Fluorescence correlation spectroscopy allows investigating molecular dynamics in highly diluted sample solutions. The researcher yields information about chemical reaction rates, flow rates, diffusion coefficients, molecular weights and molecular aggregates. In confocal FCS a collimated laser beam is concentrated through a high numerical aperture objective lens irradiating a cone shaped area of the sample solution. A volume of approximately one femtoliter [1µm³] is precisely in focus, comprising the region of measurement. Fluorescent signals from this area enter the objective, pass the confocal optics and are detected, while out of focus blur is blocked.
Intensity fluctuations arising from the number or quantum yield of the few fluorescent molecules occupying the measurement volume as a function of time are recorded. While small fluorophores diffuse rapidly and at random paths through the illuminated volume generating short, randomised bursts of intensity, larger molecular complexes (fluorescently labelled macromolecules) move more slowly and produce a longer, more sustained time-dependent fluorescence intensity pattern. The mean diffusion time which is affected by the molecular weight can be calculated on the basis of a temporal correlation of the signals of many single molecules.