Imaging of protein cluster sizes by means of confocal time-gated fluorescence anisotropy microscopy

A time-resolved fluorescence anisotropy imaging method for studying nanoscale clustering of proteins or lipids was developed and evaluated. It is based on FRET between the identical fluorophores (homo-FRET), which results in a rapid depolarization of the fluorescence. The method employs the time-resolved fluorescence anisotropy decays recorded in a confocal microscope equipped with pulsed excitation and time-gated detection. From the decay the limiting anisotropy r(inf) was derived, which is a direct measure for the number of fluorophores per cluster. The method was evaluated by imaging GPI-GFP, a lipid raft marker. Small clusters were observed in the plasma membrane while the cytoplasm and the Golgi contained predominantly monomers.