Fluorescence correlation spectroscopy (FCS) is a fluctuation method established three decades ago, whose application to cellular systems became popular in the last decade. Fluctuations of fluorescence emission are observed from a small, femtoliter to sub-femtoliter, usually confocal volume at high time resolution. A time-dependent autocorrelation function is generated and evaluated to obtain time constants of photophysical and photochemical reactions, as well as of molecular diffusion and in the observation volume. Molecules in various subcellular compartments-including the nucleus, the cytoplasm, and the membrane-can be observed after labeling them with antibodies, ligands, or fluorescent proteins. The anomaly of diffusion, the local concentration, and the average fluorescence per diffusing particle can also be determined, all of which can be characteristic of molecular interactions. A two-color version of FCS, fluorescence cross-correlation spectroscopy, can also be applied to observe co-diffusion, i.e., stable association of two distinct molecular species in their cellular environment.
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