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]
Chen H, Ma N, Kagawa K, Kawahito S, Digman M, Gratton E.
Widefield multi-frequency fluorescence lifetime imaging using a two-tap CMOS camera with lateral electric field charge modulators.
J Biophotonics. 2018; [Epub ahead of print], 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 (CMOS) camera equipped with two-tap true correlated double sampling (CDS) lock-in pixels and lateral electric field charge modulators. Due to the fast intrinsic response and modulation of the camera, our system allows parallel multi-frequency Fluorescence Lifetime Imaging Microscopy (FLIM) in one measurement via Fast Fourier Transform. We demonstrate that at a fundamental frequency of 20MHz, 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 (FRET) 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 ... [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]
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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Analyses of the cellular signaling responses to DNA damage and DNA repair factor recruitment using fluorescence lifetimes and fluctuations.
PhD in Biomedical Engineering, University of California, Irvine, 2018.
Advisor: Michelle A Digman
Genome integrity is continually challenged by threats including DNA replication errors, toxic metabolic byproducts, and exposure to exogenous genotoxins. Responding to and repairing damaged DNA requires coordinating a number of critical cellular events including activating DNA repair, facilitating chromatin rearrangement, and delaying cell cycle progression. Although the factors important for DNA repair have been identified, how these activities are coordinated in the nucleus and the long-term cellular-wide consequences of DNA repair are not well characterized.
Nicotinamide adenine dinucleotide (NAD) is a coenzyme involved in both cellular metabolism and DNA repair. Thus, analyses of NAD species could provide new insight into DNA damage signaling as well as damage-induced metabolic responses to various types of DNA lesions. Here, we use the phasor approach to fluorescence lifetime imaging microscopy in combination with genetically encoded fluorescent biosensors to measure metabolic changes in single cells with high spatiotemporal resolution. We found that the absence of 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.
Hadraba D, Lopot F, Paesen R, Ameloot M, Jelen K, Digman MA.
Mechanical testing of connective tissue from nano to macro.
19th Annual UC Systemwide Bioengineering Symposium. Riverside, CA. June 21-23, 2018.
UCR BIEN Symposium Complete ABSTRACT Book. 2018; 1: 108.Introduction: Connective tissue is an inhomogeneous composite structure with high variability in mechanical properties. The mechanical properties originate in a hierarchical organization of proteins from which type I collagen plays the most evident role. This role seems to differ at each hierarchical level. For example, type I collagen triple helix returns the highest Young’s modulus but only fibers are aligned into a zig-zag wavy pattern, termed the crimp pattern. The mechanical properties are also not stationary and alter with age or disease, and therefore, conducting an experiment on the crimp pattern behavior under mechanical load simultaneously with the measurement on the helical pitch angle of type I collagen molecule can provide a new insight into the clinical classification of connective tissue.
Materials and Methods: The rabbit tendons were gripped in an in-house built tensile instrument that was placed at the microscope stage. The tendons were imaged using polarized second harmonic generation ... [truncated at 150 words]
Trinh AL, Chen H, Chen Y, Hu Y, Li Z, Siegel ER, Linskey ME, Wang PH, Zhou YH, Digman MA.
Distinguishing stem-like tumor-initiating cells in malignant glioma by phasor fluorescence lifetime imaging.
19th Annual UC Systemwide Bioengineering Symposium. Riverside, CA. June 21-23, 2018.
UCR BIEN Symposium Complete ABSTRACT Book. 2018; 1: 113.Introduction: Intra-tumoral heterogeneity is associated with therapeutic resistance of cancer. In particular, stem-like tumor initiating cell (STIC) subpopulations within malignant gliomas tumors have been shown to be responsible for treatment resistance and tumor recurrence. Therefore, there exists a need to monitor functional tumor subpopulations during treatment and cancer progression. Reduced nicotinamide adenine dinucleotide (NADH) is a metabolic coenzyme essential in cellular respiration. Fluorescence lifetime imaging microscopy (FLIM) of NADH has been demonstrated to be a powerful label-free indicator for inferring metabolic states of living cells. In this study, FLIM of NADH was applied to distinguish between malignant glioma subpopulations of STIC and tumor mass-forming cells (TMC). In addition, changes in the fluorescence lifetimes of NADH were compared to cellular respiration (oxidative phosphorylation and glycolysis), EGFR expression, and cell-growth rates.
