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Fluorescence Correlation Spectroscopy with Photobleaching Correction in Slowly Diffusing Systems

Fluorescence Correlation Spectroscopy with Photobleaching Correction in Slowly Diffusing Systems Fluorescence correlation spectroscopy (FCS) is a powerful tool to quantitatively study the diffusion of fluorescently labeled molecules. It allows in principle important questions of macromolecular transport and supramolecular aggregation in living cells to be addressed. However, the crowded environment inside the cells slows diffusion and limits the reservoir of labeled molecules, causing artifacts that arise especially from photobleaching and limit the utility of FCS in these applications. We present a method to compute the time correlation function from weighted photon arrival times, which compensates computationally during the data analysis for the effect of photobleaching. We demonstrate the performance of this method using numerical simulations and experimental data from model solutions. Using this technique, we obtain correlation functions in which the effect of photobleaching has been removed and in turn recover quantitatively accurate mean-square displacements of the fluorophores, especially when deviations from an ideal Gaussian excitation volume are accounted for by using a reference calibration correlation function. This allows quantitative FCS studies of transport processes in challenging environments with substantial photobleaching like in living cells in the future. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Fluorescence Springer Journals

Fluorescence Correlation Spectroscopy with Photobleaching Correction in Slowly Diffusing Systems

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Publisher
Springer Journals
Copyright
Copyright © 2018 by Springer Science+Business Media, LLC, part of Springer Nature
Subject
Biomedicine; Biomedicine, general; Biological and Medical Physics, Biophysics; Biotechnology; Biochemistry, general; Analytical Chemistry
ISSN
1053-0509
eISSN
1573-4994
DOI
10.1007/s10895-018-2210-y
pmid
29368157
Publisher site
See Article on Publisher Site

Abstract

Fluorescence correlation spectroscopy (FCS) is a powerful tool to quantitatively study the diffusion of fluorescently labeled molecules. It allows in principle important questions of macromolecular transport and supramolecular aggregation in living cells to be addressed. However, the crowded environment inside the cells slows diffusion and limits the reservoir of labeled molecules, causing artifacts that arise especially from photobleaching and limit the utility of FCS in these applications. We present a method to compute the time correlation function from weighted photon arrival times, which compensates computationally during the data analysis for the effect of photobleaching. We demonstrate the performance of this method using numerical simulations and experimental data from model solutions. Using this technique, we obtain correlation functions in which the effect of photobleaching has been removed and in turn recover quantitatively accurate mean-square displacements of the fluorophores, especially when deviations from an ideal Gaussian excitation volume are accounted for by using a reference calibration correlation function. This allows quantitative FCS studies of transport processes in challenging environments with substantial photobleaching like in living cells in the future.

Journal

Journal of Fluorescence – Springer Journals

Published: Jan 24, 2018

References

  • Thermodynamic fluctuations in a reacting system – measurement by fluorescence correlation spectroscopy
    Magde, D; Elson, E; Webb, WW
  • Fluorescence correlation spectroscopy: the technique and its applications
    Krichevsky, O; Bonnet, G
  • Fluorescence correlation spectroscopy in membrane structure elucidation
    Chiantia, S; Ries, J; Schwille, P
  • Correct diffusion coefficients of proteins in fluorescence correlation spectroscopy. Application to Tubulin Oligomers induced by Mg2+ and Paclitaxil
    Krouglova, T; Vercammen, J; Engelborghs, Y
  • Measuring and imaging diffusion with multiple scan speed image correlation spectroscopy
    Gröner, N; Capoulade, J; Cremer, C; Wachsmuth, M
  • A study of the dynamics of PTEN proteins in living cells using in vivo fluorescence correlation spectroscopy
    Du, ZX; Dong, CQ; Ren, JC
  • Direct measurement of association and disassociation rates of DNA binding in live cells by fluorescence correlation spectroscopy
    Michelman-Ribeiro, A; Mazza, D; Rosales, T; Stasevich, TJ; Boukari, H; Rishi, V; Vinson, C; Knutson, JR; McNally, JG
  • Direct evidence of amyloid precursor-like protein 1 trans interactions in cell-cell adhesion platforms investigated via fluorescence fluctuation spectroscopy
    Dunsig, V; Mayer, M; Liebsch, F; Multhaup, G; Chiantia, S
  • FCS cell surface measurements – photophysical limitations and consequences on molecular ensembles with heterogenic mobilities
    Widengren, J; Thyberg, P
  • Fast calculation of fluorescence correlation data with asynchronous time-correlated single photon counting
    Wahl, M; Gregor, I; Patting, M; Enderlein, J
  • Nonthermal ATP-dependent fluctuations contribute to the in vivo motion of chromosomal loci
    Weber, SC; Spakowitz, AJ; Theriot, JA
  • Lateral diffusion of membrane proteins
    Ramadurai, S; Holt, A; Krasnikov, V; Bogaart, G; Killian, JA; Poolman, B
  • Application of fluorescence correlation spectroscopy to the measurement of local temperature in solutions under optical trapping conditions
    Ito, S; Sugiyama, T; Toitani, N; Katayama, G; Miyasaka, H
  • Mean squared displacement from fluorescence correlation spectroscopy
    Kubečka, J; Uhlik, F; Košovan, P