A comprehensive framework for fluorescence cross-correlation spectroscopy

• Verfasst von: Ries, Jonas [VerfasserIn]
• Verfasst von: Petrášek, Zdeněk [VerfasserIn]
• Verfasst von: García-Sáez, Ana J. [VerfasserIn]
• Verfasst von: Schwille, Petra [VerfasserIn]
• Titel: A comprehensive framework for fluorescence cross-correlation spectroscopy
• Verf.angabe: Jonas Ries, Zdeněk Petrášek, Anna J García-Sáez and Petra Schwille
• E-Jahr: 2010
• Jahr: 4 November 2010
• Umfang: 32 S.
• Fussnoten: Gesehen am 24.05.2023
• Titel Quelle: Enthalten in: New journal of physics
• Ort Quelle: [Bad Honnef] : Dt. Physikalische Ges., 1999
• Jahr Quelle: 2010
• Band/Heft Quelle: 12(2010), 11 vom: Nov., Artikel-ID 113009, Seite 1-32
• ISSN Quelle: 1367-2630
• DOI: doi:10.1088/1367-2630/12/11/113009
• Datenträger: Online-Ressource
• Sprache: eng
• K10plus-PPN: 1846076048

Abstract

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.