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A comprehensive framework for fluorescence cross-correlation spectroscopy

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...

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Hauptverfasser: Ries, Jonas (VerfasserIn) , Petrášek, Zdeněk (VerfasserIn) , García-Sáez, Ana J. (VerfasserIn) , Schwille, Petra (VerfasserIn)
Dokumenttyp: Article (Journal)
Sprache:Englisch
Veröffentlicht: 4 November 2010
In: New journal of physics
Year: 2010, Jahrgang: 12, Heft: 11, Pages: 1-32
ISSN:1367-2630
DOI:10.1088/1367-2630/12/11/113009
Online-Zugang:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1088/1367-2630/12/11/113009
Verlag, lizenzpflichtig, Volltext: https://dx.doi.org/10.1088/1367-2630/12/11/113009
Volltext
Verfasserangaben:Jonas Ries, Zdeněk Petrášek, Anna J García-Sáez and Petra Schwille
Beschreibung
Zusammenfassung: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.
Beschreibung:Gesehen am 24.05.2023
Beschreibung:Online Resource
ISSN:1367-2630
DOI:10.1088/1367-2630/12/11/113009

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