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ABSTRACT
Measurement of fluorescence resonance energy transfer (FRET) efficiency and the relative concentration of donor and acceptor fluorophores in living cells using the three-filter cube approach requires the determination of two constants: 1), the ratio of sensitized acceptor emission to donor fluorescence quenching (G factor) and 2), the ratio of donor/acceptor fluorescence intensity for equimolar concentrations in the absence of FRET (k factor). We have developed a method to determine G and k that utilizes two donor-acceptor fusion proteins with differing FRET efficiencies-the value of which need not be known. We validated the method by measuring the FRET efficiency and concentration ratio of the fluorescent proteins Cerulean and Venus in mammalian cells expressing a series of fusion proteins with varying stoichiometries. The method greatly simplifies quantitative FRET measurement in living cells as it does not require cell fixation, acceptor photobleaching, protein purification, or specialized equipment for determining fluorescence spectra or lifetime.
Fluorescence resonance energy transfer (FRET) occurs when a donor tluorophore in the excited state transfers energy nonradiatively to an acceptor fluorophore in the ground state (1). FRET efficiency, defined as the proportion of the donor molecules that have transferred excitation state energy to the acceptor molecules, increases with decreasing intermolecluar distance (typically over the range 1-10 nm for lluorescent proteins). Thus FRET-hased imaging can be used to assess fluorophore proximity, and by inference, protein-protein interaction, in living cells.
FRET measurements in living cells using "three-cube FRET" fluorescence microscopy (2-5) has become increasingly popular as the method is fast, simple, nondestructive, and requires only a standard fluorescence imaging microscope. With this method, images are acquired using three different fluorescence filter cubes: 1), the donor channel (I^sub DD^, donor excitation and emission), 2), the FRET channel (I^sub DA^, donor excitation, acceptor emission), and 3), the acceptor channel (I^sub AA^, acceptor excitation and emission). Because of spectral overlap between donor and acceptor fluorescent proteins (FP), procedures ((3-6), see Supplementary Materials) are used to isolate the donor (I^sub dd^), sensitized acceptor (F^sub c^, i.e., fraction of I^sub DA^ resulting from FRET), and direct acceptor (I^sub aa^) fluorescence intensities from the uncorrected intensity images (I^sub DD^, I^sub DA^, and I^sub AA^).
FRET indices based on normalized F^sub c^ are often used to report experimental results. Unfortunately, such indices...