The Experts below are selected from a list of 34230 Experts worldwide ranked by ideXlab platform
Enrico Gratton - One of the best experts on this subject based on the ideXlab platform.
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fit free Analysis of fluorescence lifetime imaging data using the Phasor approach
Nature Protocols, 2018Co-Authors: Suman Ranjit, Leonel Malacrida, David M Jameson, Enrico GrattonAbstract: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 fluorescence. Undoubtedly, Phasor plots will be increasingly important in the future Analysis and understanding of FLIM and hyperspectral confocal imaging. This protocol presents the principle of the method and guides users through one of the popular interfaces for FLIM Phasor Analysis, namely, the SimFCS software. Implementation of the Analysis takes only minutes to complete for a dataset containing hundreds of files.
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A multidimensional Phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes
Scientific Reports, 2017Co-Authors: Leonel Malacrida, David M Jameson, Enrico GrattonAbstract:Mammalian 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 of the image is associated with a Phasor and each Phasor maps to pixels and features in the image. In this paper the lifetime and spectral fluorescence data are used simultaneously to determine the contribution of polarity and dipolar relaxations of LAURDAN in each pixel of an image.
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spectral Phasor Analysis of laurdan fluorescence in live a549 lung cells to study the hydration and time evolution of intracellular lamellar body like structures
Biochimica et Biophysica Acta, 2016Co-Authors: Leonel Malacrida, Soledad Astrada, Arturo Briva, Mariela Bollatifogolin, Enrico Gratton, Luis A BagatolliAbstract:Abstract Using 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.
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characterizing fibrosis in uuo mice model using multiparametric Analysis of Phasor distribution from flim images
Biomedical Optics Express, 2016Co-Authors: Suman Ranjit, Alexander Dvornikov, Moshe Levi, Seth B Furgeson, Enrico GrattonAbstract:Phasor 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 fibrosis with time is detected using this multiparametric approach to Phasor Analysis.
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Phasor Analysis of local ics detects heterogeneity in size and number of intracellular vesicles
Biophysical Journal, 2016Co-Authors: Enrico Gratton, Lorenzo Scipioni, Alberto Diaspro, Luca LanzanoAbstract:Organelles 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 in the average properties of a single correlation function. We apply the method to quantify the spatial and temporal heterogeneity in the size and number of intracellular vesicles formed after endocytosis in live cells.
Conor L Evans - One of the best experts on this subject based on the ideXlab platform.
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Time-resolved fluorescence microscopy with Phasor Analysis for visualizing multicomponent topical drug distribution within human skin
Scientific Reports, 2020Co-Authors: Sinyoung Jeong, Maiko Hermsmeier, Kin F Chan, Daniel A. Greenfield, Akira Yamamoto, Xin Chen, Conor L EvansAbstract:Understanding a drug candidate’s pharmacokinetic (PK) parameters is a challenging but essential aspect of drug development. Investigating the penetration and distribution of a topical drug’s active pharmaceutical ingredient (API) allows for evaluating drug delivery and efficacy, which is necessary to ensure drug viability. A topical gel (BPX-05) was recently developed to treat moderate to severe acne vulgaris by directly delivering the combination of the topical antibiotic minocycline and the retinoid tazarotene to the pilosebaceous unit of the dermis. In order to evaluate the uptake of APIs within human facial skin and confirm accurate drug delivery, a selective visualization method to monitor and quantify local drug distributions within the skin was developed. This approach uses fluorescence lifetime imaging microscopy (FLIM) paired with a multicomponent Phasor Analysis algorithm to visualize drug localization. As minocycline and tazarotene have distinct fluorescence lifetimes from the lifetime of the skin’s autofluorescence, these two APIs are viable targets for distinct visualization via FLIM. Here, we demonstrate that the Analysis of the resulting FLIM output can be used to determine local distributions of minocycline and tazarotene within the skin. This approach is generalizable and can be applied to many multicomponent fluorescence lifetime imaging targets that require cellular resolution and molecular specificity.
