Accessing molecular dynamics in cells by fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) analyzes spontaneous fluctuatio ns in the fluorescence emission of small molecular ensembles, thus providin g information about a multitude of parameters, such as concentrations, mole cular mobility and dynamics of fluorescently labeled molecules. Performed w ithin diffraction-limited confocal volume elements, FCS provides an attract ive alternative to photobleaching recovery methods for determining intracel lular mobility parameters of very low quantities of fluorophores. Due to it s high sensitivity sufficient for single molecule detection, the method is subject to certain artifact hazards that must be carefully controlled, such as photobleaching and intramolecular dynamics, which introduce fluorescenc e flickering. Furthermore, if molecular mobility is to be probed, nonspecif ic interactions of the labeling dye with cellular structures can introduce systematic errors. In cytosolic measurements, lipophilic dyes, such as cert ain rhodamines that bind to intracellular membranes, should be avoided. To study free diffusion, genetically encoded fluorescent labels such as green fluorescent protein (GFP) or DsRed are preferable since they are less likel y to nonspecifically interact with cellular substructures.