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Fluorescence Fluctuation Spectroscopy in the Perinuclear Space
Tuesday, March 1, 2016 understanding and manipulating their photophysics. In particular, the nonradiative and optically-induced recovery of photo-excited FPs trapped from long-lived dark states forms the basis for many super-resolution imaging techniques. We have employed time and frequency-domain technique to capture the dynamics of dark sate conversion (DSC) and ground state recovery (GSR) processes and investigate the molecular mechanisms of these phenomena. In our time-domain method, FPs are excited with millisecond time-scale pulses with a variable inter-pulse delay, to directly obtain the GSR rate in a modelfree manner. The complementary frequency-domain technique, which relies on the sinusoidal modulation of excitation intensity and measurement of ‘‘phase advance’’ resulting from the asymmetry of the fluorescence signals, is suitable for high-throughput DSC rate measurements. The GSR and DSC time-constants of mCherry, TagRFP-T and Kriek FPs measured using those techniques fall in the ms-ms range of time-scales, suggesting that these phenomena might be associated with conformational change or protonation of the chromophore. Building on these developments, we capitalized on a high-throughput flowcytometry platform for simultaneous characterization of the DSC and irreversible photobleaching of large FP libraries generated by site-directed or random mutagenesis. We employed the frequency-domain technique for DSC rate characterization whereas the determination of irreversible photobleaching is based on pulsed bleaching and subsequent recovery. Our results provide evidence for a correlation between those above-mentioned processes that might reveal the pathways of photo-bleaching processes of the FPs. This unique capability of characterizing the dark states of large FP libraries has potential of finding new mutants with novel photo-physical properties for diverse applications in, for example, optogenetics and super-resolution imaging. 2430-Pos Board B574 Experimental Determination of Single- and Two-Photon Excitation Transition Moments in Representative Fluorescent Proteins Josef Lazar1, Prakash Shukla2, Richard Chazal2, Alexey Bondar1, David von Stetten3, Antoine Royant3. 1 Institute of Nanobiology and Structural Biology, Nove Hrady, Czech Republic, 2Institute of Organic Chemistry and Biochemistry AS CR, Prague, Czech Republic, 3European Synchrotron Radiation Facility, Grenoble, France. Fluorescent proteins are the workhorses of biological molecular imaging. Important imaging modalities (such as polarization microscopy or FRET imaging) exploit anisotropic optical properties of fluorescent proteins. The anisotropy of optical properties of fluorescent proteins is described by a vector (the transition dipole moment, TDM) for single-photon absorption, and by a tensor (the absorptivity tensor) for two-photon absorption. Despite the importance of anisotropy of light absorption in fluorescent proteins for quantitative structural interpretation of many imaging experiments, relevant experimental data is very limited. Here we present the results of our optical measurements on crystals of representative fluorescent proteins, as well as mathematical interpretation of these results, yielding information on the orientation of TDMs and properties of absorptivity tensors of the investigated fluorescent protein molecules. 2431-Pos Board B575 Fluorescence Fluctuation Spectroscopy in the Perinuclear Space Jared Hennen1, Cosmo Saunders2, G.W. Gant Luxton2, Joachim D. Mueller1. 1 School of Physics and Astronomy, University of Minnesota, Minneapolis, MN, USA, 2Department of Genetics, Cell Biology, and Development, University of Minnesota, Minneapolis, MN, USA. The importance of the nuclear envelope extends well beyond separating the cytoplasm and nucleoplasm. The perinuclear space (PS) located within the nuclear envelope contains proteins involved in many cellular functions including cellular signaling, force transduction, and mechanical properties of the cell. Protein oligomerization plays a central role in the control of these biological processes. Currently, there is no technique that allows for quantitative, real time exploration of the oligomeric state of proteins inside the PS. We previously used fluorescence fluctuation spectroscopy (FFS) with brightness analysis to measure the oligomeric state of protein complexes in the cytoplasm, the nucleus, and the plasma membrane. To extend these measurements to the PS we performed z-scan FFS to account for the layered structure of the nuclear envelope within the cell. However, the conventional analysis tools that allowed us to quantify protein complexes in the cytoplasm and at the plasma membrane failed in the PS. To overcome this obstacle we explored an alternative analysis approach and tested it on fluorescent proteins that were co-translationally targeted to the PS. We compared the recovered diffusion and brightness of these
