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Practical guidelines for dual-color fluorescence cross-correlation spectroscopy

Nature Protocols volume 2, pages 2842–2856 (2007)Cite this article

Abstract

Dual-color fluorescence cross-correlation spectroscopy (FCCS) allows for the determination of molecular mobility and concentrations and for the quantitative analysis of molecular interactions such as binding or cleavage at very low concentrations. This protocol discusses considerations for preparing a biological system for FCCS experiments and offers practical advice for performing FCCS on a commercially available setup. Although FCCS is closely related to two-color confocal microscopy, critical adjustments and test measurements are necessary to establish successful FCCS measurements, which are described in a step-by-step manner. Moreover, we discuss control experiments for a negative cross-correlation artifact, arising from a lack of detection volume overlap, and a positive artifact, arising from cross-talk. FCCS has been applied to follow molecular interactions in solutions, on membranes and in cells and to analyze dynamic colocalization during intracellular transport. It is a technique that is expected to see new applications in various fields of biochemical and cell biological research.

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Figure 1: FCCS setup.
Figure 2: The importance of axial detection volume overlap.
Figure 3: Applying FCCS to protein–protein interactions in solution.
Figure 4: Applying FCCS to intracellular cargo transport.

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Acknowledgements

We thank Sally A. Kim for critical reading of the manuscript, Elke Haustein and Jonas Ries for helpful comments and former and current members of the Schwille lab for materials and discussions. This work was supported by the Volkswagen foundation, the German Ministry of Education and Research, the Human Frontiers Science Program and the European Regional Development Fund.

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  1. Kirsten Bacia

    Present address: Present address: Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California 94720-3202, USA.,

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  1. Department of Biophysics, BIOTEC, Dresden University of Technology, Tatzberg 47-51, Dresden, D-01307, Germany

    Kirsten Bacia & Petra Schwille

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Correspondence to Petra Schwille.

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Bacia, K., Schwille, P. Practical guidelines for dual-color fluorescence cross-correlation spectroscopy. Nat Protoc 2, 2842–2856 (2007). https://doi.org/10.1038/nprot.2007.410

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