Fluorescence resonance energy transfer (FRET) is a spectroscopic tool widely used in the detection of biological events and in particular of molecular interactions.
In numerous cases, the FRET, which requires bringing close together the donor and acceptor fluorescent molecules which will be involved in the energy transfer, proves to be a powerful tool in the detection of biological interactions. It can be used in fields as varied as molecular biology, the in-vitro or in-cellulo detection of enzymatic phenomena (peptide cleavage, phosphorylation) or interactions between proteins (1, 2, 3).
Detection of the FRET phenomenon can be carried out by measuring different parameters of the fluorescence signal emitted either by the donor, or by the acceptor, or by both molecules. Among the most common techniques, there can in particular be mentioned:                measurement of the reduction in the donor's fluorescence induced by the FRET phenomenon (4),        measurement of the increase in the acceptor's fluorescence induced by the energy originating from the donor via the FRET (5),        determination of the [(acceptor fluorescence increase)/(donor fluorescence reduction)] ratio (6),        measurement of the reduction in the lifetime of the donor's fluorescence induced by the FRET phenomenon (7). The latter is in particular measured by the “Fluorescence Lifetime Imaging Microscopy” (FLIM) technique,        measurement of the increase in the fluorescence of the donor involved in a FRET after the photobleaching of the acceptor (8); this photobleaching technique is known as Fluorescence Recovery After Photobleaching (FRAP).        
Leaving aside techniques combining the FRET and time-resolved detection made possible by the use of fluorescence donors with a long lifetime (e.g.: HTRF), the FRET phenomenon proves complex to detect in numerous applications based on fluorescence intensity measurements. The need for significant energy compatibility between the donor and the acceptor often leads to the use of molecules possessing relatively similar fluorescence emission spectra. The resulting overlap of the donor's and acceptor's spectra make it very difficult to precisely measure variations in signals recorded on the donor or on the acceptor (9).
This is particularly true when fluorescent proteins derived from Green Fluorescent Protein (GFP) are used in the FRET experiments, such as the Cyan Fluorescent Protein (CFP)/Yellow Fluorescent Protein (YFP) donor/acceptor pair which is the most used. These molecules, which are capable of being expressed in fluorescent form in numerous types of cell, allow the detection of numerous intracellular events. However, a significant overlap of fluorescence spectra exists between the latter, resulting in the direct parasitic excitation of the acceptor by the donor molecule's excitation beam. Therefore, the signal/noise ratio of the FRET experiments carried out with this donor/acceptor pair is low, often less than 1.5 (1). As a result, it is necessary to implement complex experimentation protocols comprising numerous experimental controls in order to be able to interpret the results obtained.
The technical problem to be resolved therefore involves providing a simple and reproducible method for correcting the FRET measurement, in particular by improving the signal/noise ratio.
It has now been found that the impact of the strong overlap of the donor's and acceptor's fluorescence emission spectra could be significantly reduced using the polarization properties of these compounds in order to correct the FRET measurement.
It has been described that the appearance of an energy transfer between two fluorescent molecules caused polarization modifications both at the level of the donor and at the level of the acceptor: the polarization of the donor increases when it is involved in a FRET (10), whereas that of the acceptor involved in the FRET decreases (11).
The influence of the FRET on the relative polarization of the donor and the acceptor has thus been used in different molecular systems in order to detect this energy transfer between two fluorescent probes.
In particular, a homoFRET between two GFP molecules has been detected by measuring their depolarization (12). Measurement of the depolarization of rhodamine coupled to a lectin was used to detect a FRET being produced between the fluorescein and the rhodamine (5). Also, measurement of the increase in the polarization of a Concanavilin A-Fluorescein donor made it possible to detect a FRET indicating the formation of a molecular cluster in the lymphocyte membranes (10).
The polarization measurements used thus far therefore had the purpose of detecting the existence of a FRET between two molecules.