Fluorescence Cross-Correlation Spectroscopy with Pulsed Interleaved Excitation (PIE-FCCS)

Fluorescence Cross-Correlation Spectroscopy (FCCS) is a development based on Fluorescence Correlation Spectroscopy (FCS) for the examination of the interaction of fluorescent molecules.
Molecules of interest diffuse through a confocal volume, where they are excited by focused laser light of more than one color at the same spot. The order of magnitude of such a volume lies roughly around a micrometer. Parameters that can be measured with FCS are the diffusion constant of a fluorescent molecule species and its concentration in the given sample solution. Due to the different excitation light colors, with FCCS this can be done for more than one fluorescent species at the same time. At the same time, FCCS gains the additional information whether fluorescent molecules of different species collocalize, that is, if they diffuse together (e.g. when they are chemically bound), or independently from each other.
Pulsed interleaved excitation (PIE) further enhances the method by the use of pulsed laser excitation sources. These are synchronized with the detectors for the photons emitted by the fluorescent molecules. This way it is possible to determine for each photon the fluorescent molecule species that has emitted it. It is then possible to use this information to remove spectral crosstalk artifacts from the data, or to detect and quantify a possible Förster Resonance Energy Transfer (FRET) between two different molecules.