Fluorescence correlation spectroscopy and its impact on single molecule biophysics

Single protein and nucleic acid molecules are believed to be the smallest functional units in biological systems, their dynamic analysis in vitro, but also in living cells and organisms remaining one of the major goals of current biophysical research. Among the most sensitive techniques presently available and frequently employed in biophysical single molecule analysis is fluorescence spectroscopy, due to its non-invasiveness being applicable to a wide variety of biological specimens. Fluorescence correlation spectroscopy (FCS) is one of the many different modes of high-resolution spatial and temporal analysis of single or sparse biomolecules. Its basic principle is the consideration of minute spontaneous deviations of small systems from thermodynamic equilibrium that give rise to fluctuations in the fluorescence emission. Among a multitude of physical parameters which are in principle accessible by FCS, it most conveniently allows to determine local concentrations, mobility coefficients, and characteristic rate constants of inter- and intramolecular reactions of fluorescently labeled biomolecules at very low (nanomolar) concentrations, under equilibrium conditions and without physical separation.