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Scanning Fluorescence Correlation Spectroscopy on Biomembranes

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Methods in Membrane Lipids

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1232))

Abstract

Fluorescence correlation spectroscopy (FCS) is a powerful quantitative method to study dynamical properties of biophysical systems. It exploits the temporal autocorrelation of fluorescence intensity fluctuations originating from a tiny volume (~fL). A theoretical model function can be then fitted to the measured auto-correlation curve to obtain physical parameters such as local concentration and diffusion time. However, the application of FCS on membranes is coupled to several difficulties like accurate positioning and stability of the set-up.

In this book chapter, we explain the theoretical framework of point FCS and Scanning FCS (SFCS), which is a variation especially suitable for membrane studies. We present a list of materials necessary for SFCS studies on Giant Unilamellar Vesicles (GUVs). Finally, we provide simple protocols for the preparation of GUVs, calibration of the microscope setup, and acquisition and analysis of SFCS data to determine diffusion coefficients and concentrations of fluorescent particles embedded in lipid membranes.

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Author information

Authors and Affiliations

  1. Max Planck Institute for Intelligent Systems, Heisenbergstr 3, 70569, Stuttgart, Germany

    Eduard Hermann & Ana J. García-Sáez

  2. German Cancer Research Center, BioQuant, Im Neuenheimer Feld 267, 69120, Heidelberg, Germany

    Eduard Hermann & Ana J. García-Sáez

  3. European Molecular Biology Laboratory, Meyerhofstr. 1, 69117, Heidelberg, Germany

    Jonas Ries

Authors
  1. Eduard Hermann
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  2. Jonas Ries
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  3. Ana J. García-Sáez
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Corresponding author

Correspondence to Ana J. García-Sáez .

Editor information

Editors and Affiliations

  1. Department of Physics and Randall Division of Cell and Molecular Biophysics, King’s College London, London, United Kingdom

    Dylan M. Owen

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Hermann, E., Ries, J., García-Sáez, A.J. (2015). Scanning Fluorescence Correlation Spectroscopy on Biomembranes. In: Owen, D. (eds) Methods in Membrane Lipids. Methods in Molecular Biology, vol 1232. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-1752-5_15

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  • DOI: https://doi.org/10.1007/978-1-4939-1752-5_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-1751-8

  • Online ISBN: 978-1-4939-1752-5

  • eBook Packages: Springer Protocols

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