approximately 40 microL for blood. The imager readily detected five subcortical hematomas and two large bilateral subarachnoid hemorrhages. The imager detected a global decrease in brain ... [truncated at 150 words]
Chen Y, Müller JD, So PTC, Gratton E.
The photon counting histogram in fluorescence fluctuation spectroscopy.
Biophys J. 1999; 77(1): 553-67. PMCID: PMC1300352Fluorescence correlation spectroscopy (FCS) is generally used to obtain information about the number of fluorescent particles in a small volume and the diffusion coefficient from the autocorrelation function of the fluorescence signal. Here we demonstrate that photon counting histogram (PCH) analysis constitutes a novel tool for extracting quantities from fluorescence fluctuation data, i.e., the measured photon counts per molecule and the average number of molecules within the observation volume. The photon counting histogram of fluorescence fluctuation experiments, in which few molecules are present in the excitation volume, exhibits a super-Poissonian behavior. The additional broadening of the PCH compared to a Poisson distribution is due to fluorescence intensity fluctuations. For diffusing particles these intensity fluctuations are caused by an inhomogeneous excitation profile and the fluctuations in the number of particles in the observation volume. The quantitative relationship between the detected photon counts and the fluorescence intensity reaching the detector is given ... [truncated at 150 words]
Franceschini MA, Gratton E, Fantini S.
Noninvasive optical method of measuring tissue and arterial saturation:an application to absolute pulse oximetry of the brain.
Opt Lett. 1999; 24(12): 829-831.We present a frequency-domain optical method for real-time noninvasive measurement of absolute tissue and arterial saturation. This method is based on quantitative measurement of the tissue absorption spectrum (for tissue saturation) and of the amplitude of the arterial-pulsation-induced absorption oscillations (for arterial saturation) at eight wavelengths in the range 633–841 nm. We report results obtained from readings taken from the forehead of a healthy volunteer, showing baseline saturation values of 74.7 6 0.2% (tissue) and 96.9 6 0.5% (arterial). These values dropped to minimum values of 71.6 6 0.2% and 90.0 6 0.2%, respectively, after 1 min of reduced inspired oxygen concentration [10% (by volume) O2 from a baseline value of 21% O2].
Fantini S, Hueber DM, Franceschini MA, Gratton E, Rosenfeld W, Stubblefield PG, Maulik D, Stankovic MR.
Non-invasive optical monitoring of the newborn piglet brain using continuous-wave and frequency-domain spectroscopy.
Phys Med Biol. 1999; 44(6): 1543-63.We have used continuous-wave (CW) and frequency-domain spectroscopy to investigate the optical properties of the newborn piglet brain in vivo and non-invasively. Three anaesthetized, intubated, ventilated and instrumented newborn piglets were placed into a stereotaxic instrument for optimal experimental stability, reproducible probe-to-scalp optical contact and 3D adjustment of the optical probe. By measuring the absolute values of the brain absorption and reduced scattering coefficients at two wavelengths (758 and 830 nm), frequency-domain spectroscopy provided absolute readings (in contrast to the relative readings of CW spectroscopy) of cerebral haemoglobin concentration and saturation during experimentally induced perturbations in cerebral haemodynamics and oxygenation. Such perturbations included a modulation of the inspired oxygen concentration, transient brain asphyxia, carotid artery occlusion and terminal brain asphyxia. The baseline cerebral haemoglobin saturation and concentration, measured with frequency-domain spectroscopy, were about 60% and 42 microM respectively. The cerebral saturation values ranged from a minimum of 17% (during transient ... [truncated at 150 words]
Fantini S, Franceschini MA, Gratton E, Hueber DM, Rosenfeld W, Maulik D, Stubblefield PG, Stankovic MR.
Non-invasive optical mapping of the piglet brain in real time.
Opt Express. 1999; 4(8): 308-314.We have performed non-invasive, real-time optical mapping of the piglet brain during a subcortical injection of autologous blood. The time resolution of the optical maps is 192 ms, thus allowing us to generate a real-time video of the growing subcortical hematoma. The increased absorption at the site of blood injection is accompanied by a decreased absorption at the contralateral brain side. This contralateral decrease in the optical absorption and the corresponding time traces of the cerebral hemoglobin parameters are consistent with a reduced cerebral blood flow caused by the increased intracranial pressure.
Casavola C, Paunescu LA, Fantini S, Franceschini MA, Lugarà PM, Gratton E.
Application of near-infrared tissue oxymetry to the diagnosis of peripheral vascular disease.