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fluorescence lifetime imaging microscopy along with non euclidean Phasor Analysis for visualization of drug distribution of a topical drug in human facial skin conference presentation
Visualizing and Quantifying Drug Distribution in Tissue III, 2019Co-Authors: Sinyoung Jeong, Alexander Fast, Sam Osseiran, Maiko Hermsmeier, Tanvee Sawant, Kin F Chan, Conor L EvansAbstract:We recently proposed a method for selective visualization of topical drug distribution within human facial skin using two-photon fluorescence lifetime imaging along with non-Euclidean Phasor Analysis as a pharmacokinetics and pharmacodynamics imaging toolkit. In order to improve the efficacy of topical drug delivery toward the treatment of inflammatory acne, we have now developed a combination topical gel containing both minocycline and a retinoid. Since both drugs have unique fluorescence lifetimes compared to skin, we were able to selectively visualize the distribution of minocycline and the retinoid within ex vivo human facial skin while isolating the contributions of the three components.
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Enhanced quantification of metabolic activity for individual adipocytes by label-free FLIM
Scientific Reports, 2018Co-Authors: Michael Evers, Sam Osseiran, Conor L Evans, Nunciada Salma, Malte Casper, Reginald Birngruber, Dieter MansteinAbstract:Fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) allows for label-free quantification of metabolic activity of individual cells over time and in response to various stimuli, which is not feasible using traditional methods due to their destructive nature and lack of spatial information. This study uses FLIM to measure pharmacologically induced metabolic changes that occur during the browning of white fat. Adipocyte browning increases energy expenditure, making it a desirable prospect for treating obesity and related disorders. Expanding from the traditional two-lifetime model of NADH to a four-lifetime model using exponential fitting and Phasor Analysis of the fluorescence decay results in superior metabolic assessment compared to traditional FLIM Analysis. The four lifetime components can also be mapped to specific cellular compartments to create a novel optical ratio that quantitatively reflects changes in mitochondrial and cytosolic NADH concentrations and binding states. This widely applicable approach constitutes a powerful tool for studies where monitoring cellular metabolism is of key interest.
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Phasor approach to fluorescence lifetime imaging microscopy for visualization and quantification of drug distribution of a topical minocycline gel in human facial skin conference presentation
Visualizing and Quantifying Drug Distribution in Tissue II, 2018Co-Authors: Sinyoung Jeong, Sam Osseiran, Maiko Hermsmeier, Kin F Chan, Akira Yamamoto, Usha Nagavarapu, Conor L EvansAbstract:Acne vulgaris is a common chronic skin disease in teenagers and young adults. Minocycline, an antibiotic, has thus far been widely utilized to treat acne, but only via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline to the epidermis and pilosebaceous unit to achieve localized treatment with lower doses of drug. In order to evaluate the effectiveness of topical drug delivery in terms of pharmacokinetics and pharmacodynamics, visualization and quantification of drug within a biological tissue is essential. As minocycline is a known fluorophore, we demonstrate a method for visualization and quantification of minocycline within human skin tissue by utilizing a Phasor approach to fluorescence lifetime microscopy (FLIM). In Phasor Analysis of FLIM, the fluorescence decay trace from each pixel in the FLIM image is plotted as a single point in the Phasor plot. Since every fluorophore has a specific decay trace, we can identify a specific molecule by its position in the Phasor plot. To demonstrate the feasibility of this visualization and quantification method, the human facial skin samples treated with various concentrations of BPX-01 were investigated using the Phasor approach to FLIM. The unique signature of minocycline in FLIM Phasor Analysis was successfully differentiated from the endogenous fluorescence of human tissue. Furthermore, by sorting the individual pixels of minocycline signature in FLIM image, the distribution of minocycline within human facial skin can be visualized and quantified. Based on these results, we believe that the visualization and quantification method using a Phasor approach to FLIM can play an important role in future pharmacokinetics and pharmacodynamics analyses.
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non euclidean Phasor Analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy
Journal of Biomedical Optics, 2017Co-Authors: Sam Osseiran, M Elisabeth M D Roider, Hequn Wang, Yusuke Suita, Michael Murphy, David E Fisher, Conor L EvansAbstract:Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin.
Sam Osseiran - One of the best experts on this subject based on the ideXlab platform.
