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Total internal reflection with fluorescence correlation spectroscopy

Nature Protocols volume 2, pages 878–890 (2007)Cite this article

Abstract

Total internal reflection–fluorescence correlation spectroscopy (TIR-FCS) is an emerging technique that is used to measure events at or near an interface, including local fluorophore concentrations, local translational mobilities and the kinetic rate constants that describe the association and dissociation of fluorophores at the interface. TIR-FCS is also an extremely promising method for studying dynamics at or near the basal membranes of living cells. This protocol gives a general overview of the steps necessary to construct and test a TIR-FCS system using either through-prism or through-objective internal reflection geometry adapted for FCS. The expected forms of the autocorrelation function are discussed for the cases in which fluorescent molecules in solution diffuse through the depth of the evanescent field, but do not bind to the surface of interest, and in which reversible binding to the surface also occurs.

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Figure 1: Apparatus.
Figure 2: Apparatus.
Figure 3: Expected results for TIR-FCS.

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Acknowledgements

We thank Randall C. Cush, Emily M. Haas, Alena M. Lieto, Jamie K. Pero and Tammy E. Starr for their contributions to the development and implementation of TIR-FCS in our laboratory. This work was supported by NIH grant HL073937 and by NSF grant MCB-0641087.

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  1. Department of Chemistry, University of North Carolina at Chapel Hill, North Carolina, 27599, USA

    Nancy L Thompson & Bridgett L Steele

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Correspondence to Nancy L Thompson.

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Thompson, N., Steele, B. Total internal reflection with fluorescence correlation spectroscopy. Nat Protoc 2, 878–890 (2007). https://doi.org/10.1038/nprot.2007.110

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