The Experts below are selected from a list of 333 Experts worldwide ranked by ideXlab platform
Yale E Goldman - One of the best experts on this subject based on the ideXlab platform.
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simultaneous position and orientation imaging of Polarized Fluorescence from rod in rod semiconductor nanoparticles on cytoplasmic dynein
Biophysical Journal, 2014Co-Authors: Lisa G Lippert, Tali Dadosh, Benjamin T Diroll, Christopher B Murray, Samara L Reckpeterson, Yale E GoldmanAbstract:Sub-pixel particle tracking and Polarized total internal reflection Fluorescence (polTIRF) microscopy have been instrumental in understanding of motor protein function. We have developed a method combining sub-pixel tracking and polTIRF microscopy to simultaneously track position and orientation of single fluorescent particles. The sample is illuminated with circularly Polarized light, and the emission is split into four polarizations and imaged with an EMCCD camera. The Yanagida lab recently published such a technique (Ohmachi et al, PNAS vol. 109, 2012). Our lab has developed a similar method, incorporating calibration routines to compensate for depolarization by phase shifts at reflecting surfaces and wavelength- and polarization-dependent detection efficiencies of the camera channels. The application of a 4x4 calibration matrix, generated by measuring deviations from theoretical Polarized Fluorescence intensities, compensates for depolarization caused by reflecting surfaces and channel crosstalk. Normalizing to an unPolarized sample at the relevant emission wavelength accounts for wavelength-dependent differential sensitivity of the four detection channels. Rod-in-rod CdSe core, CdS shell particles exhibit high polarization ratios; rods with dimensions of only ∼5x20 nm display an average polarization ratio of 0.81. This position and orientation tracking method is currently being used to observe rotations of streptavidin-conjugated quantum rods bound to the cytoplasmic dynein AAA ring. Correlation of angular and translocation information may provide insight into mechanisms of fluctuating dynein stepping. Supported by NIH grant P01GM087253.
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rotational motions of domains in elongation factor g detected by single molecule Polarized Fluorescence microscopy
Biophysical Journal, 2014Co-Authors: Chunlai Chen, John F Beausang, Xiaonan Cui, Barry S Cooperman, Yale E GoldmanAbstract:During the elongation cycle of protein synthesis, translocation of tRNAs and mRNA is catalyzed by the GTPase elongation factor G (EF-G) with high precision and speed. Conversion of the GTP to the GDP form of EF-G is considered essential for translocation, but the structural dynamics on the ribosome have not been reported. We used single molecule Polarized total internal reflection Fluorescence (polTIRF) microscopy to characterize tilting and rotational fluctuations within specific domains of EF-G. When EF-G binds to the ribosomal pre-translocation (PRE) complex, domains I and IV of EF-G undergo small rotations (10-15°) in conjunction with translocation, whereas domain III shows a much greater angular change, averaging 50°. Viomycin (Vio), which prevents translocation, reduces the rotational motions of domain III to 10-15° but has virtually no effect on the other domains. Spectinomycin also reduces domain III motions but less strongly than Vio. EF-G binding to ribosomal initiation complexes lacking A-site tRNA gives a similar pattern of domain rotations, but with shorter dwell times. In this case, the large rotation of domain III is barely inhibited by Vio. Irrespective of completion of translocation or presence of A-site tRNA, the initial 10-15° rotations of EF-G domains I, III and IV in the ribosome/EF-G complex indicate that the EF-G initially shifts the minimum of the free energy profile in the direction of translocation, suggesting that EF-G generates a force on the ribosome and/or the mRNA and tRNAs. Near the end of translocation, domain III completes its rotation either to push the mRNA and tRNAs (a working stroke) or to prevent reversal of translocation driven by thermal fluctuations (a ratchet). Supported by NIH grant GM080376 to YEG and BSC and AHA fellowship 12POST8910014 to CC.