Materials and Methods: Human glioblastoma-derived cell line U251 were enriched for STIC and TMC subpopulations by established cell culture conditions. U251 STIC ... [truncated at 150 words]
Rahim MK, Gratton E, Haun JB.
Transforming FLIM into a high-content molecular analysis platform.
19th Annual UC Systemwide Bioengineering Symposium. Riverside, CA. June 21-23, 2018.
UCR BIEN Symposium Complete ABSTRACT Book. 2018; 1: 69.Introduction: Tumors are complex and heterogeneous, and therefore diagnostic techniques will have to comprehensively assess molecular features across diverse cell types and functional states. Current methods that can provide both spatially-resolved and quantitative molecular interpretation of tissues are limited in multiplexing capacity or are complex and time-consuming. To address these limitations, we have developed a multiplexed imaging platform that utilizes fluorescence lifetime imaging microscopy (FLIM). Fluorescence lifetime refers to the duration of fluorescence light emission after excitation, and it can be leveraged to resolve fluorescent probes that have the same color. Moreover, the phasor approach to FLIM greatly simplifies lifetime analysis. Instead of fitting complicated exponential functions, the phasor approach provides a graphical representation that enables the unmixing of more than one lifetime using simple geometrical considerations. In this study, we are developing new methodologies to quantitatively detect 4 molecular probes within the same spectral window using FLIM and the ... [truncated at 150 words]
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Nanoscale changes for macroscale results: modulation of cancer metabolism and adhesion by the substrate.
PhD in Chemical and Biochemical Engineering, University of California, Irvine, 2018.
Advisor: Michelle A Digman
Cell-to-substrate interactions are known to regulate important cellular processes such as migration, proliferation, and intercellular signaling. Cancer cells remodel the surrounding collagen matrix and promote collagen fibrillogenesis for invasion. This increases substrate density and activates the mechanosensing pathway for events such as adhesion and migration. Recent studies indicate that metabolism is able to affect cancer invasion and that mitochondria are recruited towards the invading edges of the cell. There seems to be a link between the mechanosensing and metabolism pathway in promoting invasion; however, it is unclear how these are regulated and affects mitochondria trafficking towards the protruding edges. In this thesis, focal adhesion (FA) dynamics within NIH3T3 cells were studied using the Number and Molecular Brightness analysis and were found to adapt to the underlying nanotopography by modulating their adhesion size and turnover dynamics leading to a change in cell speed. This laid the foundational studies within cancer cells. ... [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]
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Multiphoton imaging and phasor approach to identify new biomarkers in Huntington Disease.
PhD in Biomedical Engineering, University of California, Irvine, 2018.
Advisor: Michelle A Digman
Neurodegenerative diseases occur when brain cells (neurons) start to deteriorate. Changes in these cells will lead to dysfunction and eventual cell death. This will lead to mild symptoms like problems with coordination, psychiatric disorders, or memory loss; and as more neurons die the symptoms also progressively worsen. World Health Organization (WHO) indicates up to 1 billion people worldwide are affected by various types of neurodegenerative diseases. In this study, I focused on Huntington disease (HD), a model to study neurodegeneration that is caused by a glitch in a single gene called huntingtin gene (HTT). Huntington disease is an autosomal dominant inherited neurodegenerative disease characterized by movement, cognitive and emotional disorders. We all carry HTT; however, the normal length of DNA trinucleotide, CAG, that codes for glutamine are between 10–35. The expanded repeats of above 40 or more will lead to HD. Using advanced functional imaging technique called Two-Photon Fluorescence Lifetime ... [truncated at 150 words]
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Spectroscopic signatures of cells metabolism and extracellular species using phasor-FLIM (Conference Presentation).
BiOS 2018. Part of SPIE Photonics West. San Francisco, California, January 27 - February 1, 2018.
