Total internal reflection energy transfer (TIRET) microscopy for analysis of focal adhesions in living cells

Total internal reflection fluorescence microscopy (TIRFM) is used to measure non-radiative energy transfer between membrane associated proteins in living cells. Measurements are concentrated on focal contacts and their associated proteins focal adhesion kinase (FAK) and Paxillin (Pax) which play major roles with respect to cell migration, growth, and survival. These proteins are visualized after fusion with variants of green fluorescent protein (ECFP and EYFP), and an intermolecular energy transfer ECFP -> EYFP is deduced from fluorescence spectra as well as from fluorescence decay kinetics of single cells.

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