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tilting and wobble of myosin v by high speed single molecule Polarized Fluorescence microscopy
Biophysical Journal, 2013Co-Authors: John F Beausang, Deborah Y Shroder, Philip C Nelson, Yale E GoldmanAbstract:Myosin V is biomolecular motor with two actin-binding domains (heads) that take multiple steps along actin by a hand-over-hand mechanism. We used high-speed Polarized total internal reflection Fluorescence (polTIRF) microscopy to study the structural dynamics of single myosin V molecules that had been labeled with bifunctional rhodamine linked to one of the calmodulins along the lever arm. With the use of time-correlated single-photon counting technology, the temporal resolution of the polTIRF microscope was improved ∼50-fold relative to earlier studies, and a maximum-likelihood, multitrace change-point algorithm was used to objectively determine the times when structural changes occurred. Short-lived substeps that displayed an abrupt increase in rotational mobility were detected during stepping, likely corresponding to random thermal fluctuations of the stepping head while it searched for its next actin-binding site. Thus, myosin V harnesses its fluctuating environment to extend its reach. Additional, less frequent angle changes, probably not directly associated with steps, were detected in both leading and trailing heads. The high-speed polTIRF method and change-point analysis may be applicable to single-molecule studies of other biological systems.
Rudolf Oldenbourg - One of the best experts on this subject based on the ideXlab platform.
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Analysis of Septin Reorganization at Cytokinesis Using Polarized Fluorescence Microscopy
Frontiers Media S.A., 2017Co-Authors: Molly Mcquilken, Shalin B Mehta, Amitabh Verma, Rudolf Oldenbourg, Amy S Gladfelter, Maximilian S. JentzschAbstract:Septins are conserved filament-forming proteins that act in diverse cellular processes. They closely associate with membranes and, in some systems, components of the cytoskeleton. It is not well understood how filaments assemble into higher-order structures in vivo or how they are remodeled throughout the cell cycle. In the budding yeast S. cerevisiae, septins are found through most of the cell cycle in an hourglass organization at the mother-bud neck until cytokinesis when the collar splits into two rings that disassemble prior to the next cell cycle. Experiments using Polarized Fluorescence microscopy have suggested that septins are arranged in ordered, paired filaments in the hourglass and undergo a coordinated 90° reorientation during splitting at cytokinesis. This apparent reorganization could be due to two orthogonal populations of filaments disassembling and reassembling or being preferentially retained at cytokinesis. In support of this idea, we report a decrease in septin concentration at the mother-bud neck during cytokinesis consistent with other reports and the timing of the decrease depends on known septin regulators including the Gin4 kinase. We took a candidate-based approach to examine what factors control reorientation during splitting and used Polarized Fluorescence microscopy to screen mutant yeast strains deficient in septin interacting proteins. Using this method, we have linked known septin regulators to different aspects of the assembly, stability, and reorganization of septin assemblies. The data support that ring splitting requires Gin4 activity and an anillin-like protein Bud4, and normal accumulation of septins at the ring requires phosphorylation of Shs1. We found distinct regulatory requirements for septin organization in the hourglass compared to split rings. We propose that septin subpopulations can vary in their localization and assembly/disassembly behavior in a cell-cycle dependent manner at cytokinesis
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dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Shalin B Mehta, Molly Mcquilken, Amitabh Verma, Rudolf Oldenbourg, Patrick J La Riviere, Patricia Occhipinti, Amy S GladfelterAbstract:Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous Fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of Polarized Fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.