Clin Hemorheol Microcirc. 1999; 21(3-4): 389-393.Near-infrared spectroscopy (NIRS) is a noninvasive technique to measure the tissue oxygenation in real time. This optical method has many advantages over the invasive analysis currently used for clinical tests. Among the possible applications of near-infrared oxymetry, we report three protocols (exercise, venous occlusion and tilting table) in conjunction with NIRS, and discuss their applicability in the diagnosis of peripheral vascular disease (PVD).
Lasagna M, Gratton E, Jameson DM, Brunet JE.
Apohorseradish peroxidase unfolding and refolding: intrinsic tryptophan fluorescence studies.
Biophys J. 1999; 76(1): 443-450. PMCID: PMC1302533The unfolding and refolding of apohorseradish peroxidase, as a function of guanidinium chloride concentration, were monitored by the intrinsic fluorescence intensity, polarization, and lifetime of the single tryptophan residue. The unfolding was reversible and characterized by at least three distinct stages-the intensity and lifetime data, for example, were both characterized by an initial increase followed by a decrease and then a plateau region. The lifetime data, in the absence and presence of guanidinium chloride, were heterogeneous and fit best to a model consisting of a major Gaussian distribution component and a minor, short discrete component. The observed increase in intensity in the initial stage of the unfolding process is attributed to the conversion of this short component into the longer, distributed component as the guanidinium chloride concentration increases. Our results clarify and amplify previous studies on the unfolding of apohorseradish peroxidase by guanidinium chloride.
Parasassi T, Krasnowska EK, Bagatolli LA, Gratton E.
Laurdan and Prodan as polarity-sensitive fluorescent membrane probes.
J Fluoresc. 1998; 8(4): 365-373.The steady-state and dynamic fluorescence spectral properties of 2-dimethylamino-6-lauroylnaphthalene (LAURDAN) and several other naphthalene derivatives are summarized to illustrate their sensitivity to the polarity of the environment. Results obtained both in solvents of different polarity and in phospholipid vesicles in two phase states are presented. The emission red shift observed in polar solvents and in the phospholipid liquid–crystalline phase is explained on the basis of dipolar relaxation of solvent molecules surrounding the fluorescent naphthalene moiety of these probes. In phospholipid environments, experimental evidence is shown that excludes the intramolecular relative reorientation of the dimethylamino and carbonyl groups in the naphthalene and the reorientation of the entire fluorescent moiety. The solvent dipolar relaxation observed for LAURDAN and PRODAN in phospholipid bilayers has been attributed to a small number of water molecules present at the membrane interface. A comparison between LAURDAN emission in phospholipid vesicles prepared in D2O and in H2O is ... [truncated at 150 words]
Franceschini MA, Fantini S, Paunescu LA, Maier JS, Gratton E.
Influence of a superficial layer in the quantitative spectroscopic study of strongly scattering media.
Appl Opt. 1998; 37(31): 7447-7458.We have experimentally investigated the meaning of the effective optical absorption a (eff) and the reduced scattering s (eff) coefficients measured on the surfaces of two-layered turbid media, using the diffusion equation for homogeneous, semi-infinite media. We performed frequency-domain spectroscopy in a reflectance geometry, using source detector distances in the range 1.5 4.5 cm. We measured 100 samples, each made of one layer (thickness in the range 0.08 1.6 cm) on top of one semi-infinite block. The optical properties of the samples were similar to those of soft tissues in the near infrared. We found that the measured effective optical coefficients are representative of the underlying block if the superficial layer is less than 0.4 cm thick, whereas they are representative of the superficial layer if it is more than 1.3 cm thick.
di Venere A, Mei G, Gilardi G, Rosato N, de Matteis F, McKay R, Gratton E, Finazzi-Agrò A.
Resolution of the heterogeneous fluorescence in multi-tryptophan proteins: ascorbate oxidase.
Eur J Biochem. 1998; 257(2): 337-43.Ascorbate oxidase is a copper-containing enzyme which catalyzes a redox reaction between vitamin C and molecular oxygen. The protein, which shows a complex tertiary structure, is an homodimer of monomers, each containing three domains and 14 tryptophan residues. Recently, we have demonstrated by spectroscopic and ultracentrifugation techniques the existence of a stable dimeric intermediate along the unfolding pathway of this enzyme [Mei, G., Di Venere, A., Buganza, M., Vecchini, P., Rosato, N. & Finazzi Agrò, A. (1997) Biochemistry 36, 10917-10922]. In this study, the steady-state and dynamic fluorescence features of ascorbate oxidase have been exploited in order to find a way of monitoring the individual subsystems of the protein. The fluorescence intensity and anisotropy upon excitation at 295 nm are extremely sensitive functions of the emission wavelength, indicating a great heterogeneity of the system. The emission decay collected through a cut-off filter can be analyzed in terms of two continuous ... [truncated at 150 words]
Helms MK, Hazlett TL, Mizuguchi H, Hasemann CA, Uyeda K, Jameson DM.
