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Handbook of Single-Molecule Biophysics pp 217–241Cite as

Fluorescence Correlation Spectroscopy in Living Cells

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Abstract

In fluorescence correlation spectroscopy (FCS), information about molecular dynamics is extracted by recording the fluctuating signal of a pico- to micromolar concentration of fluorescent molecules in solution. As primary parameters, FCS provides time constants of the fluorescence emission, as well as numbers and dwell times of the observed particles diffusing through the open volume. A biochemical reaction or macromolecular rearrangement causing changes in these parameters, when linked to fluorescence readout, can be quantified by FCS. Since the measurements are now routinely performed in a laser-illuminated confocal spot, making measurements in living cells is straightforward. Different cellular compartments, such as the nucleus, the cytoplasm, or the plasma membrane, can be easily distinguished and addressed. In addition to biochemical reactions, the local environment of macromolecules, for example, ion concentrations, pH, or viscosity, can be probed. Thus, FCS is a versatile and attractive technique for researchers striving for a quantitative understanding of interactions and dynamics of biological and in particular cellular systems.

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Notes

  1. 1.

    The acronym ACF can denote both the measured autocorrelated data and the model function that is used for fitting. We try to specify these according to the context.

  2. 2.

    The issue of calibrating diffusion times has been recently overcome by scanning FCS (S-FCS).

  3. 3.

    Relations that are important for the application-oriented user are indicated by an asterisk.

  4. 4.

    Vectors are written in boldface type.

  5. 5.

    M. Hintersteiner personal communication.

  6. 6.

    This avoids accumulation of large amounts of data and allows on-line inspection of the measured curves. However, with the constant advances in computer performance, software-based correlators also have come into use.

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Acknowledgments

TW is indebted to Konstantin Klenin and Malte Wachsmuth for their contributions in deriving FCS theory. TW and PS thank Eugene Petrov for helpful discussions.

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  1. BIOTEC, Biophysics, Technische Universität Dresden, Tatzberg 47-51, 01307 Dresden, Germany

    Thomas Weidemann & Petra Schwille

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  1. Institut für Biophysik, Universität Linz, Altenberger Straße 69, Linz, 4040, Austria

    Peter Hinterdorfer

  2. Harvard Medical School, Harvard University, Longwood Ave. 250, Boston, 02115, U.S.A.

    Antoine Oijen

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Weidemann, T., Schwille, P. (2009). Fluorescence Correlation Spectroscopy in Living Cells. In: Hinterdorfer, P., Oijen, A. (eds) Handbook of Single-Molecule Biophysics. Springer, New York, NY. https://doi.org/10.1007/978-0-387-76497-9_8

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