Multiphoton Microscopy in the Biomedical Sciences XVIII (Vol. 10498). 1049803, 2018. Fluorescence lifetime imaging has become a common technique in microscopy. The information contained in the fluorescence decay curve can be obtained with different methods. A major issue is that the fluorescence decay must be measured at many pixels either sequentially or in parallel, which generally results in relatively noisy curves. Another major issue is data analysis of the decay at each point of an image and the visualization of the results. Nonetheless, methods of data analysis and visualization have become relatively common and today we can analyze an image for different molecular species and molecular processes. In this talk I will discuss a method, called the phasor that was developed in my lab and that is now used in several commercial instruments. Using the phasor method applied to cell autofluorescence it has become possible to construct quantitative indices of cell metabolism and to identify molecular species characteristic to some diseases.
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]
Leemans SW, Dvornikov A, Gratton E.
Adaptive optics in deep tissue microscopy.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 533a, 2638-Pos.Multiphoton fluorescence microscopy is a powerful technique to visualize the form and function of the cells in tissue at sub-micron scales. To diminish fluorescence emission losses due to light scattering in the sample, our lab has developed a transmission fluorescence microscope to recover more of the emitted fluorescence from biological samples. Multiphoton fluorescence is sensitive to the peak power of the light, so the efficiency of the process improves when pulse duration is decreased and the focal volume is minimized. The DIVER (Deep Imaging Via Emission Recovery) microscope uses a pulse compressor that pre-compensates for dispersion in the excitation path to minimize pulse duration. Spatial variations in the index of refraction in tissue distort the focal volume, resulting in decreased resolution as imaging depth is increased. We compensate for spatial variations in index by conjugating the wavefront of the excitation light to the optical aberrations that are introduced by the ... [truncated at 150 words]
Scipioni L, Diaspro A, Lanzanò L, Gratton E.
AiryScan comprehensive superresolution correlation analysis.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 533a, 2641-Pos.AiryScan is a detector array developed by Zeiss that consists in 32 GaAs PMT detectors arranged in a hexagonal pattern. The development of single point detector arrays is recently increasing also thanks to the possibility of achieving super-resolution without signal loss by means of Image Scanning Microscopy (ISM, Sheppard et al. 2013). Furthermore, super-resolution techniques applied to fluorescence correlation techniques have shown to be effective in retrieving information that is not accessible with diffraction-limited methods, giving new valuable tools to biological research. Being essentially a high-speed nano-camera, AiryScan allows, in principle, for the implementation of most of the state-of-the-art correlation techniques at super-resolution in a single analysis. In this work we will present the parallel implementation of fluorescence correlation spectroscopy (FCS, Digman et al. 2011), pair-correlation function (pCF, Hinde et al. 2010) and connectivity analysis, image-derived mean square displacement (iMSD, Di Rienzo et al. 2014), number and brightness (N&B, Digman ... [truncated at 150 words]
Malacrida LS, Hedde PN, Ranjit S, Cardarelli F, Gratton E.
Elucidating invisible barriers and obstacles to molecular diffusion in live cells by the spatial pair-correlation function: a connectivity view of the cell.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 166a, 835-Pos.Despite recent advances in optical super-resolution, we lack of methods to visualize the path followed by diffusing molecules in the cell. Fluorescence correlation spectroscopy (FCS) provides molecular dynamics at the single molecule level; however, the classical single point FCS lacks spatial resolution. Methods based on image analysis like raster-scanning and spatiotemporal image correlation still needs spatial averaging over relatively large areas; hence the spatial resolution is compromised. Here, we develop a new method based on the spatial pair-cross-correlation in two dimensions (2D-pCF) to obtain relatively high-resolution images of molecular diffusion dynamics and transport in live cells. The 2D-pCF method measures the time for a particle to go from one location to another by cross-correlating the intensity fluctuations at specific points in an image. Hence, a visual map of connectivity for the average path followed by molecules is created. To test our approach we use a nanometer channel simulation and a ... [truncated at 150 words]
Torrado B, Malacrida L, Badano JL, Irigoín F, Gratton E.