Site-directed mutants of rat testis fructose 6-phosphate, 2-kinase/fructose 2,6-bisphosphatase: localization of conformational alterations induced by ligand binding.
Biochemistry. 1998; 37(40): 14057-64.Site-directed mutagenesis was utilized to construct mutants, containing one or two tryptophan residues, of the bifunctional enzyme fructose 6-phosphate,2-kinase-fructose 2,6-bisphosphatase. Two of the single-tryptophan mutants (W15 and W64) had the tryptophan residue located in the kinase domain, which is in the N-terminal half, and two (W299 and W320) had the tryptophan residue located in the phosphatase domain, which is in the C-terminal half. The double-tryptophan mutants were W15/W64, W15/W299, W64/W299, and W299/W320. Dynamic polarization data indicated that these tryptophan residues had varying degrees of local mobility. Steady-state polarization data revealed energy transfer between the tryptophan residues in the double mutant W299/W320 but not in the W15/W64, W15/W299, or W64/W299 mutants, indicating the proximity of the W299 and W320 residues. The binding of fructose-6-phosphate resulted in a significant increase in the anisotropy of the W15 mutants, but did not affect the anisotropies of any of the other single-tryptophan mutants. Binding of ... [truncated at 150 words]
Sánchez SA, Hazlett TL, Brunet JE, Jameson DM.
Aggregation states of mitochondrial malate dehydrogenase.
Protein Sci. 1998; 7(10): 2184-9. PMCID: PMC2143840The oligomeric state of fluorescein-labeled mitochondrial malate dehydrogenase (L-malate NAD+ oxidoreductase; mMDH; EC 1.1.1.37), as a function of protein concentration, has been examined using steady-state and dynamic polarization methodologies. A "global" rotational relaxation time of 103 ± 7 ns was found for micromolar concentrations of mMDH-fluorescein, which is consistent with the reported size and shape of mMDH. Dilution of the mMDH-fluorescein conjugates, prepared using a phosphate buffer protocol, to nanomolar concentrations had no significant effect on the rotational relaxation time of the adduct, indicating that the dimer-monomer dissociation constant for mMDH is below 10(-9) M. In contrast to reports in the literature suggesting a pH-dependent dissociation of mMDH, the oligomeric state of this mMDH-fluorescein preparation remained unchanged between pH 5.0 and 8.0. Application of hydrostatic pressure up to 2.5 kilobars was ineffective in dissociating the mMDH dimer. However, the mMDH dimer was completely dissociated in 1.5 M guanidinium hydrochloride. Dilution ... [truncated at 150 words]
Chance B, Cope M, Gratton E, Ramanujam N, Tromberg BJ.
Phase measurement of light absorption and scatter in human tissue.
Rev Sci Instrum. 1998; 69(10): 3457-3481.Analog and digital technologies are presented for precise measurement of propagation delay of photons from source and detector placed on portions of the human body. The goal of the apparatus design is to quantify absorption (µa) and scattering (µs) induced by biological pigments and biological structures, respectively. Body tissues are highly scattering with a mean distance between scatterers of less than a mm (at 700–850 nm). Significant absorption is mainly due to 5%–10% of the tissue volume occupied by blood. Measurement of µa and µs is done by both time and frequency domain equipment. This article focuses upon frequency domain equipment because of its simplicity, reduced noise bandwidth, versatility, and the strong analogy to very high frequency/ultrahigh frequency communication devices, particularly those using phase modulation. Comparisons are made of homodyne and heterodyne systems together with evaluation of single and multiple side band systems, with particular emphasis on methods for multiplexed ... [truncated at 150 words]
Gilmore AM, Shinkarev VP, Hazlett TL, Govindjee.
Quantitative analysis of the effects of intrathylakoid pH and xanthophyll cycle pigments on Chlorophyll A fluorescence lifetime distributions and intensity in thylakoids.
