LAUSR

AS a result of heightened interest in elucidating protein–protein interactions, techniques for mapping the protein interactome are frequently used by biologists. In an era when a multitude of molecular biological and superresolved microscopy techniques are available for this purpose, Förster resonance energy transfer (FRET) preserved its status as a fashionable, accurate and relatively easy-to-use approach (1). FRET is a nonradiative energy transfer process from an excited fluorophore (donor) to an acceptor by dipole–dipole coupling. Its sensitivity to molecular interactions and conformations stems from the fact that its efficiency steeply decreases with the donor–acceptor separation in the 2–10 nm range. Although it is already valuable as a standalone approach, its usefulness can be further increased by combining with other modalities, like confocal microscopy (2). Its flexibility and versatility are enhanced by the generation of sensors, that is, donor–acceptor fluorescence protein constructs connected by a linker, whose conformation responds to certain properties of the environment. The FRET efficiency of such sensors can be used as a readout parameter for a multitude of biological processes (3,4). Although a large number of approaches are available for the determination of FRET, intensity-based (ratiometric) measurements remain the cornerstone in its biological applications since these experiments are the easiest to implement and they usually provide the answer to the questions asked about protein clustering by ordinary biologists (1). The method typically involves the measurement of fluorescence intensities in three channels corresponding to donor fluorescence, directly excited and FRET-sensitized acceptor emissions. In order to solve the equation set, two different kinds of correction parameters are required: