Analysis of Ligand Binding by Two-Colour Fluorescence Cross-Correlation Spectroscopy

✍ Scribed by Thomas Weidemann; Malte Wachsmuth; Michael Tewes; Karsten Rippe; Jörg Langowski

• Publisher: John Wiley and Sons
• Year: 2002
• Tongue: English
• Weight: 251 KB
• Volume: 3
• Category: Article
• ISSN: 1438-5163
• DOI: 10.1002/1438-5171(200204)3:1<49::aid-simo49>3.0.co;2-t

✦ Synopsis

Fluorescence correlation spectroscopy (FCS) is a well-established method for the analysis of freely diffusing fluorescent particles in solution. In a two-colour setup, simultaneous detection of two different dyes allows the acquisition of both the autocorrelation of the signal of each channel and the cross-correlation of the two channels (fluorescence cross-correlation spectroscopy, FCCS). The cross-correlation function is related to the amount of diffusing particles carrying both dyes and can be used for monitoring a binding reaction. Here we develop a formalism for a quantitative analysis of ligand binding from a combination of the auto-and the cross-correlation amplitudes. Technical constraints, like the focal geometry, background signal and cross-talk between the detection channels as well as photophysical and biochemical effects which modulate the brightness of the particles are included in the analysis. Based on this framework a comprehensive treatment for the determination of two-component binding equilibria by FCS/FCCS is presented.