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: | , , , |
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| Dokumenttyp: | Article (Journal) |
| Sprache: | Englisch |
| Veröffentlicht: |
4 November 2010
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| 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 
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| Verfasserangaben: | Jonas Ries, Zdeněk Petrášek, Anna J García-Sáez and Petra Schwille |
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| 520 | |a 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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