Fluorescence cross-correlation spectroscopy

Fluorescence cross-correlation spectroscopy (FCCS) is a spectroscopic technique that examines the interactions of fluorescent particles of different colours as they randomly diffuse through a microscopic detection volume over time, under steady conditions. Eigen and Rigler first introduced the fluorescence cross-correlation spectroscopy (FCCS) method in 1994. Later, in 1997, Schwille experimentally implemented this method. FCCS is an extension of the fluorescence correlation spectroscopy (FCS) method that uses two fluorescent molecules instead of one that emit different colours. The technique measures coincident green and red intensity fluctuations of distinct molecules that correlate if green and red labelled particles move together through a predefined confocal volume. FCCS utilizes two species that are independently labeled with two different fluorescent probes of different colours. These fluorescent probes are excited and detected by two different laser light sources and detectors typically labeled as "green" and "red." By combining FCCS with a confocal microscope, the technique's capabilities are highlighted, as it becomes possible to detect fluorescence molecules in femtoliter volumes within the nanomolar range, with a high signal-to-noise ratio, and at a microsecond time scale. The normalized cross-correlation function is defined for two fluorescent species, G and R, which are independent green and red channels, respectively: where differential fluorescent signals at a specific time, and at a delay time, later is correlated with each other. In the absence of spectral bleed-through -when the fluorescence signal from an adjacent channel is visible in the channel being observed-, the cross-correlation function is zero for non-interacting particles. In contrast to FCS, the cross-correlation function increases with increasing numbers of interacting particles. FCCS is mainly used to study bio-molecular interactions both in living cells and in vitro. It allows for measuring simple molecular stoichiometries and binding constants.