A comprehensive framework for fluorescence cross-correlation spectroscopy

Details A comprehensive framework for fluorescence cross-correlation spectroscopy A comprehensive framework for fluorescence cross-correlation spectroscopy

Nov 2010

adsabs.harvard.edu/cgi-bin/nph-data_query?bibcode=2010njph...12k3009r&link_type=abstract

New Journal of Physics, Volume 12, Issue 11, pp. 113009 (2010).

Physics

Scientific paper

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.

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