Intracellular transport characterization of the transcription factor Gli2 by fluorescence correlation spectroscopy approaches.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 630a, 3123-Pos.Gli2 is the principal transcriptional activator of the Hedgehog signalingin mammals. Upon pathway activation, Gli2 moves into the cilium before reaching the nucleus. While two conserved classical nuclear localization signals (cNLS) mediate Gli2 nuclear localization via importin (Imp)-α/β1, these sequences are not required for Gli2 ciliary import (Torrado et al, 2016. PLoSONE ;11(8)). However, Importin-β2 is involved in Gli2 ciliary entry, and might also collaborate in Gli2 nuclear entry (Torrado et al, 2016. PLoSONE ;11(8)). Several questions about Gli2 intracellular traffic still remain opened. To analyze the nucleo-cytoplasmic transport of Gli2 we used fluorescence correlation spectroscopy methods, in particular pair correlation function and Number & Brightness approaches. We compared Gli2 nuclear transport when the pathway is on and off. Gli2 ciliary transport was analyzed by fluorescence correlation spectroscopy approaches combined with 3D-particle tracking so we could avoid the out of focus produced by the movement of the cilium. We applied ... [truncated at 150 words]
Ranjit S, Dvornikov A, Levi M, Gratton E.
Multicomponent analysis of phasor plot to decipher changes in metabolic trajectory of biological systems.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 167a, 843-Pos.Recent developments of phasor approach to Fluorescence Lifetime Imaging (FLIM) in cells undergoing oxidative stress or cultured in oleic acid enriched media and in mice liver sections having high fat diet, have shown presence of a third autofluorescent component indicative of lipid droplets besides the phasor components from free and enzyme bound NADH in the 400-450 nm emission wavelength. This third component changes the position and shape of the phasor distribution away from the metabolic trajectory, defined by the line joining the phasor position of free and enzyme bound NADH, and makes the study of changes in metabolism using the ratio of free to bound NADH difficult. However, the phasor rule of addition applies to any number of components and this was exploited to create a multicomponent analysis. A change in phasor distribution towards free or bound NADH can then be separated and analyzed independent of the third individual. Law ... [truncated at 150 words]
Digman MA, Palomba F, Kong X, Yokomori K, Gratton E.
Pair correlation analysis of Ku dynamics upon DNA damage.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 351a, 1740-Pos.We used the pair correlation function (pCF) analysis developed for fast frame acquisition to measure the changes in molecular connectivity of the Ku protein following double strand DNA damage. Ku protein binds to the ends of double-strand breaks and is required in DNA-repair for non-homologous end joining (NHEJ). Before damage, Ku diffuses freely with low anisotropy in the entire nucleus. After damage, we observe the formation of barriers of Ku at the damage site. In the rest of the nucleus the diffusion coefficient of Ku increases that persists four hours post damage. The diffusion anisotropy tensor of Ku in the nucleus was measured and an increase in anisotropy compared to the control cells without damage was observed. Given that DNA-PKcs play a critical role in NHEJ, loss of their activity can lead to a reduced repair rate and would alter Ku motility at the damage sites. To test this, we ... [truncated at 150 words]
Hedde PN, Malacrida L, Ahrar S, Siryaporn A, Gratton E.
SideSPIM - a flexible multipurpose platform for light sheet microscopy.
62nd Annual Meeting of the Biophysical Society. San Francisco, California. February 17–21, 2018.
Biophys J. 2018; 114(3, Suppl 1): 187a, 945-Plat.We devised a flexible multipurpose platform for light sheet microscopy. Based on a regular inverted microscope, a custom designed chamber is used to allow selective plane illumination from the side (sideSPIM). This eliminates the need of dipping into the sample container, hence small sample volumes (~100 µl) can be realized and objective lenses can be interchanged quickly. We demonstrate that this flexible multipurpose platform is suitable for a large variety of applications involving samples of substantially different sizes and geometries. For example, we captured the microcirculation of erythrocytes in a zebrafish embryo at 5 stacks/s (40 planes), studied the growth of bacteria biofilms under flow using a fluidic device, and probed the diffusion of EGFP tagged proteins in mammalian cells by acquiring fast time series (200 fps) and subjecting them to fluorescence fluctuation based image mean square displacement (iMSD) and two-dimensional pair cross-correlation function (2D-pCF) analysis. We further evaluated the ... [truncated at 150 words]