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Polarized Fluorescence microscopy to study cytoskeleton assembly and organization in live cells
Current protocols in pharmacology, 2015Co-Authors: Molly Mcquilken, Shalin B Mehta, Amitabh Verma, Grant Harris, Rudolf Oldenbourg, Amy S GladfelterAbstract:The measurement of not only the location but also the organization of molecules in live cells is crucial to understanding diverse biological processes. Polarized light microscopy provides a nondestructive means to evaluate order within subcellular domains. When combined with Fluorescence microscopy and GFP-tagged proteins, the approach can reveal organization within specific populations of molecules. This unit describes a protocol for measuring the architectural dynamics of cytoskeletal components using Polarized Fluorescence microscopy and OpenPolScope open-access software (http://www.openpolscope.org). The protocol describes installation of linear polarizers or a liquid crystal (LC) universal compensator, calibration of the system, Polarized Fluorescence imaging, and analysis. The use of OpenPolScope software and hardware allows for reliable, user-friendly image acquisition to measure and analyze Polarized Fluorescence. © 2015 by John Wiley & Sons, Inc. Keywords: Polarized Fluorescence; cytoskeleton; image analysis; OpenPolScope
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rapid and quantitative imaging of excitation Polarized Fluorescence reveals ordered septin dynamics in live yeast
Biophysical Journal, 2011Co-Authors: Bradley S Demay, Rudolf Oldenbourg, Amy S Gladfelter, Naoki NodaAbstract:We report an imaging method for fast, sensitive analysis of the orientation of fluorescent molecules by employing a liquid-crystal based universal polarizer in the optical path of a wide-field light microscope. We developed specific acquisition and processing algorithms for measuring the anisotropy and for correcting artifacts caused by Fluorescence bleaching, background light, and differential transmission of optical components. We call this approach the Fluorescence LC-PolScope and we used it to analyze the architectural dynamics of septin-green fluorescent protein (septin-GFP) constructs in the neck region of budding yeast. We describe three different states of highly anisotropic septin arrays in which the prevailing orientation of GFP dipoles was either parallel or perpendicular to the mother-bud axis. The transitions between these ordered states were characterized by transient isotropic states. To analyze the patterns of Polarized Fluorescence, we modeled the alignment of septin-GFP constructs in different stages of septin ring formation. Based on our model, our experimental data are consistent with the formation of paired rather than single filaments and the axis of the α-helical septin terminus linked to a GFP molecule is likely oriented normal to the cell surface. The Fluorescence LC-PolScope combines the molecular specificity of Fluorescence tagging with the structural specificity of Polarized light analysis.
Asima Pradhan - One of the best experts on this subject based on the ideXlab platform.
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characteristic spectral features of the Polarized Fluorescence of human breast cancer in the wavelet domain
Applied Spectroscopy, 2012Co-Authors: Anita H Gharekhan, Nrusingh C Biswal, Sharad Gupta, Prasanta K Panigrahi, Asima PradhanAbstract:Wavelet transform of Polarized Fluorescence spectra of human breast tissues is found to localize spectral features that can reliably differentiate normal and malignant tissue types. The intensity differences of parallel and perpendicularly Polarized Fluorescence spectra are subjected to investigation, since they are relatively free of diffusive background. A number of parameters, capturing spectral variations and subtle changes in the diseased tissues in the visible wavelength regime, are clearly identifiable in the wavelet domain. These manifest both in the average low-pass and high frequency high-pass wavelet coefficients.
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characteristic spectral features of the Polarized Fluorescence of human breast cancer in the wavelet domain
arXiv: Medical Physics, 2012Co-Authors: Anita H Gharekhan, Nrusingh C Biswal, Sharad Gupta, Prasanta K Panigrahi, Asima PradhanAbstract:Wavelet transform of Polarized Fluorescence spectra of human breast tissues is found to localize spectral features that can reliably differentiate normal and malignant tissue types. The intensity differences of parallel and perpendicularly Polarized Fluorescence spectra are subjected to investigation, since the same is relatively free of the diffusive background. A number of parameters, capturing spectral variations and subtle changes in the diseased tissues in the visible wavelength regime, are clearly identifiable in the wavelet domain. These manifest both in the average low pass and high frequency high pass wavelet coefficients.