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proteins in the PS and in the cytoplasm. We extended our investigation to functional proteins, including SUN2, and demonstrate that measurements of the oligomeric state of protein complexes in the PS are feasible. This work has been supported by a grant from the National Institutes of Health (R01 GM64589). 2432-Pos Board B576 A Comparison of Libs for the Quantification of Au Nanoparticles using 1064 nm, 532 nm, and 266 nm Excitation Komal Vig1, Aaleyah Joe2, Cleon M. Barnett1. 1 Center for Nanobiotechnology Research, Alabama State University, Montgomery, AL, USA, 22Mary Baldwin College, Staunton, VA, USA. Nanoparticles have emerged as a tool for wide range of medical and industrial applications. There are several methods that are used to characterize the size and distribution of the nanoparticles in cells and tissues. Laser-Induced Breakdown Spectroscopy (LIBS) is a versatile technique that has found its place in several analytical applications. Through short laser pulses, LIBS creates a plasma on the sample surface which yields critical elemental information. In this study we explore its use as method to estimate the concentration of gold nanoparticles in cells. We compare limit of detection (L.O.D.) obtained using plasmas produced by 15 mJ of laser energy at 1064 nm, 532 nm and 266 nm. Gold nanoparticles were incubated in human epithelial (HEp-2) cells at concentrations ranging from 0.30 to 1.25 mg/ml. Cells were trypsinzed and collected in HBSS after 24 hours incubation. 1.0 ml of cell incubated with nanoparticles were deposited on to pure silicon wafers and were analyzed using LIBS. The gold nanoparticle emission was monitored using the atomic emission line (Au I) at 267.59 nm. Under the current experimental conditions, the emission intensity emerging from the 266 nm laser produced plasmas was higher than the other two excitation wavelengths. In addition, using 266 nm as the excitation source led to improved L.O.D as compared to the other two wavelengths. Our results are promising towards using LIBS as a detection and quantification tools for nanoparticles in biological matrix. This work was supported by NSFREU (DBI-1358923) to Dr. Komal Vig (PI) 2433-Pos Board B577 Direct Label-Free Measurement of the Distribution of Small Molecular Weight Compound Inside Thick Biological Tissue using Coherent Raman Microspectroscopy Masahiko Kawagishi1, Yuki Obara2, Takayuki Suzuki2, Masumi Hayashi3, Kazuhiko Misawa2, Sumio Terada1. 1 Neuroanat/Cell Neurobiol, Tokyo Med Dent Univ (TMDU) Grad Sch Med Dent Sci, Bunkyo-ku, Tokyo, Japan, 2Appl Phys, Tokyo Univ Agr Tech (TUAT), Koganei, Japan, 3Wired Co Ltd, Komae, Japan. Distributions of small molecular weight (less than 300 Da) compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterise the localisation of large biomolecules, they cannot be easily applied to the cases with small molecule compounds. We used CARS spectroscopy to detect and identify a label-free small molecule compound. To facilitate detection in aqueous environment, we utilised time-resolved and phase-sensitive techniques to reduce non-resonant background generated from water. We applied this technique to detect small molecular weight compound, taurine, inside mouse cornea tissue immersed in taurine solution as an initial model experiment. We detected a Raman peak of taurine near wavenumber 1033 /cm inside cornea and successfully characterised its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualise the distribution of small bio-related compounds in biological background without using any labeling, paving the way for new cell biological analysis in various disciplines. Sci. Rep. 5, 13868; http://dx.doi.org/10.1038/srep13868 (2015). 2434-Pos Board B578 Comparison between Autofluorescence and Reflectance-Based Hyperspectral Imaging for Visualization of Atrial Ablation Lesions Huda Asfour1, Mohammed Aljishi1, Tigran Chahbazian2, Luther Swift1, Narine Muselimyan1, Daniel Gil3, Narine Sarvazyan1. 1 Pharmacology and Physiology, George Washington University, Washington, DC, USA, 2Strasbourg Medical University, Strasbourg, France, 3 Vanderbilt University, Nashville, TN, USA. Rationale: Atrial fibrillation (AF) is the most common cardiac arrhythmia. A primary cause of AF is abnormal electrical activity stemming from ectopic foci located near muscle sleeves of the pulmonary veins. One of the most common and effective ways to treat AF is radiofrequency ablation (RFA) to electrically isolate the pulmonary veins from the left atrium or to directly eliminate ectopic foci. Because endocardial surface of left atrium is covered by thick interwoven layers of collagen and elastin, sites of RF ablation are hard to see.
