Fluorescence Cross Correlation Spectroscopy (FCCS) / Fluorescence Lifetime Cross Correlation Spectroscopy (FLCCS)

This employs the same principle as FCS except that the fluorescence signal from two different fluorescence species is measured in two separate channels simultaneously. A correlation analysis is performed between the intensity time trace from the two channels (as opposed to an auto-correlation analysis in the case of FCS). This will yield information on any correlated motion of the two-fluorescence species, such that if they are bound one observes a strong (cross-) correlation.

Unlike FLIM-FRET which may also be used to measure binding dynamics, there is no constraint on the relative orientations or distances between the two molecules (i.e. they may be separated by >10nm), not on there being any spectral overlap in the emission and absorption spectra of the two fluorescent species being studied.

Examples of use:

• Binding (dynamics) or colocalization of two fluorescently-labelled molecules separated (especially by more >10nm).

Useful references:

• Fluorescence Lifetime Imaging (FLIM) in Confocal Microscopy Applications: An Overview (Application Note by Picoquant GmbH)
• Fluorescence lifetime imaging microscopy: Spatial resolution of biochemical processes in the cell Bastiaens PIH, Squire A (1999)Trends in Cell Biology, 9:48-52.