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characterizing breast cancer tissues through the spectral correlation properties of Polarized Fluorescence
Journal of Biomedical Optics, 2008Co-Authors: Anita H Gharekhan, Prasanta K Panigrahi, Siddharth Arora, K B K Mayya, M B Sureshkumar, Asima PradhanAbstract:We study the spectral correlation properties of the Polarized Fluorescence spectra of normal and cancerous human breast tissues, corresponding to patients belonging to diverse age groups and socioeconomic backgrounds. The emission range in the visible wavelength regime of 500 to 700 nm is analyzed, with the excitation wavelength at 488 nm, where flavin is one of the active fluorophores. The correlation matrices for parallel and perpendicularly Polarized Fluorescence spectra reveal correlated domains, differing significantly in normal and cancerous tissues. These domains can be ascribed to different fluorophores and absorbers in the tissue medium. The spectral fluctuations in the perpendicular component of the cancerous tissue clearly reveal randomization not present in the normal channel. Random matrix-based predictions for the spectral correlations match quite well with the observed behavior. The eigenvectors of the correlation matrices corresponding to large eigenvalues clearly separate out different tissue types and identify the dominant wavelengths, which are active in cancerous tissues.
Amy S Gladfelter - One of the best experts on this subject based on the ideXlab platform.
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Analysis of Septin Reorganization at Cytokinesis Using Polarized Fluorescence Microscopy
Frontiers Media S.A., 2017Co-Authors: Molly Mcquilken, Shalin B Mehta, Amitabh Verma, Rudolf Oldenbourg, Amy S Gladfelter, Maximilian S. JentzschAbstract:Septins are conserved filament-forming proteins that act in diverse cellular processes. They closely associate with membranes and, in some systems, components of the cytoskeleton. It is not well understood how filaments assemble into higher-order structures in vivo or how they are remodeled throughout the cell cycle. In the budding yeast S. cerevisiae, septins are found through most of the cell cycle in an hourglass organization at the mother-bud neck until cytokinesis when the collar splits into two rings that disassemble prior to the next cell cycle. Experiments using Polarized Fluorescence microscopy have suggested that septins are arranged in ordered, paired filaments in the hourglass and undergo a coordinated 90° reorientation during splitting at cytokinesis. This apparent reorganization could be due to two orthogonal populations of filaments disassembling and reassembling or being preferentially retained at cytokinesis. In support of this idea, we report a decrease in septin concentration at the mother-bud neck during cytokinesis consistent with other reports and the timing of the decrease depends on known septin regulators including the Gin4 kinase. We took a candidate-based approach to examine what factors control reorientation during splitting and used Polarized Fluorescence microscopy to screen mutant yeast strains deficient in septin interacting proteins. Using this method, we have linked known septin regulators to different aspects of the assembly, stability, and reorganization of septin assemblies. The data support that ring splitting requires Gin4 activity and an anillin-like protein Bud4, and normal accumulation of septins at the ring requires phosphorylation of Shs1. We found distinct regulatory requirements for septin organization in the hourglass compared to split rings. We propose that septin subpopulations can vary in their localization and assembly/disassembly behavior in a cell-cycle dependent manner at cytokinesis
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dissection of molecular assembly dynamics by tracking orientation and position of single molecules in live cells
Proceedings of the National Academy of Sciences of the United States of America, 2016Co-Authors: Shalin B Mehta, Molly Mcquilken, Amitabh Verma, Rudolf Oldenbourg, Patrick J La Riviere, Patricia Occhipinti, Amy S GladfelterAbstract:Regulation of order, such as orientation and conformation, drives the function of most molecular assemblies in living cells but remains difficult to measure accurately through space and time. We built an instantaneous Fluorescence polarization microscope, which simultaneously images position and orientation of fluorophores in living cells with single-molecule sensitivity and a time resolution of 100 ms. We developed image acquisition and analysis methods to track single particles that interact with higher-order assemblies of molecules. We tracked the fluctuations in position and orientation of molecules from the level of an ensemble of fluorophores down to single fluorophores. We tested our system in vitro using fluorescently labeled DNA and F-actin, in which the ensemble orientation of Polarized Fluorescence is known. We then tracked the orientation of sparsely labeled F-actin network at the leading edge of migrating human keratinocytes, revealing the anisotropic distribution of actin filaments relative to the local retrograde flow of the F-actin network. Additionally, we analyzed the position and orientation of septin-GFP molecules incorporated in septin bundles in growing hyphae of a filamentous fungus. Our data indicate that septin-GFP molecules undergo positional fluctuations within ∼350 nm of the binding site and angular fluctuations within ∼30° of the central orientation of the bundle. By reporting position and orientation of molecules while they form dynamic higher-order structures, our approach can provide insights into how micrometer-scale ordered assemblies emerge from nanoscale molecules in living cells.
