Förster resonance energy transfer, or FRET, provides a means to study the temporal dynamics of protein interactions by measuring the transfer of energy between donor and acceptor molecules. Researchers label two molecules of interest with a donor-acceptor pair, and by monitoring the acceptor emission, they can tell when the molecules are in proximity.For the most part, researchers have used only single donor-acceptor pairs in their FRET investigations. The ability to image multiple such pairs has been constrained by the considerable spectral overlap of the commonly used fluorophores, which makes it difficult to separate the emissions from different pairs.Researchers have achieved multiplexed FRET imaging by using a recently introduced red protein called TagRFP as a donor, thus avoiding spectral overlap with the other donor-acceptor pair. Shown here are multiplexed FRET images of calcium flux and Ras activation. The top row (A) shows spectral ratiometric images of the first donor-acceptor pair between 0 and 100 s. The middle row (B) shows lifetime maps of TagRFP between 0 and 250 s, as well as lifetime merged with intensity (bottom row). Reprinted with permission of Biophysical Journal. But multispectral imaging with FRET “is in fact possible,” said Marina K. Kuimova, a researcher in the chemistry department at Imperial College London who uses the technique and follows trends in its development. By carefully managing the spectral overlap, developing novel acceptors and using fluorescence lifetime imaging (FLIM) to monitor FRET, she said, researchers can achieve imaging of multiple FRET pairs at the same time, thus allowing investigators to build complex bioassays that will help answer a host of questions.One of the ways this can be achieved, she continued, is to have one pair in the green or blue end of the spectrum and another shifted far into the red. In a Biophysical Journal article published online Aug. 29, another group at Imperial College London reported just such an approach. The researchers used a recently introduced red protein called TagRFP as a donor. This in turn called for a further red-shifted protein to be used as an acceptor. The investigators found this in mPlum, which offers the longest emission spectrum among currently available fluorescent proteins.Because of its low quantum yield, mPlum isn’t necessarily well-suited for ratiometric FRET measurements – in which researchers compare the emission intensities of the donor and the acceptor following excitation of the donor. This is one reason the TagRFP/mPlum pair has not previously been used for FRET, noted the authors of the paper. They were able to sidestep this issue, though, by measuring FRET with FLIM, which relies on the donor emission signal only. Thus the authors of the Biophysical Journal paper were able to demonstrate multiplexed FRET by combining FLIM FRET of the TagRFP/mPlum pair with ratiometric imaging of an enhanced CFP/Venus pair, and thus to look at two events occurring within the cell at the same time.Kuimova said that another important development – one that has taken hold in the past few years – was the introduction of a nonfluorescent (“dark”) chromoprotein known as REACh (resonance energy-accepting chromoprotein). First reported in a 2006 PNAS paper by investigators from European Neuroscience Institute-Göttingen in Germany; from Gray Cancer Institute, Mount Vernon Hospital in Middlesex, UK; and from King’s College London, REACh acts by quenching the donor fluorescence. Researchers can image FRET with a REACh acceptor using one of several means: for example, by visualizing the changes in donor emission; by measuring with FLIM the reduced lifetime of the donor; or by observing quenched emission with respect to a reference fluorophore.Eliminating the acceptor fluorescence by using the dark chromoprotein allows researchers to optimize the spectral overlap in a FRET pair comprised of REACh and GFP, for instance. This facilitates detection of photons in the entire spectral window of green fluorescent protein emission and furthermore opens up the spectral range to allow simultaneous imaging with an additional FRET pair.