Techniques utilizing Forster-type FRET offer convenient tools for mapping the spatial distribution and molecular vicinity relations of membrane proteins on live cells in situ, without any major interference with the physiological condition of the cells. Several techniques have been developed to measure FRET on cell surfaces [5,41-43].
FRET is a process wherein energy is transferred non-radiatively from an excited donor fluorophor to a nearby acceptor via dipole-dipole coupling . In order for FRET to occur, it is necessary that the dipole moments of the dyes have a proper relative orientation and the emission spectrum of the donor and the absorption spectrum of the acceptor molecule overlap with each other . From the point of view of biological applications, the most important property of FRET is that its rate is inversely proportional to the sixth power of the donor-acceptor distance; hence, it is a sensitive tool for the determination of inter- or intramolecular distances in the 1- to 10-nm range, and can be applied as a "spectroscopic ruler" . The efficiency of FRET, E, is defined as the fraction of excitation quanta transferred from the donor to the acceptor, which can be expressed as:
where R is the donor-acceptor distance, Ro is the so-called Förster distance, at which the FRET efficiency is 50% for the given donor-acceptor pair, and kFRET, kf and knf are the rate constants of de-excitation by FRET, fluorescence emission and non-radiative processes other than FRET, respectively. Ro is usually 5-10 nm, which defines the distance range for which FRET is applicable. In practice, the detection of FRET is based on the measurement of one or more of the following physical parameters: a) the decreased intensity of the donor (donor quenching); b) the enhanced emission of the acceptor (sensitized emission); c) the decreased fluorescence lifetime of the donor; d) the increased fluorescence anisotropy of the donor; or e) the decreased photobleaching rate of the donor.
Measurement of FRET by microscopy provides subcellular mapping of proteinprotein interactions, allowing the visual identification of compartments/organelles where the interactions of interest take place (for a review, see ). Without going into detail, the most commonly used FRET microscopic methods are described below.
Based on the simultaneous detection of three (or in the case of using autofluorescence correction, four) fluorescence intensities (autofluorescence, donor and acceptor channels and FRET channel) at each pixel, and using appropriate controls for determining the spectral spillover between the channels, FRET efficiency can be measured even at rather low expression levels .
With the acceptor photobleaching technique, the extent of donor quenching due to FRET is detected in a fairly simple way [49,50]. If the acceptor is irreversibly photodestroyed by selective illumination at the acceptor's absorption wavelength, the intensity of the donor increases, and from the extent of increase E can be calculated:
i d where IDA and ID are the background-corrected donor fluorescence intensities with and without acceptor - that is, before and after photobleaching. The advantage of the method is its simplicity and its requirement for only donor-acceptor double-labeled samples.
The donor photobleaching (pbFRET) method exploits the increased resistivity of the donor to photobleaching in the presence of acceptor. Photobleaching is initiated from the excited state. FRET reduces the fluorescence lifetime of the donor; that is, the dye spends less time in the excited state, resulting in an elongated photobleaching time constant:
where Td and Tda are the photobleaching time constants of the donor in the absence and presence of acceptor, respectively [50,51].
The fluorescence lifetime of the donor can be directly measured by using the imaging version of phase fluorimetry, fluorescence lifetime imaging microscopy
(FLIM) . Lifetime is a fairly robust parameter, which makes FLIM an attractive method in spite of its relatively complex instrumentation.
Although FRET microscopy has a clear advantage in providing subcellular information on interactions at microscopic resolution, it requires large numbers of cells to be evaluated to provide statistically valid data, and this is both time-consuming and labor-intensive. To determine small quantitative changes in proteinprotein interactions, at least several hundreds or thousands of cells must be evaluated. A suitable alternative is that of flow cytometric cell-by-cell FRET measurement, which provides the mean FRET efficiency for each cell in the analyzed population [53,54]. The high number of cells that can be analyzed by flow cyto-metry provides excellent statistics, high accuracy, and reproducibility.
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