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.

FCS and FCCS measurements provide information about protein concentration, diffusion and protein-protein interactions. These measurements can be conducted in vitro or in vivo.

Such measurements can be done using commercial equipment based on confocal microscopes. These permit single point measurements inside individual cells. Principal set up of a confocal FCS/FCCS system. Fluorescence fluctuations of mobile and fluorescently labelled proteins are acquired from a chosen location within a cell using a parked beam. Typical FCS/FCCS measurements take a few seconds to 1-2 minutes.

Such measurements can be done using commercial equipment based on confocal microscopes. These permit single point measurements inside individual cells. Principal set up of a confocal FCS/FCCS system. Fluorescence fluctuations of mobile and fluorescently labelled proteins are acquired from a chosen location within a cell using a parked beam. Typical FCS/FCCS measurements take a few seconds to 1-2 minutes.

Custom made instruments are now able to measure in full 2D, thus providing spatial resolution to FCS (see our paper on this).

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 have organized an EMBO course in spring 2009. More courses may be organized in the future.

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

                                                        
Our wiede field FCCS system. IT is based on a Nikon Ti-Eclipse and a MicroTime 200 with pusled 440 and 488 nm laser for life time filtering FCCS along with a green laser. The system is fully automated and enable automatized FCCS, controlled using the open source software Micro-Manager.
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