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fluorescence lifetime imaging microscopy along with non euclidean Phasor Analysis for visualization of drug distribution of a topical drug in human facial skin conference presentation
Visualizing and Quantifying Drug Distribution in Tissue III, 2019Co-Authors: Sinyoung Jeong, Alexander Fast, Sam Osseiran, Maiko Hermsmeier, Tanvee Sawant, Kin F Chan, Conor L EvansAbstract:We recently proposed a method for selective visualization of topical drug distribution within human facial skin using two-photon fluorescence lifetime imaging along with non-Euclidean Phasor Analysis as a pharmacokinetics and pharmacodynamics imaging toolkit. In order to improve the efficacy of topical drug delivery toward the treatment of inflammatory acne, we have now developed a combination topical gel containing both minocycline and a retinoid. Since both drugs have unique fluorescence lifetimes compared to skin, we were able to selectively visualize the distribution of minocycline and the retinoid within ex vivo human facial skin while isolating the contributions of the three components.
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Enhanced quantification of metabolic activity for individual adipocytes by label-free FLIM
Scientific Reports, 2018Co-Authors: Michael Evers, Sam Osseiran, Conor L Evans, Nunciada Salma, Malte Casper, Reginald Birngruber, Dieter MansteinAbstract:Fluorescence lifetime imaging microscopy (FLIM) of intrinsic fluorophores such as nicotinamide adenine dinucleotide (NADH) allows for label-free quantification of metabolic activity of individual cells over time and in response to various stimuli, which is not feasible using traditional methods due to their destructive nature and lack of spatial information. This study uses FLIM to measure pharmacologically induced metabolic changes that occur during the browning of white fat. Adipocyte browning increases energy expenditure, making it a desirable prospect for treating obesity and related disorders. Expanding from the traditional two-lifetime model of NADH to a four-lifetime model using exponential fitting and Phasor Analysis of the fluorescence decay results in superior metabolic assessment compared to traditional FLIM Analysis. The four lifetime components can also be mapped to specific cellular compartments to create a novel optical ratio that quantitatively reflects changes in mitochondrial and cytosolic NADH concentrations and binding states. This widely applicable approach constitutes a powerful tool for studies where monitoring cellular metabolism is of key interest.
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Phasor approach to fluorescence lifetime imaging microscopy for visualization and quantification of drug distribution of a topical minocycline gel in human facial skin conference presentation
Visualizing and Quantifying Drug Distribution in Tissue II, 2018Co-Authors: Sinyoung Jeong, Sam Osseiran, Maiko Hermsmeier, Kin F Chan, Akira Yamamoto, Usha Nagavarapu, Conor L EvansAbstract:Acne vulgaris is a common chronic skin disease in teenagers and young adults. Minocycline, an antibiotic, has thus far been widely utilized to treat acne, but only via oral administration. Recently, a topical minocycline gel (BPX-01) was developed to directly deliver minocycline to the epidermis and pilosebaceous unit to achieve localized treatment with lower doses of drug. In order to evaluate the effectiveness of topical drug delivery in terms of pharmacokinetics and pharmacodynamics, visualization and quantification of drug within a biological tissue is essential. As minocycline is a known fluorophore, we demonstrate a method for visualization and quantification of minocycline within human skin tissue by utilizing a Phasor approach to fluorescence lifetime microscopy (FLIM). In Phasor Analysis of FLIM, the fluorescence decay trace from each pixel in the FLIM image is plotted as a single point in the Phasor plot. Since every fluorophore has a specific decay trace, we can identify a specific molecule by its position in the Phasor plot. To demonstrate the feasibility of this visualization and quantification method, the human facial skin samples treated with various concentrations of BPX-01 were investigated using the Phasor approach to FLIM. The unique signature of minocycline in FLIM Phasor Analysis was successfully differentiated from the endogenous fluorescence of human tissue. Furthermore, by sorting the individual pixels of minocycline signature in FLIM image, the distribution of minocycline within human facial skin can be visualized and quantified. Based on these results, we believe that the visualization and quantification method using a Phasor approach to FLIM can play an important role in future pharmacokinetics and pharmacodynamics analyses.
