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Enrico Gratton - One of the best experts on this subject based on the ideXlab platform.
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Shedding light on melanins within in situ human eye melanocytes using 2-photon microscopy profiling techniques
Scientific Reports, 2019Co-Authors: Ephrem Sitiwin, Enrico Gratton, Michele C. Madigan, Svetlana Cherepanoff, Robert Max Conway, Renee Whan, Alexander MacmillanAbstract:Choroidal 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 longer spectral emission (580–625 nm) and pheomelanin-enriched lighter pigmented HCM regions mapped to Phasors with longer lifetimes and shorter spectral emission (550–585 nm). Overall, we demonstrated that these methods can identify and quantitatively profile the heterogeneous eumelanins/pheomelanins within in situ HCMs, and visualize melanosome spatial distributions, not previously reported for these cells.
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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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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:This protocol describes fit-free analysis of fluorescence lifetime imaging microscopy (FLIM) data using the Phasor approach. Pixel-by-pixel decays are transformed to the Phasor space, and then the clusters can be connected to the image by the reciprocity rules of the Phasor plots. 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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Phasor plots and spectral Phasor analysis of laurdan and prodan for membrane heterogeneity studies new frontiers in membrane biophysics
Biophysical Journal, 2014Co-Authors: Leonel Malacrida, Arturo Briva, Carrisa M Vetromile, Ana Denicola, Enrico Gratton, David M JamesonAbstract:449-Pos Board B204 Phasor Plots and Spectral Phasor Analysis of Laurdan and Prodan for Membrane Heterogeneity Studies: New Frontiers in Membrane Biophysics Leonel S. Malacrida 1 , Arturo Briva 1 , Carrisa M. Vetromile 2 , Enrico Gratton 3 , Ana Denicola 4 , David M. Jameson 2 . Departamento de Fisiopatologi´a, Hospital de Cli´nicas, Universidad de la Republica, Montevideo, Uruguay, 2 Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI, USA, 3 Laboratory for Fluorescence Dynamics, University of California, Irvine, CA, USA, 4 Laboratorio de Fisicoqui´mica Biolo´gica, Facultad de Ciencias, Universidad de la Republica, Montevideo, Uruguay. Since its introduction by Weber [1], fluorophores in the PRODAN series have contributed to our understanding of hydration and packing in biological mem- branes. Here we apply methods based on lifetime determinations and Phasor plots as well as steady-state measurements using the spectral Phasor approach, for analysis of the behavior of LAURDAN and PRODAN in vesicles. The life- time Phasor approach (Jameson et al., [2]) uses a plot of M.sin(F) versus M.cos(F), where M is the modulation ratio and F is the phase angle taken from frequency domain fluorometry. With Spectral Phasors, introduced by Fer- eidouni et al [3], the steady-state fluorescence spectrum is Fourier transformed, resulting in two coordinates in x and y used for a scatter plot (Spectral Phasor). The temporal Phasor approach shows significant improvement compared with older methods as regards discrimination of the effects of temperature, choles- terol content and drug addition, in our membrane model systems. This approach is very convenient for characterization of complex systems wherein lifetime heterogeneity and relaxation processes are present. The Spectral Phasor approach is a very useful method for characterization of subtle changes in membrane hydration and packing. The major advantage of both methods is that they provide a model-less approach, which is relevant to complex studies on native systems, where endogenous fluorescence can introduce undesired mistakes. Examples of the application of both methods to membrane systems will be given. [1] Weber et al, Biochemistry, 1979. [2] Jameson et al, Appl. Spectosc. Rev., 1991. [3] Fereidouni et al, Opt. Express, 2012.
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flim Phasor analysis for time domain and frequency domain data
Biophysical Journal, 2013Co-Authors: Enrico Gratton, Chiara Stringari, Michelle A Digman, 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
Dusmanta Kumar Mohanta - One of the best experts on this subject based on the ideXlab platform.
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Real-Time Wide-Area Disturbance Monitoring and Protection Methodology for EHV Transmission lines
INAE Letters, 2018Co-Authors: Balimidi Mallikarjuna, Debjyoti Chatterjee, Maddikara Jaya Bharata Reddy, Dusmanta Kumar MohantaAbstract:This paper presents a real-time wide-area disturbance monitoring and protection (RWADMP) methodology for the backup protection of EHV transmission lines in smart grids (SG). The proposed methodology employs harmonic Phasors (1st, 2nd, and 3rd order) of 3-Φ current signals estimated using multi-Phasor measurement unit (MPMU) for fault detection, classification, and discrimination of faulty line accurately. Further, the stressed condition such as load encroachment is discriminated from fault. The Phasors which were estimated at MPMU are communicated to Phasor data concentrator (PDC) at system protection center. MPMU is realized in hardware using NI cRIO, current sensor and GPS module in conjunction with LabVIEW software. Performance of the proposed methodology has been validated on the scale down laboratory models of 400 kV EHV transmission systems such as two-generator two-bus and two-generator three-bus systems. The experimental results show the efficacy of the proposed methodology in improving the overall reliability of the protection system. Also, the proposed methodology is less sensitive to the different fault conditions like fault resistance (FR) and fault distance (FD). Further, the user-friendly LabVIEW monitoring system enhances the visualization of the power system state in PDC. Also, it helps the grid operator to take appropriate action in case of system stressed condition and avoids the mal-operation of the protective devices.
