23.08.2010
Fluorescence correlation spectroscopy (FCS) is a method to examine local fluctuations of molecules and their dynamic properties such as, e.g., diffusion. Fluctuations in the fluorescence intensity of a dye molecule are usually assessed to this end. Using time-correlated single photon counting, it is possible to measure not only the fluorescence intensity, but also the fluorescence lifetime. Fluorescence Lifetime Correlation Spectroscopy (FLCS) takes advantage of the specific time-dependent distribution of the detected photons on a picosecond to nanosecond time scale for statistic weighting. Scattered excitation light, for example, can be detected only during the excitation pulse, whereas fluorescence photons are detected by means of a time-dependent distribution which is typical for the fluorophore. It is thus possible to differentiate not only between scattered light and fluorescence, but also between different fluorophores if these differ with regard to their fluorescence lifetime, even if it is difficult to differentiate their spectra.
Within the scope of a cooperation project with PicoQuant GmbH in Berlin which was fostered by the BMWi (Federal Ministry of Economics and Technology), it was proved that FLCS provides correct concentrations of molecules in solution from the picomolecular to micromolar range, and FLCS enabled to reliably extract concentrations and diffusion coefficients, even in the case of binary mixtures of spectrally undistinguishable dyes having different fluorescence lifetimes.