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proteins in the PS and in the cytoplasm. We extended our investigation to functional proteins, including SUN2, and demonstrate that measurements of the oligomeric state of protein complexes in the PS are feasible. This work has been supported by a grant from the National Institutes of Health (R01 GM64589). 2432-Pos Board B576 A Comparison of Libs for the Quantification of Au Nanoparticles using 1064 nm, 532 nm, and 266 nm Excitation Komal Vig1, Aaleyah Joe2, Cleon M. Barnett1. 1 Center for Nanobiotechnology Research, Alabama State University, Montgomery, AL, USA, 22Mary Baldwin College, Staunton, VA, USA. Nanoparticles have emerged as a tool for wide range of medical and industrial applications. There are several methods that are used to characterize the size and distribution of the nanoparticles in cells and tissues. Laser-Induced Breakdown Spectroscopy (LIBS) is a versatile technique that has found its place in several analytical applications. Through short laser pulses, LIBS creates a plasma on the sample surface which yields critical elemental information. In this study we explore its use as method to estimate the concentration of gold nanoparticles in cells. We compare limit of detection (L.O.D.) obtained using plasmas produced by 15 mJ of laser energy at 1064 nm, 532 nm and 266 nm. Gold nanoparticles were incubated in human epithelial (HEp-2) cells at concentrations ranging from 0.30 to 1.25 mg/ml. Cells were trypsinzed and collected in HBSS after 24 hours incubation. 1.0 ml of cell incubated with nanoparticles were deposited on to pure silicon wafers and were analyzed using LIBS. The gold nanoparticle emission was monitored using the atomic emission line (Au I) at 267.59 nm. Under the current experimental conditions, the emission intensity emerging from the 266 nm laser produced plasmas was higher than the other two excitation wavelengths. In addition, using 266 nm as the excitation source led to improved L.O.D as compared to the other two wavelengths. Our results are promising towards using LIBS as a detection and quantification tools for nanoparticles in biological matrix. This work was supported by NSFREU (DBI-1358923) to Dr. Komal Vig (PI) 2433-Pos Board B577 Direct Label-Free Measurement of the Distribution of Small Molecular Weight Compound Inside Thick Biological Tissue using Coherent Raman Microspectroscopy Masahiko Kawagishi1, Yuki Obara2, Takayuki Suzuki2, Masumi Hayashi3, Kazuhiko Misawa2, Sumio Terada1. 1 Neuroanat/Cell Neurobiol, Tokyo Med Dent Univ (TMDU) Grad Sch Med Dent Sci, Bunkyo-ku, Tokyo, Japan, 2Appl Phys, Tokyo Univ Agr Tech (TUAT), Koganei, Japan, 3Wired Co Ltd, Komae, Japan. Distributions of small molecular weight (less than 300 Da) compounds inside biological tissue have been obscure because of the lack of appropriate methods to measure them. Although fluorescence techniques are widely used to characterise the localisation of large biomolecules, they cannot be easily applied to the cases with small molecule compounds. We used CARS spectroscopy to detect and identify a label-free small molecule compound. To facilitate detection in aqueous environment, we utilised time-resolved and phase-sensitive techniques to reduce non-resonant background generated from water. We applied this technique to detect small molecular weight compound, taurine, inside mouse cornea tissue immersed in taurine solution as an initial model experiment. We detected a Raman peak of taurine near wavenumber 1033 /cm inside cornea and successfully characterised its depth profile in the tissue. Our CARS spectra measurement can be a promising method to measure and visualise the distribution of small bio-related compounds in biological background without using any labeling, paving the way for new cell biological analysis in various disciplines. Sci. Rep. 5, 13868; http://dx.doi.org/10.1038/srep13868 (2015). 2434-Pos Board B578 Comparison between Autofluorescence and Reflectance-Based Hyperspectral Imaging for Visualization of Atrial Ablation Lesions Huda Asfour1, Mohammed Aljishi1, Tigran Chahbazian2, Luther Swift1, Narine Muselimyan1, Daniel Gil3, Narine Sarvazyan1. 1 Pharmacology and Physiology, George Washington University, Washington, DC, USA, 2Strasbourg Medical University, Strasbourg, France, 3 Vanderbilt University, Nashville, TN, USA. Rationale: Atrial fibrillation (AF) is the most common cardiac arrhythmia. A primary cause of AF is abnormal electrical activity stemming from ectopic foci located near muscle sleeves of the pulmonary veins. One of the most common and effective ways to treat AF is radiofrequency ablation (RFA) to electrically isolate the pulmonary veins from the left atrium or to directly eliminate ectopic foci. Because endocardial surface of left atrium is covered by thick interwoven layers of collagen and elastin, sites of RF ablation are hard to see.