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PMU based adaptive zone settings of distance relays for protection of multi-terminal transmission lines
Protection and Control of Modern Power Systems, 2018Co-Authors: Balimidi Mallikarjuna, Pudi Shanmukesh, Dwivedi Anmol, Maddikara Jaya Bharata Reddy, Dusmanta Kumar MohantaAbstract:This paper proposes Phasor Measurement Unit (PMU) based adaptive zone settings of distance relays (PAZSD) methodology for protection of multi-terminal transmission lines (MTL). The PAZSD methodology employs current coefficients to adjust the zone settings of the relays during infeed situation. These coefficients are calculated in Phasor data concentrator (PDC) at system protection center (SPC) using the current Phasors obtained from PMUs. The functioning of the distance relays during infeed condition with and without the proposed methodology has been illustrated through a four-bus model implemented in PSCAD/EMTDC environment. Further, the performance of the proposed methodology has been validated in real-time, on a laboratory prototype of Extra High Voltage multi-terminal transmission lines (EHV MTL). The Phasors are estimated in PMUs using NI cRIO-9063 chassis embedded with data acquisition sensors in conjunction with LabVIEW software. The simulation and hardware results prove the efficacy of the proposed methodology in enhancing the performance and reliability of conventional distance protection system in real-time EHV MTLs.
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An adaptive supervised wide-area backup protection scheme for transmission lines protection
Protection and Control of Modern Power Systems, 2017Co-Authors: Balimidi Mallikarjuna, P. V. Vardhan Varma, S. D. Samir, M. Jaya Bharata Reddy, Dusmanta Kumar MohantaAbstract:Maloperation of conventional relays is becoming prevalent due to ever increase in complexity of conventional power grids. They are dominant during power system contingencies like power swing, load encroachment, voltage instability, unbalanced loading, etc. In these situations, adaptive supervised wide-area backup protection (ASWABP) plays a major role in enhancing the power system reliability. A balance between security and dependability of protection is essential to maintain the reliability. This paper proposes multi-Phasor measurement units (MPMU) based ASWABP scheme that can function effectively during faults besides power system contingencies. MPMU is an extended version of Phasor Measurement Unit (PMU). It is an intelligent electronic device which estimates the synchronized predominant harmonic Phasors (100Hz and 150Hz) along with the fundamental Phasors (50Hz) of three phase voltages and currents with high precision. The proposed ASWABP scheme can detect the fault, identify the parent bus, determine the faulty branch and classify the faults using MPMU measurements at System Protection Center (SPC). Based on these MPMU measurements (received at Phasor data concentrator (PDC) at SPC) the appropriate relays will be supervised to enhance the overall reliability of the power grid. Numerous case studies are conducted on WSCC-9 bus and IEEE-14 bus systems to illustrate the security and dependability attributes of proposed ASWABP scheme in MATLAB/Simulink environment. Also, comparative studies are performed with the existing conventional distance protection (Mho relays) for corroborating the superiority of the proposed scheme regarding security and dependability. Comparative studies have shown that the proposed scheme can be used as adaptive supervised wide-area backup protection of conventional distance protection.
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Situational Awareness Enhancement of Smart Grids Using Intelligent Maintenance Scheduling of Phasor Measurement Sensors
IEEE Sensors Journal, 2017Co-Authors: Soumita Ghosh, Debomita Ghosh, Dusmanta Kumar MohantaAbstract:Phasor measurement units (PMUs) have emerged as the sensors for smart grids because of their inherent capability to provide with synchronized current and voltage Phasors at buses sprawling over large geographical area. Thus, PMUs or Phasor measurement sensors (PMSs) constitute the edifice for wide area measurement system to provide with situational awareness (SA) for a complex interconnected electric grid by providing with real-time data. They assist in predicting future disturbances and also for decision-making of control actions. The PMS outage has adverse impact on grid operation. Therefore, their periodic maintenance must be carried out to ensure healthy and reliable operation to enhance SA. In this paper, optimized routine and self-supervision test intervals for ensuring maximum observability of the grid have been proposed. Since PMSs are taken out of service during maintenance, an intelligent maintenance scheduling algorithm has been proposed such that SA is maximum. Case studies pertaining to an example nine-bus, IEEE 14, 24, 30 and new England 39-bus system validate the efficacy of proposed approach. To prove the efficacy of results, general confidence intervals are reported for observability.