Biochemistry. 1998; 37(39): 13582-93.The xanthophyll cycle-dependent dissipation of excitation energy in higher plants is one of the most important regulatory and photoprotective mechanisms in photosynthesis. Using parallel time-resolved and pulse-amplitude modulation fluorometry, we studied the influence of the intrathylakoid pH and the xanthophyll cycle carotenoids on the PSII chlorophyll (Chl) a fluorescence yield in thylakoids of Arabidopsis, spinach, and barley. Increases in concentrations of dithiothreitol in thylakoids, which have a trans-thylakoid membrane pH gradient and are known to have decreased conversion of violaxanthin (V) to zeaxanthin (Z), lead to (1) decreases in the fractional intensity of the approximately 0.5 ns Chl a fluorescence lifetime (tau) distribution component and simultaneous increases in a 1.6-1.8 ns fluorescence component and (2) increases in the maximal fluorescence intensity. These effects disappear when the pH gradient is eliminated by the addition of nigericin. To quantitatively explain these results, we present a new mathematical model that describes the simultaneous ... [truncated at 150 words]
Toronov VY, Filiaci ME, Fantini S, Gratton E.
Photon-density wave correlation spectroscopy detects large-scale fluctuations in turbid media.
Phys Rev E. 1998; 58(2): 2288-2297.We study the fluctuations in the photon-density wave parameters [average intensity (dc), modulation amplitude, and phase] caused by macroscopic fluctuations in the optical properties of turbid media. We present both a theoretical analysis based on diffusion theory and its experimental verification on a strongly scattering suspension containing absorbing particles (1-1.6 mm effective diameter) in turbulent motion. The photon-density waves are induced by the laser diode output (750 nm), which is intensity-modulated at 110 MHz. The dc, amplitude, and phase are acquired with an acquisition time per data point of 8 ms, which corresponds to a frequency bandwidth of 62.5 Hz. We have found that in the presence of the absorbing particles, the dc and phase average values and power spectra are in good agreement with our theoretical predictions. We have verified that our instrument can extend the measured frequency band up to the kHz region, which is appropriate for the ... [truncated at 150 words]
Brune M, Hunter JL, Howell SA, Martin SR, Hazlett TL, Corrie JE, Webb MR.
Mechanism of inorganic phosphate interaction with phosphate binding protein from Escherichia coli.
Biochemistry. 1998; 37(29): 10370-80.The mechanism of Pi interaction with phosphate binding protein of Escherichia coli has been investigated using the A197C mutant protein labeled with a coumarin fluorophore (MDCC-PBP), which gives a fluorescence change on binding Pi. A pure preparation of MDCC-PBP was obtained, in which the only significant inhomogeneity is the presence of equal amounts of two diastereoisomers due to the chiral center formed on reaction of the cysteine with the maleimide. These diastereoisomers could not be separated, but Pi binding data suggest that they differ in affinity and fluorescence change. When Pi binds to MDCC-PBP, the fluorescence quantum yield increases 8-fold and the fluorescence intensity at 465 nm increases 13-fold. The kinetics of Pi binding show saturation of the rate at high Pi concentrations, and this together with other information suggests a two-step mechanism with the fluorescence change after binding, concomitant with a conformational change of the protein that closes the ... [truncated at 150 words]
So PTC, König K, Berland KM, Dong CY, French TE, Buehler C, Ragan TM, Gratton E.
New time-resolved techniques in two-photon microscopy.
Cell Mol Biol (Noisy-le-grand). 1998; 44(5): 771-793.Microscopy is traditionally a tool for determining biological structures. Many recent advances in optical microscopy involves the incorporation of spectroscopy techniques to monitor biochemical states of microscopic structures in living cells and tissues. By minimizing tissue photodamage, two-photon excitation microscopy provides a new opportunity to study the dynamics of biological systems on time scales from nanoseconds to hours. This review will focus on a number of these new methods: two-photon time-lapse microscopy, two-photon photoactivation, two-photon correlated spectroscopy, two-photon single particle tracking and two-photon lifetime microscopy.
Bagatolli LA, Gratton E, Fidelio GD.
Water dynamics in glycosphingolipid aggregates studied by LAURDAN fluorescence.
Biophys J. 1998; 75(1): 331-41. PMCID: PMC1299702We have characterized the fluorescence properties of 6-dodecanoyl-2-dimethylamine-naphthalene (LAURDAN) in pure interfaces formed by sphingomyelin and 10 chemically related glycosphingolipids (GSLs).1 The GSLs contain neutral and anionic carbohydrate residues in their oligosaccharide chain. These systems were studied at temperatures below, at, or above the main phase transition temperature of the pure lipid aggregates. The extent of solvent dipolar relaxation around the excited fluorescence probe in the GSLs series increases with the magnitude of the glycosphingolipid polar headgroup below the transition temperature. This conclusion is based on LAURDAN's excitation generalized polarization (GPex) and fluorescence lifetime values found in the different interfaces. A linear dependence between the LAURDAN GPex and the intermolecular spacing among the lipid molecules was found for both neutral and anionic lipids in the GSLs series. This relationship was also followed by phospholipids. We conclude that LAURDAN in these lipid aggregates resides in sites containing different amounts of water. ... [truncated at 150 words]
Krasnowska EK, Gratton E, Parasassi T.