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Polarized Fluorescence microscopy to study cytoskeleton assembly and organization in live cells
Current protocols in pharmacology, 2015Co-Authors: Molly Mcquilken, Shalin B Mehta, Amitabh Verma, Grant Harris, Rudolf Oldenbourg, Amy S GladfelterAbstract:The measurement of not only the location but also the organization of molecules in live cells is crucial to understanding diverse biological processes. Polarized light microscopy provides a nondestructive means to evaluate order within subcellular domains. When combined with Fluorescence microscopy and GFP-tagged proteins, the approach can reveal organization within specific populations of molecules. This unit describes a protocol for measuring the architectural dynamics of cytoskeletal components using Polarized Fluorescence microscopy and OpenPolScope open-access software (http://www.openpolscope.org). The protocol describes installation of linear polarizers or a liquid crystal (LC) universal compensator, calibration of the system, Polarized Fluorescence imaging, and analysis. The use of OpenPolScope software and hardware allows for reliable, user-friendly image acquisition to measure and analyze Polarized Fluorescence. © 2015 by John Wiley & Sons, Inc. Keywords: Polarized Fluorescence; cytoskeleton; image analysis; OpenPolScope
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rapid and quantitative imaging of excitation Polarized Fluorescence reveals ordered septin dynamics in live yeast
Biophysical Journal, 2011Co-Authors: Bradley S Demay, Rudolf Oldenbourg, Amy S Gladfelter, Naoki NodaAbstract:We report an imaging method for fast, sensitive analysis of the orientation of fluorescent molecules by employing a liquid-crystal based universal polarizer in the optical path of a wide-field light microscope. We developed specific acquisition and processing algorithms for measuring the anisotropy and for correcting artifacts caused by Fluorescence bleaching, background light, and differential transmission of optical components. We call this approach the Fluorescence LC-PolScope and we used it to analyze the architectural dynamics of septin-green fluorescent protein (septin-GFP) constructs in the neck region of budding yeast. We describe three different states of highly anisotropic septin arrays in which the prevailing orientation of GFP dipoles was either parallel or perpendicular to the mother-bud axis. The transitions between these ordered states were characterized by transient isotropic states. To analyze the patterns of Polarized Fluorescence, we modeled the alignment of septin-GFP constructs in different stages of septin ring formation. Based on our model, our experimental data are consistent with the formation of paired rather than single filaments and the axis of the α-helical septin terminus linked to a GFP molecule is likely oriented normal to the cell surface. The Fluorescence LC-PolScope combines the molecular specificity of Fluorescence tagging with the structural specificity of Polarized light analysis.
