Cytometry of fluorescence resonance energy transfer.

Publisher Summary luorescence resonance energy transfer (FRET) is a phenomenon in fluorescence spectroscopy where energy is transferred from an excited donor molecule to an acceptor molecule under favorable spectral, proximity, and orientational conditions. Applying donor- and acceptor-labeled antibodies, lipids, and various types of fluorescent proteins (such as green fluorescent protein [GFP] analogs), the FRET technique can be used to determine intermolecular and intramolecular distances of cell surface components in biological membranes and molecular associations within live cells. The chapter provides an introduction to the theory of FRET, and explains the relevant protocols of FRET techniques. The various approaches that have been used to quantitate FRET can be categorized based on the spectrofluorometric parameter detected and if the donor or the acceptor is investigated. Specific advantages and limitations of FRET approaches are also discussed, together with perspectives and advances that may provide new approaches for the detection of FRET in the future. Advances in developing new fluorescent probes, instrumentation, and methodologies have greatly improved the applicability of FRET to the systematic exploration of the localization, translocation, and association of signaling proteins in living or intact cells.