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Journal Article

A comprehensive framework for fluorescence cross-correlation spectroscopy


Schwille,  Petra
Max Planck Institute of Molecular Cell Biology and Genetics, Max Planck Society;

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Ries, J., Petrásek, Z., Garcia-Sáez, A. J., & Schwille, P. (2010). A comprehensive framework for fluorescence cross-correlation spectroscopy. New Journal of Physics, 12: 113009. doi:10.1088/1367-2630/12/11/113009.

Cite as: http://hdl.handle.net/21.11116/0000-0008-238E-1
Dual-colour fluorescence cross-correlation spectroscopy is a powerful method of studying binding between labelled biomolecules in vitro as well as in vivo. However, numerous artefacts and experimental complexities complicate quantitative measurements. Here, we show that a combination of dual-colour fluorescence correlation spectroscopy (FCS) with dual-focus FCS avoids artefacts due to chromatic aberrations or saturation and circumvents the calibration of the detection volumes. In addition, we present a comprehensive mathematical framework that allows us to accurately analyse correlation curves even in the presence of spectral cross-talk, incomplete or stochastic labelling, multiple binding sites, a fluorescent background and depletion due to photobleaching. We demonstrate the merits of this approach using dual-colour dual-focus scanning FCS, which allows binding measurements on membranes not affected by membrane movements.