Open AccessAnalysis of Ligand Binding by Two‐Colour Fluorescence Cross‐Correlation Spectroscopy
Author(s) -
Weidemann Thomas,
Wachsmuth Malte,
Tewes Michael,
Rippe Karsten,
Langowski Jörg
Publication year - 2002
Publication title -
single molecules
Language(s) - English
Resource type - Journals
eISSN - 1438-5171
pISSN - 1438-5163
DOI - 10.1002/1438-5171(200204)3:1<49::aid-simo49>3.0.co;2-t
Subject(s) - fluorescence correlation spectroscopy , fluorescence , autocorrelation , chemistry , cross correlation , fluorescence cross correlation spectroscopy , spectroscopy , fluorescence spectroscopy , biological system , correlation , analytical chemistry (journal) , biophysics , physics , optics , mathematics , chromatography , geometry , biology , mathematical analysis , statistics , quantum mechanics
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.