Prodan as a membrane surface fluorescence probe: partitioning between water and phospholipid phases.
Biophys J. 1998; 74(4): 1984-93. PMCID: PMC1299539Fluorescence spectral features of 6-propionyl-2-dimethylaminonaphthalene (Prodan) in phospholipid vesicles of different phase states are investigated. Like the spectra of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), the steady-state excitation and emission spectra of Prodan are sensitive to the polarity of the environment, showing a relevant shift due to the dipolar relaxation phenomenon. Because of the different lengths of their acyl residues, the partitioning of the two probes between water and the membrane bilayer differs profoundly. To account for the contribution of Prodan fluorescence arising from water, we introduce a three-wavelength generalized polarization method that makes it possible to separate the spectral properties of Prodan in the lipid phase and in water, and to determine the probe partitioning between phospholipid and water and between the gel and the liquid-crystalline phases of phospholipids. In contrast to Laurdan, Prodan preferentially partitions in the liquid-crystalline phase with respect to the gel and is sensitive to the polar head pretransition, ... [truncated at 150 words]
Walker SA, Boas DA, Gratton E.
Photon density waves scattered from cylindrical inhomogeneities: theory and experiments.
Appl Opt. 1998; 37(10): 1935-1944.We present an analytical solution for the scattering of diffuse photon density waves from an infinite circular, cylindrical inhomogeneity embedded in a homogeneous highly scattering turbid medium. The analytical solution, based on the diffusion approximation of the Boltzmann transport equation, represents the contribution of the cylindrical inhomogeneity as a series of modified Bessel functions integrated from zero to infinity and weighted by different angular dependencies. This series is truncated at the desired precision, similar to the Mie theory. We introduce new boundary conditions that account for specular reflections at the interface between the background medium and the cylindrical inhomogeneity. These new boundary conditions allow the separate recovery of the index of refraction of an object from its absorption and reduced scattering coefficients. The analytical solution is compared with data obtained experimentally to evaluate the predictive capability of the model. Optical properties of known cylindrical objects are recovered accurately. However, as ... [truncated at 150 words]
Fantini S, Walker SA, Franceschini MA, Kaschke M, Schlag PM, Moesta KT.
Assessment of the size, position, and optical properties of breast tumors in vivo by noninvasive optical methods.
Appl Opt. 1998; 37(10): 1982-1989.We present a method for the noninvasive determination of the size, position, and optical properties
(absorption and reduced scattering coefficients) of tumors in the human breast. The tumor is first detected by frequency-domain optical mammography. It is then sized, located, and optically characterized by use of diffusion theory as a model for the propagation of near-infrared light in breast tissue. Our method assumes that the tumor is a spherical inhomogeneity embedded in an otherwise homogeneous tissue. We report the results obtained on a 55-year-old patient with a papillary cancer in the right breast. We found that the tumor absorbs and scatters near-infrared light more strongly than the surrounding healthy tissue. Our method has yielded a tumor diameter of 2.1 ± 0.2 cm, which is comparable with the actual size of 1.6 cm, determined after surgery. From the tumor absorption coefficients at two wavelengths (690 and 825 nm), we calculated the total ... [truncated at 150 words]
Masters BR, So PTC, Gratton E.
Optical biopsy of In vivo human skin: multi-photon excitation microscopy.
Lasers Med Sci. 1998; 13(3): 197-204.A technique for functional and morphological optical biopsy of in vivo human skin is presented. NAD(P)H is an intrinsic probe of cellular metabolism and is a major source of in vivo human skin autofluorescence as characterized with fluorescence spectroscopy techniques [1–5]. Two-photon excitation microscopy with excitation at 730 nm can be used to monitor cellular metabolism, based on NAD(P)H fluorescence, in thick, highly scattering tissues such as in vivo human skin. Non-invasive measurements of tissue metabolism is finding applications in a number of important biomedical areas such as the diagnosis of cancer and the monitoring of wound healing. For cancer diagnosis, the change in NAD(P)H fluorescence has been linked to melanoma in skin and cancer in breast tissues [6–8]. Since the oxidative process is closely related to tissue health, the ability to image cellular NAD(P)H fluorescence in thick tissue may find applications in accessing the performance of various tissue grafts ... [truncated at 150 words]
