Datensatz

Zusammenfassung

Fluorescence correlation spectroscopy (FCS) ana-lyzes spontaneous fluctuations in the fluorescenceemission of small molecular ensembles, thus provid-ing information about a multitude of parameters,such as concentrations, molecular mobility and dy-namics of fluorescently labeled molecules. Per-formed within diffraction-limited confocal volume el-ements, FCS provides an attractive alternative tophotobleaching recovery methods for determining in-tracellular mobility parameters of very low quantitiesof fluorophores. Due to its high sensitivity sufficientfor single molecule detection, the method is subjectto certain artifact hazards that must be carefully con-trolled, such as photobleaching and intramoleculardynamics, which introduce fluorescence flickering.Furthermore, if molecular mobility is to be probed,nonspecific interactions of the labeling dye with cel-lular structures can introduce systematic errors. Incytosolic measurements, lipophilic dyes, such as cer-tain rhodamines that bind to intracellular mem-branes, should be avoided. To study free diffusion,genetically encoded fluorescent labels such as greenfluorescent protein (GFP) or DsRed are preferablesince they are less likely to nonspecifically interactwith cellular substructures.