FCS measures interactions between biomolecules, fluctuations within biomolecules, as well as their concentration, size and mobility. Measurements can be performed in solution or in living cells.
FCS analyses interactions between biomolecules in solution or in living cells at pM to uM concentrations. The technique detects biomolecules randomly diffusing through a femtoliter excitation focus and can in addition to molecular interactions detect absolute concentrations, diffusion coefficients, rotational motion, and conformational fluctuations. In living cells measurements can be performed for example in the plasma membrane, in the cytoplasm or in the nucleus. Samples are usually tagged either with fluorescent proteins or fluorescent antibodies.
Most commonly used FCS-variants:
• Single color FCS – Detects concentrations and diffusion coefficients, as well as molecular
interactions when binding partners differ in size.
• Dual color FCCS – Detects molecular interactions very effectively and independently on the
size of the binding partners.
• FRET - FCS – Detects rare oligomers that constitute only a few percent or less
compared to the monomers.
• Inverse - FCS – Detects the absolute size of mobile clusters and oligomers in membranes,
potentially of objects smaller than 10 nm.
Figure 1. Autocorrelation FCS-curves (blue and red) and cross-correlation curves (white and green) recorded in the plasma membrane of living cells. The clear amplitude of the cross-correlation curves is the signature of binding, and thus correlated movement, between the fluorescent proteins EGFP and mCherry.
Figure 2. Autocorrelation FCS-curves (blue and red) and cross-correlation curves (white and green) recorded in the plasma membrane of living cells. Here the EGFP- and mCherry-tagged proteins do not interact, resulting in a low amplitude of the cross-correlation curves (white and green) relative to that of the autocorrelation curves (blue and red).