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non euclidean Phasor Analysis for quantification of oxidative stress in ex vivo human skin exposed to sun filters using fluorescence lifetime imaging microscopy
Journal of Biomedical Optics, 2017Co-Authors: Sam Osseiran, M Elisabeth M D Roider, Hequn Wang, Yusuke Suita, Michael Murphy, David E Fisher, Conor L EvansAbstract:Chemical sun filters are commonly used as active ingredients in sunscreens due to their efficient absorption of ultraviolet (UV) radiation. Yet, it is known that these compounds can photochemically react with UV light and generate reactive oxygen species and oxidative stress in vitro, though this has yet to be validated in vivo. One label-free approach to probe oxidative stress is to measure and compare the relative endogenous fluorescence generated by cellular coenzymes nicotinamide adenine dinucleotides and flavin adenine dinucleotides. However, chemical sun filters are fluorescent, with emissive properties that contaminate endogenous fluorescent signals. To accurately distinguish the source of fluorescence in ex vivo skin samples treated with chemical sun filters, fluorescence lifetime imaging microscopy data were processed on a pixel-by-pixel basis using a non-Euclidean separation algorithm based on Mahalanobis distance and validated on simulated data. Applying this method, ex vivo samples exhibited a small oxidative shift when exposed to sun filters alone, though this shift was much smaller than that imparted by UV irradiation. Given the need for investigative tools to further study the clinical impact of chemical sun filters in patients, the reported methodology may be applied to visualize chemical sun filters and measure oxidative stress in patients' skin.
Leonel Malacrida - One of the best experts on this subject based on the ideXlab platform.
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fit free Analysis of fluorescence lifetime imaging data using the Phasor approach
Nature Protocols, 2018Co-Authors: Suman Ranjit, Leonel Malacrida, David M Jameson, Enrico GrattonAbstract: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 fluorescence. Undoubtedly, Phasor plots will be increasingly important in the future Analysis and understanding of FLIM and hyperspectral confocal imaging. This protocol presents the principle of the method and guides users through one of the popular interfaces for FLIM Phasor Analysis, namely, the SimFCS software. Implementation of the Analysis takes only minutes to complete for a dataset containing hundreds of files.
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Publisher Correction: Alteration in Fluidity of Cell Plasma Membrane in Huntington Disease Revealed by Spectral Phasor Analysis
Scientific Reports, 2018Co-Authors: Sara Sameni, Leonel Malacrida, Michelle A DigmanAbstract:A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Alteration in Fluidity of Cell Plasma Membrane in Huntington Disease Revealed by Spectral Phasor Analysis
Scientific Reports, 2018Co-Authors: Sara Sameni, Leonel Malacrida, Michelle A DigmanAbstract:Huntington 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 in the cells expressing expanded polyQ shows a shift toward increased membrane fluidity revealed by both LAURDAN and NR spectral Phasors. This finding brings a new perspective in the understanding of the early stages of HD that can be used as a target for drug screening.
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spectral Phasor Analysis reveals altered membrane order and function of root hair cells in arabidopsis dry2 sqe1 5 drought hypersensitive mutant
Plant Physiology and Biochemistry, 2017Co-Authors: Florencia Sena, Leonel Malacrida, Soledad Astrada, Mariana Sotelosilveira, Miguel A Botella, Omar BorsaniAbstract:Abstract Biological membranes allow the regulation of numerous cellular processes, which are affected when unfavorable environmental factors are perceived. Lipids and proteins are the principal components of biological membranes. Each lipid has unique biophysical properties, and, therefore the lipid composition of the membrane is critical to maintaining the bilayer structure and functionality. Membrane composition and integrity are becoming the focus of studies aiming to understand how plants adapt to its environment. In this study, using a combination of di-4-ANEPPDHQ fluorescence and spectral Phasor Analysis, we report that the drought hypersensitive/squalene epoxidase (dry2/sqe1-5) mutant with reduced major sterols such as sitosterol and stigmasterol in roots presented higher membrane fluidity than the wild type. Moreover, Analysis of endomembrane dynamics showed that vesicle formation was affected in dry2/sqe1-5. Further Analysis of proteins associated with sterol rich micro domains showed that dry2/sqe1-5 presented micro domains function altered.
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A multidimensional Phasor approach reveals LAURDAN photophysics in NIH-3T3 cell membranes
Scientific Reports, 2017Co-Authors: Leonel Malacrida, David M Jameson, Enrico GrattonAbstract:Mammalian 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 of the image is associated with a Phasor and each Phasor maps to pixels and features in the image. In this paper the lifetime and spectral fluorescence data are used simultaneously to determine the contribution of polarity and dipolar relaxations of LAURDAN in each pixel of an image.
Michelle A Digman - One of the best experts on this subject based on the ideXlab platform.
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Publisher Correction: Alteration in Fluidity of Cell Plasma Membrane in Huntington Disease Revealed by Spectral Phasor Analysis
Scientific Reports, 2018Co-Authors: Sara Sameni, Leonel Malacrida, Michelle A DigmanAbstract:A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has been fixed in the paper.