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A Brief Review of Phasor Measurement Units as Sensors for Smart Grid
Electric Power Components and Systems, 2016Co-Authors: Dusmanta Kumar Mohanta, Cherukuri Murthy, Diptendu Sinha RoyAbstract:AbstractBasic Phasor measurement units are the sensing devices that capture power system Phasors synchronously over wide geographical areas with high precision by means of global positioning system enabled time-stamping. The data obtained from Phasor measurement units form the basis of all monitoring and control actions for wide-area measurement systems, and hence, Phasor measurement units are the edifice of a wide-area measurement system. Within the purview of Phasor measurement units, there have been diverse research contributions made in past decades. This article presents a brief review of Phasor measurement units performing their functions as smart sensors. The review includes their evolution, optimal placement, applications, and reliability assessment. The main contributions pertain to segregation of research findings into broad domains with corroborations to validate Phasor measurement units as the efficient sensors for the smart grid.
David M Jameson - 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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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:This protocol describes fit-free analysis of fluorescence lifetime imaging microscopy (FLIM) data using the Phasor approach. Pixel-by-pixel decays are transformed to the Phasor space, and then the clusters can be connected to the image by the reciprocity rules of the Phasor plots. 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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Phasor plots and spectral Phasor analysis of laurdan and prodan for membrane heterogeneity studies new frontiers in membrane biophysics
Biophysical Journal, 2014Co-Authors: Leonel Malacrida, Arturo Briva, Carrisa M Vetromile, Ana Denicola, Enrico Gratton, David M JamesonAbstract:449-Pos Board B204 Phasor Plots and Spectral Phasor Analysis of Laurdan and Prodan for Membrane Heterogeneity Studies: New Frontiers in Membrane Biophysics Leonel S. Malacrida 1 , Arturo Briva 1 , Carrisa M. Vetromile 2 , Enrico Gratton 3 , Ana Denicola 4 , David M. Jameson 2 . Departamento de Fisiopatologi´a, Hospital de Cli´nicas, Universidad de la Republica, Montevideo, Uruguay, 2 Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI, USA, 3 Laboratory for Fluorescence Dynamics, University of California, Irvine, CA, USA, 4 Laboratorio de Fisicoqui´mica Biolo´gica, Facultad de Ciencias, Universidad de la Republica, Montevideo, Uruguay. Since its introduction by Weber [1], fluorophores in the PRODAN series have contributed to our understanding of hydration and packing in biological mem- branes. Here we apply methods based on lifetime determinations and Phasor plots as well as steady-state measurements using the spectral Phasor approach, for analysis of the behavior of LAURDAN and PRODAN in vesicles. The life- time Phasor approach (Jameson et al., [2]) uses a plot of M.sin(F) versus M.cos(F), where M is the modulation ratio and F is the phase angle taken from frequency domain fluorometry. With Spectral Phasors, introduced by Fer- eidouni et al [3], the steady-state fluorescence spectrum is Fourier transformed, resulting in two coordinates in x and y used for a scatter plot (Spectral Phasor). The temporal Phasor approach shows significant improvement compared with older methods as regards discrimination of the effects of temperature, choles- terol content and drug addition, in our membrane model systems. This approach is very convenient for characterization of complex systems wherein lifetime heterogeneity and relaxation processes are present. The Spectral Phasor approach is a very useful method for characterization of subtle changes in membrane hydration and packing. The major advantage of both methods is that they provide a model-less approach, which is relevant to complex studies on native systems, where endogenous fluorescence can introduce undesired mistakes. Examples of the application of both methods to membrane systems will be given. [1] Weber et al, Biochemistry, 1979. [2] Jameson et al, Appl. Spectosc. Rev., 1991. [3] Fereidouni et al, Opt. Express, 2012.
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Applications of Phasors to in vitro time-resolved fluorescence measurements
Analytical Biochemistry, 2011Co-Authors: Martin Štefl, Justin A. Ross, Nicholas G. James, David M JamesonAbstract:The Phasor method of treating fluorescence lifetime data provides a facile and convenient approach to characterize lifetime heterogeneity and to detect the presence of excited state reactions such as solvent relaxation and Förster resonance energy transfer. The method uses a plot of M sin(Φ) versus M cos(Φ), where M is the modulation ratio and Φ is the phase angle taken from frequency domain fluorometry. A principal advantage of the Phasor method is that it provides a model-less approach to time-resolved data amenable to visual inspection. Although the Phasor approach has been recently applied to fluorescence lifetime imaging microscopy, it has not been used extensively for cuvette studies. In the current study, we explore the applications of the method to in vitro samples. The Phasors of binary and ternary mixtures of fluorescent dyes demonstrate the utility of the method for investigating complex mixtures. Data from excited state reactions, such as dipolar relaxation in membrane and protein systems and also energy transfer from the tryptophan residue to the chromophore in enhanced green fluorescent protein, are also presented. © 2010 Elsevier Inc. All rights reserved.
