Biological and chemical applications of fluorescence correlation spectroscopy: a review.

The mathematical concept of fluorescence correlation spectroscopy (FCS)1 (1) emerged from quasi-elastic light scattering (QELS) spectroscopy (2) in the early 1970s. Compared to light scattering, the enhanced sensitivity of fluorescence to changes in molecular structure, chemistry, and local environment makes FCS a superior analytical tool for chemical kinetics studies (1, 3-5). The primary motivation for the invention of FCS was the study of chemical kinetics at very dilute concentrations in biological systems, such as the reversible binding reaction between ethidium bromide, a fluorescent nucleic acid synthesis inhibitor, and DNA (1). Theoretical and experimental studies (3, 4, 6) soon established that FCS could measure not only diffusion coefficients but also chemical rate constants, concentration, aggregation, and rotational dynamics (3-9). Building on this foundation, significant advances in the understanding of lipid diffusion in membranes were made soon after the birth of FCS (10) using a confocal microscope geometry (11) introduced into FCS by Koppel et al. (7) and still used today. Recently, technological advances in detectors, autocorrelation electronics, and confocal microscopy were incorporated into FCS, mainly in the laboratories of R. Rigler and M. Eigen (12, 13). A detailed theoretical framework on the effects of translational and rotational motion of a fluorescent molecule undergoing chemical reactions, in a three-dimensional (3D) Gaussian observation volume, has been introduced (14). Statistical analysis provided the basis for optimizing the signal-to-noise ratio (S/N) in FCS (15). Furthermore, analytic functions describing molecular translation (16), shot noise effects on higher-order fluorescence fluctuation moments (17), and the effect of the observation volume (18) on the S/N were explored. Finally, the ability of FCS to resolve multiple species with equivalent diffusion properties was extended by probability analysis of fluorescence intensity distributions (19-22). These advances extend the horizon of FCS in biological and chemical studies (for a recent review, see ref 23).