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Fluorescence Correlation Spectroscopy: Principles and Applications


Schwille,  Petra
Schwille, Petra / Cellular and Molecular Biophysics, Max Planck Institute of Biochemistry, Max Planck Society;

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Bacia, K., Haustein, E., & Schwille, P. (2014). Fluorescence Correlation Spectroscopy: Principles and Applications. Cold Spring Harbor Protocols, 709-725. doi:10.1101/pdb.top081802.

Fluorescence correlation spectroscopy (FCS) is used to study the movements and the interactions of biomolecules at extremely dilute concentrations, yielding results with good spatial and temporal resolutions. Using a number of technical developments, FCS has become a versatile technique that can be used to study a variety of sample types and can be advantageously combined with other methods. Unlike other fl uorescence-based techniques, the analysis of FCS data is not based on the average intensity of the fl uorescence emission but examines the minute intensity fl uctuations caused by sponta- neous deviations from the mean at thermal equilibrium. These fl uctuations can result from variations in local concentrations owing to molecular mobility or from characteristic intermolecular or intramo- lecular reactions of fl uorescently labeled biomolecules present at low concentrations. Here, we provide a basic introduction to FCS, including its technical development and theoretical basis, experimental setup of an FCS system, adjustment of a setup, data acquisition, and analysis of FCS measurements. Finally, the application of FCS to the study of lipid bilayer membranes and to living cells is discussed.