the postgenomic era, the characterization of protein net-works has become a major focus of scientific interest. To inter-pret signal transduction through membrane receptors and tobuild a model of a signaling network, it is essential to obtainthe spatiotemporal activities of participating molecules.Although a plethora of different approaches is available fordissecting the protein interactome such molecular interactionsare often determined by modern biophysical methods such asForster resonance energy transfer (FRET, which is often inter-€preted to stand for fluorescence resonance energy transfer)providing more quantitative and statistically more robustinformation than classical molecular biological approacheslike coprecipitation, chemical crosslinking, yeast two-hybrid-type approaches, and so on (1,2).FRET is a process by which radiationless transfer ofenergy occurs from a donor fluorophore to an acceptor placedin close proximity to the donor. The probability of the energytransfer is characterized by the FRETefficiency (E), which tellsus what fraction of excited donor molecules relaxes by trans-ferring their excitation energy to the acceptor. The rate of theFRET process depends on the negative 6th power of the do-nor–acceptor distance, so the FRET efficiency can serve as aspectroscopic ruler allowing sensitive determination of inter-molecular and intramolecular distances under ideal conditions(3). FRET is the consequence of the interaction between thedonor and acceptor dipoles and the dependence of its rate onthe negative 6th power of the donor–acceptor separation dis-tance is the result of the ‘‘resonance’’ between the donor andthe acceptor dipoles which is distributed over a wide spectralrange characterized by the overlap integral (4). Because of theFRET process, among others, the fluorescence intensity andlifetime of the donor dye decreases and at the same time thefluorescence intensity of the acceptor increases (sensitizedemission). The FRETefficiency can be determined by variousmethods taking advantage of one or more of these spectro-scopic changes (2,5). We can classify them, somewhat arbitra-rily, into (a) lifetime-based methods determining the fluores-cence lifetime components directly or instead, a spectroscopicquantity related to the lifetime (such as the characteristic do-nor photobleaching rate in a donor photobleaching experi-ment) and (b) intensity-based methods measuring donorquenching and/or sensitized emission. Intensity-based meth-ods are usually called ratiometric, as these approaches involvethe division of two fluorescence intensities roughly corre-sponding to those measured in the absence and presence ofFRET. Fluorescence lifetime imaging microscopy (FLIM) is asophisticated approach which is getting more and more wide-spread even though it is cumbersome, requires highly specia-lized equipment and involves long acquisition time (6). Itshould be mentioned, however, that inventive and modernevaluation methods have been developed for FLIM to reducethe necessary acquisition time (7–9). On the contrary, ratio-metric FRET methods provide higher speed of acquisition,require less sophisticated equipment, but can be challengingbecause of the significant fluorescence spectral bleed-throughoriginating from the donor and the acceptor into the FRET