QUANTITATIVE FLUORESCENCE RESONANCE ENERGY-TRANSFER MEASUREMENTS USING FLUORESCENCE MICROSCOPY

Fluorescence resonance energy transfer (FRET) is a technique used for quantifying the distance between two molecules conjugated to different fluorophores. By combining optical microscopy with FRET it is possibl e to obtain quantitative temporal and spatial information about the bi nding and interaction of proteins, lipids, enzymes, DNA, and RNA in vi vo. In conjunction with the recent development of a variety of mutant green fluorescent proteins (mtGFPs), FRET microscopy provides the pote ntial to measure the interaction of intracellular molecular species in intact living cells where the donor and acceptor fluorophores are act ually part of the molecules themselves. However, steady-state FRET mic roscopy measurements can suffer from several sources of distortion, wh ich need to be corrected, These include direct excitation of the accep tor at the donor excitation wavelengths and the dependence of FRET on the concentration of acceptor. We present a simple method for the anal ysis of FRET data obtained with standard filter sets in a fluorescence microscope. This method is corrected for cross talk (any detection of donor fluorescence with the acceptor emission filter and any detectio n of acceptor fluorescence with the donor emission filter), and for th e dependence of FRET on the concentrations of the donor and acceptor. Measurements of the interaction of the proteins Bcl-2 and Beclin (a re cently identified Bcl-2 interacting protein located on chromosome 17q2 1), are shown to document the accuracy of this approach for correction of donor and acceptor concentrations, and cross talk between the diff erent filter units.