Fluorescence Correlation Spectroscopy (FCS) and Fluorescence Cross Correlation Spectroscopy (FCCS)

FCS and FCCS (generally called fluorescence fluctuation spectroscopy) are confocal microscope-based methods that allow assessing transport and mobility properties as well as interactions of molecules (protein-protein, protein-nucleic acid, protein-compound, protein complex formation) in vitro and in living cells. These methods are based on fluorescence-labeled molecules that, in the case of in vivo studies, are either injected or genetically encoded. FCS/FCCS experiments require equipment that is commercially available. Measurements are based on the observation of fluorescence fluctuations caused by diffusion of labeled molecules in and out of a single confocal volume that is positioned at a selected location within the cell. Mathematical analysis of the data yields concentrations of the investigated molecules, dissociation constants of their interactions and their diffusion coefficients. The development of superior light detectors (avalanche photodiodes/APDs), high resolution optics and ultra-bright fluorescent proteins with different and well separated emission spectra enable FCS/FCCS applications in living cells to become within reach for cell biological studies. FCS/FCCS renders possible, in contrast to established methods such as co-immunoprecipitation, 2-hybrid etc., to investigate interactions and motilities in different locations of a cell (nucleus, cytoplasm, membranes). FCS/FCCS data is of quantitative nature (concentrations, diffusion coefficients and dissociation constants) and independent on the spatial arrangement of the chromophores (in contrast to FRET based methods). The concentration range to which FCS/FCCS is adapted matches very well the range of concentration of endogenously expressed proteins. In particular, as a single molecule method, FCS/FCCS is extremely well suited to study very low-expressed proteins (down to approx. 10-20 nM) that are difficult to detect with conventional methods. Hence, FCS/FCCS is likely to become a key technology in the future enabling measurements of parameters required for systems biology.

FCS/FCCS is particularly well suited to study protein-protein interactions in yeast (Saccharomyces cerevisiae, Schizosaccharomyces pombe), since yeasts enable the rapid construction of strains that express endogenous levels of fluorescently tagged proteins by using PCR targeting. Furthermore, yeast enables rapid testing whether the resulting fusion protein retains its biological activity.

FCS/FCCS experiments, in order to be conclusive, require a careful setting up of the method. This involves extensive knowledge about all the different aspects of the experiments: photo-physical properties of the used fluorescent proteins, analysis of FCS/FCCS data using specific software, correction factors needed to correct for systematic errors (e.g. maturation time of fluorescent proteins, volume overlap, etc.). Some hints about the requirements are described in Supplementary Methods to our paper on FCS/FCCS (Maeder et al., 2007) and in a recent publication by the Schwille lab (Bacia and Schwille, 2007). For training of Cell Biologists to use FCS/FCCS, we just received funding for an EMBO course in spring 2009.

More background on FCS/FCCS can be also found on Wikipedia.