ABSTRACT

Fluorescence correlation spectroscopy (FCS) is a confocal fluorimetric method in which the fluorescence of a single dye-labeled molecule excited by a sharp focused laser beam is observed. With this technique, initially conceived in the early 1970s (Ehrenberg and Rigler, 1974; Elson and Madge, 1974; Koppel, 1974), the detection of single molecules in the millisecond range has been made possible due to improvements in the sensitivity of FCS (Eigen and Rigler, 1994; Rigler et al., 1993; Rigler, 1995; Rigler and Metz, 1992; Rigler and Widengren, 1990). A disadvantage of conventional fluorescence spectroscopy is its limited resolution and sensitivity in recording fluorescence signals, owing to the high background noise. A relatively large volume element must be illuminated and the emitted fluorescence must be carefully separated from the following: scattered laser light, Raman scattering from the solvent, spurious fluorescence signals from the solvent and the optics and other luminescence.