A M Ranjbar - One of the best experts on this subject based on the ideXlab platform.
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an adaptive pmu based wide area backup protection scheme for power transmission lines
IEEE Transactions on Smart Grid, 2015Co-Authors: Kalantar M Neyestanaki, A M RanjbarAbstract:This paper proposes a novel adaptive wide area backup protection scheme for transmission lines. In the proposed scheme, both the faulted line and fault location are determined by a limited number of synchronized Phasor measurements. Based on Phasor measurement unit placement and network topology, subsets of lines and buses called backup protection zones (BPZs) are formed. After a fault occurs in the transmission network, the sum of zero- and/or positive-sequence currents entering the faulted BPZ highly increases, and hence, the faulted BPZ can be determined. The linear least squares method is then used to determine the faulted line, as well as the fault location by voltage and current Phasors of the faulted BPZ. Accordingly, the proposed scheme provides a closed-form and noniterative solution for the faulted line and fault location identification problem. On the other hand, it readily determines the faulted line regardless of the fault type, fault resistance, and measurement errors. To show the effectiveness of the method, it is applied to the WSCC 9-bus and IEEE 118-bus test systems. Simulation results verify successful identification of the faulted BPZ as well as the faulted line within the faulted BPZ with limited measurement points.
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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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:This protocol describes fit-free analysis of fluorescence lifetime imaging microscopy (FLIM) data using the Phasor approach. Pixel-by-pixel decays are transformed to the Phasor space, and then the clusters can be connected to the image by the reciprocity rules of the Phasor plots. 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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Phasor plots and spectral Phasor analysis of laurdan and prodan for membrane heterogeneity studies new frontiers in membrane biophysics
Biophysical Journal, 2014Co-Authors: Leonel Malacrida, Arturo Briva, Carrisa M Vetromile, Ana Denicola, Enrico Gratton, David M JamesonAbstract:449-Pos Board B204 Phasor Plots and Spectral Phasor Analysis of Laurdan and Prodan for Membrane Heterogeneity Studies: New Frontiers in Membrane Biophysics Leonel S. Malacrida 1 , Arturo Briva 1 , Carrisa M. Vetromile 2 , Enrico Gratton 3 , Ana Denicola 4 , David M. Jameson 2 . Departamento de Fisiopatologi´a, Hospital de Cli´nicas, Universidad de la Republica, Montevideo, Uruguay, 2 Department of Cell and Molecular Biology, John A. Burns School of Medicine, University of Hawaii, Honolulu, HI, USA, 3 Laboratory for Fluorescence Dynamics, University of California, Irvine, CA, USA, 4 Laboratorio de Fisicoqui´mica Biolo´gica, Facultad de Ciencias, Universidad de la Republica, Montevideo, Uruguay. Since its introduction by Weber [1], fluorophores in the PRODAN series have contributed to our understanding of hydration and packing in biological mem- branes. Here we apply methods based on lifetime determinations and Phasor plots as well as steady-state measurements using the spectral Phasor approach, for analysis of the behavior of LAURDAN and PRODAN in vesicles. The life- time Phasor approach (Jameson et al., [2]) uses a plot of M.sin(F) versus M.cos(F), where M is the modulation ratio and F is the phase angle taken from frequency domain fluorometry. With Spectral Phasors, introduced by Fer- eidouni et al [3], the steady-state fluorescence spectrum is Fourier transformed, resulting in two coordinates in x and y used for a scatter plot (Spectral Phasor). The temporal Phasor approach shows significant improvement compared with older methods as regards discrimination of the effects of temperature, choles- terol content and drug addition, in our membrane model systems. This approach is very convenient for characterization of complex systems wherein lifetime heterogeneity and relaxation processes are present. The Spectral Phasor approach is a very useful method for characterization of subtle changes in membrane hydration and packing. The major advantage of both methods is that they provide a model-less approach, which is relevant to complex studies on native systems, where endogenous fluorescence can introduce undesired mistakes. Examples of the application of both methods to membrane systems will be given. [1] Weber et al, Biochemistry, 1979. [2] Jameson et al, Appl. Spectosc. Rev., 1991. [3] Fereidouni et al, Opt. Express, 2012.