A. J. W. G. Visser - One of the best experts on this subject based on the ideXlab platform.
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maximum entropy analysis of Polarized Fluorescence decay of e gfp in aqueous solution
Methods and Applications in Fluorescence, 2017Co-Authors: Victor V Skakun, Eugene Novikov, Jan Willem Borst, A. J. W. G. VisserAbstract:The maximum entropy method (MEM) was used for the analysis of Polarized Fluorescence decays of enhanced green fluorescent protein (EGFP) in buffered water/glycerol mixtures, obtained with time-correlated single-photon counting (Visser et al 2016 Methods Appl. Fluoresc. 4 035002). To this end, we used a general-purpose software module of MEM that was earlier developed to analyze (complex) laser photolysis kinetics of ligand rebinding reactions in oxygen binding proteins. We demonstrate that the MEM software provides reliable results and is easy to use for the analysis of both total Fluorescence decay and Fluorescence anisotropy decay of aqueous solutions of EGFP. The rotational correlation times of EGFP in water/glycerol mixtures, obtained by MEM as maxima of the correlation-time distributions, are identical to the single correlation times determined by global analysis of parallel and perpendicular Polarized decay components. The MEM software is also able to determine homo-FRET in another dimeric GFP, for which the transfer correlation time is an order of magnitude shorter than the rotational correlation time. One important advantage utilizing MEM analysis is that no initial guesses of parameters are required, since MEM is able to select the least correlated solution from the feasible set of solutions.
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comparison of the dynamical structures of lipoamide dehydrogenase and glutathione reductase by time resolved Polarized flavin Fluorescence
Biochemistry, 1992Co-Authors: Philippe I. H. Bastiaens, Arie Van Hoek, Willem F Wolkers, Jean Claude Brochon, A. J. W. G. VisserAbstract:Time-resolved Polarized Fluorescence spectroscopy has been applied to the bound FAD in the structurally related flavoproteins lipoamide dehydrogenase from Azotobacter uinelandii (LipDH-AV) and glutathione reductase (GR) from human erythrocytes. The Fluorescence parameters as obtained from the maximum entropy analysis differ considerably in both enzymes, reflecting the unique properties of the flavin microenvironment. Three conformational substates are revealed in LipDH-AV and five in GR. Almost 90% of the population of GR molecules has a Fluorescence lifetime in the order of 30 ps which originates from efficient exciplex formation with Tyr197. Equilibrium fluctuations between conformational substates are observed for LipDH-AV on a nanosecond time scale in the temperature range 277-3 13 K. Intercon- version between conformational substates in GR is slow, indicating that large activation barriers exist between the states. In agreement with these results, a model is postulated which ascribes a role in catalysis to equilibrium fluctuations between conformational substates in GR and LipDH-AV. From time-resolved Fluorescence anisotropy as a function of temperature, distinction can be made between flavin reorientational motion and interflavin energy transfer. In both proteins intersubunit energy transfer between the prosthetic groups is observed. Furthermore, it is revealed that only the flavin in glutathione reductase exhibits rapid restricted reorientational motion. Geometric information concerning the relative orientation and distance of the flavins can be extracted from the parameters describing the energy-transfer process. The obtained spatial arrangement of the flavins is in excellent agreement with crystallographic data.
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energy transfer between the flavin chromophores of electron transferring flavoprotein from megasphaera elsdenii as inferred from time resolved red edge and blue edge Fluorescence spectroscopy
Journal of Fluorescence, 1991Co-Authors: Philippe I. H. Bastiaens, E M Onuallain, Annemieke Hoek, Stephen G Mayhew, A. J. W. G. VisserAbstract:Both a mode-locked argon-ion laser and synchrotron radiation were used as excitation sources to obtain time-resolved Polarized Fluorescence of the two FAD cofactors in electron transferring flavoprotein fromMegasphaera elsdenii. Red-edge excited and blue-edge detected Fluorescence anisotropy decay curves did not contain a fast relaxation process which was observed upon mainband excitation and detection. This relaxation was assigned to homo-energy transfer between the two FAD cofactors. Failure of energy transfer as observed with edge spectroscopy on this protein excludes restricted reorientational motion of the flavins as a possible mechanism of depolarization. From the global analysis of the Fluorescence anisotropy decay surface obtained at multiple excitation and detection wavelengths, the distance between and the relative orientation of the flavins could be estimated. The methodology described has general applicability in other multichromophoric biopolymers and has the potential to acquire accurate geometrical parameters in these systems.