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Alteration in Fluidity of Cell Plasma Membrane in Huntington Disease Revealed by Spectral Phasor Analysis
Scientific Reports, 2018Co-Authors: Sara Sameni, Leonel Malacrida, Michelle A DigmanAbstract:Huntington 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 in the cells expressing expanded polyQ shows a shift toward increased membrane fluidity revealed by both LAURDAN and NR spectral Phasors. This finding brings a new perspective in the understanding of the early stages of HD that can be used as a target for drug screening.
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profiling cancer cell intrinsic fluorescence with the spectral camera Phasor Analysis method
Biophysical Journal, 2014Co-Authors: Michelle A Digman, Enrico Gratton, Hongtao ChenAbstract:2030-Pos Board B760 Profiling Cancer Cell Intrinsic Fluorescence with the Spectral Camera- Phasor Analysis Method Michelle A. Digman, Enrico Gratton, Hongtao Chen. Biomedical Engineering, University of California, Irvine, Irvine, CA, USA. Advances in hyper- or multi-spectral based imaging cameras have expanded new areas of cellular research for the last 20 years. Most recently, intensity sig- nals can now be detected with a line spectrograph in which light is transmited over an imaging signal to an ultra sensitive CCD camera with fast readout speed. However, spectrally resolving these images is highly problematic given that most fluorescent emission spectra are broad and superimposed one with another. In order to spectrally resolve these images with high spectral resolution, we have developed the Phasor Analysis, used in the first and second harmonic, where each pixel in the image is used to construct a spectral profile that is Fourier trans- formed to produce the co-ordinates of the pixel in a polar plot. This graphical representation is free of fitting routines and can easily separate linear combina- tions of multiple spectral components. For this application we used the Andor’s iXon Ultra EMCCD camera to obtain spectral emission of cancer cell autofluor- escence excited with a multiphoton laser source. Using the multispectral Phasor Analysis we have been able to identify autofluorescence of cell membrane, mito- chondria, nucleus and other organelles. Given the complexity of these biochem- ical species, we treated the cells with various agents used to perturb metabolic states, membrane fluidity and cellular function to identify changes in spectro- scopic signals. A map of these chemical species can provide important informa- tion to identify proliferation, stress and dysplasia at the single cell level and can be applied to in the study of cancer and other diseases. Work supported in part by NIH grants P50 GM076516 and P41 GM103540.
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flim Phasor Analysis for time domain and frequency domain data
Biophysical Journal, 2013Co-Authors: Enrico Gratton, Michelle A Digman, Chiara Stringari, Cosimo ArnesanoAbstract:1779-Pos Board B671 FLIM Phasor Analysis for Time-Domain and Frequency-Domain Data Enrico Gratton, Michelle A. Digman, Chiara Stringari, Cosimo Arnesano. University of California, Irvine, Irvine, CA, USA. The Phasor Analysis of FLIM images provides a fit free global view of molec- ular species and their interaction in cells and tissues. Different techniques are used to collect the original data either in the time domain or in the frequency domain. The ‘‘Phasor transformation’’ which is based on the calculation of Fourier components should in principle make the Phasor plot independent of the domain of data collection. However, technical differences between the modalities of data acquisition in various instruments result in slightly different Phasor calculations. In this poster we discuss the origin of the variations be- tween the different methods of data acquisition. In particular we compare data obtained with the classical analog frequency domain instrument, data obtained with the FLIMbox principle that is based on a digital equivalent of the frequency domain instrument and data obtained with the popular time- correlated single photon counting instrument. We discuss how to minimize these differences which could results in Phasors plots that can be directly com- pared form data obtained with different instruments. We also discuss and com- pare methods of data filtering which can decrease the noise in the Phasor plot without affecting the resolution of FLIM images. Finally we compare Phasor plots obtained for different harmonics of the laser repetition frequency. We show that the Phasor plot at high harmonics from autofluorescence tissue sam- ples can distinguish between various extracellular components such as the weak fluorescence from collagen and elastin. Work supported in part by NIH-P41 P41-RRO3155, 8P41GM103540 and P50-GM076516
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spectral Phasor Analysis of pyronin y labeled rna microenvironments in living cells
Biomedical Optics Express, 2013Co-Authors: Laura M Andrews, Michelle A Digman, Mark R Jones, Enrico GrattonAbstract:We 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.