Fluorescence Correlation Spectroscopy of Molecular Motions and Kinetics
The foundations for fluorescence correlation spectroscopy (FCS) were already laid in the early 1970s, but this technique did not become widely used until single-molecule detection was established almost 20 years later with the use of diffraction-limited confocal volume element. The analysis of molecular noise from the GHz- to the Hz-region facilitates measurements over a large dynamic range covering photophysics, conformational transitions and interactions as well as transport properties of fluorescent biomolecules. From the Poissonian nature of the noise spectrum the absolute number of molecules is obtainable. Originally used for the analysis of molecular interactions in solutions, the strength of FCS lies also in its applicability to molecular processes at either the surface or interior of single cells. Examples for the analysis of surface kinetics including on and off rates of ligand– receptor interactions will be given. The possibility of obtaining this type of information by FCS will be of particular interest for cell-based drug screening.