FCS - Fluorescence Correlation Spectroscopy - FMP

FCS - Fluorescence Correlation Spectroscopy
Short description of the method
FCS is a correlation analysis of fluctuating of the fluorescence intensities. This
method is based on the detection of single molecules excited to fluorescence in
diffraction limited confocal volume elements and the time correlation of stochastic
events. A sensitive detector records single photons emitted by the dye molecules.
The result is an intensity trace representing random noise. Correlation functions are
used to extract information about diffusion times and number of molecules in the
sample. Physical models describe the source of the fluctuations and are fitted to the
correlated data to quantify these informations.
If only one type of fluorescent dye and one detector are used, the method is called
auto-correlation. To increase the flexibility of the method, two dyes and detectors can
be used. This method is called cross-correlation.

Other common methods to use fluorescence fluctuations to probe molecular
interactions include Photon Counting Histograms (PCH).
Possible applications
The measurement of translational diffusion is probably the simplest measurement
which can be performed with FCS. The most frequent application of FCS consists in
assaying molecular interactions (binding/debinding). The method is best suited to
study the binding of a small compound (e.g. a ligand) to a bigger molecular object
(e.g. the ligand’s receptor). By the use of cross-correlation protein-protein interactions
(e.g. receptor dimerization) can be examined.
When an appropriate model is known, FCS can be applied to obtain quantitative
information such as:
• diffusion coefficients,
• hydrodynamic radii,
• average concentrations,
• kinetic chemical reaction rates,
• singlet-triplet dynamics.
Examples of applications in our group
Schmid, A. et al., Traffic 10 2-15 (2009)
Schmidt V. et al., EMBO J. 31 187-200 (2011)
Ridelis I. et al., FEBS Lett. 586 784-791 (2012)
Teichmann A. et al., J Biol Chem. 287 27265-27274 (2012)