Fluorescence-correlation-spectroscopy is a highly sensitive optical measurement method. Fluctuations in the fluorescence emission intensity over time are recorded, which are caused by individual fluorophores that pass through the detection volume. With FCS, among other things, diffusion constants, concentrations and bonds between different diffusing species can be determined.
For FCS, a confocal microscope is usually used, whereby the excitation light is focused into the sample with the aid of an objective. In this way, the excitation volume is limited. If fluorescent particles diffuse into the excitation volume, they are excited to fluorescence. These can be fluorescence-labeled proteins, for example. In FCS, these absorb the photons of the excitation light and in turn emit photons of longer wavelength, i.e. lower energy. A beam splitter is used to separate the excitation light from the emitted photons. The latter can pass through the beam splitter and are then detected by a photodetector. The beam splitter, on the other hand, does not allow the excitation light to pass through.
A prerequisite here is that the readout rate of the detector is several orders of magnitude higher than the typical residence time of a particle at the focus. For this reason, today, avalanche photodiodes are mainly used for FCS as these can detect single photons.
The combination of FCS with TCSPC is called FLCS (fluorescence lifetime correlation spectroscopy). In this method, time-resolved detection of fluorescence is used to separate the contribution of different processes to the measured signal.
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