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).