Főrster Resonance Energy Transfer
FRET (Fluorecence or Főrster Resonance Energy Transfer): FRET is an established technique used to visualize the interaction and location of two proteins or structures within the cell. The method involves the fluorescent labeling of two proteins or structures within the cell using spectrally distinct (but matched) loopholes. The techniques relies upon FRET a process whereby energy is transferred from one fluorophore to another only when the two are in close proximity to each other. The process is outlined in figure 1.
The two fluorophores involved as known as the donor and acceptor when these are in close proximity to each other for example when two proteins interact, energy is transferred between them.
Of note, the energy transfer is not transferred by fluorescence but is of a nonradiative nature.
FRET is a powerful technique capable of allowing the experimenter to resolve molecular interactions of 10nm or less thus dramatically improving spatial resolution.
However, there are a number of important conditions that must be met in order for the FRET technique to be successful:
- emission spectrum of the donor must be matched (overlap) with the excitation spectra of the acceptor
- donor and acceptor fluorophores must be correctly orientated to permit the energy transfer
- two fluorophores must be close (<10nm) to each other
Commonly used FRET fluorescent pairs are CFP(donor) YFP (acceptor), FITC (donor) Rhoda mine (acceptor and Cy3 (donor) Cy5 (acceptor).
Three FRET techniques are widely used:
Acceptor photobleaching
A consequence of the FRET process is that when two fluorophores are brought into close proximity to each other the fluorescence of the donor fluorophore is quenched by the presence of the acceptor; essentially the energy that would be released as a photon is passed to the acceptor molecule. In the acceptor photobleaching technique, the acceptor fluorophore is bleached thereby reducing its ability to receive energy form the donor, this leads to an increase in fluorescence of the donor molecule. The pre and post bleach difference in donor fluorescence can be used to estimate the level of FRET.
This approach is relatively straightforward way to estimate FRET however; it is best applied to fixed samples.
Sensitised emission
A second method to estimate the level of FRET is by a technique know as sensitized emission, the principle of this technique relies upon the aforementioned transfer of energy from the donor to acceptor fluorophore and the subsequent increase in it’s fluorescence. If when the donor fluorophore is excited FRET occurs between it and the acceptor, a component of the acceptor fluorescence will result for the excitation of the donor.
To correctly estimate this, donor only, acceptor only and ‘FRET’ samples need to be imaged under different conditions:
- donor excitation- acceptor emission (acceptor only sample, gives a measure of acceptor excitation by the donor excitation)
- donor excitation acceptor emission (donor only sample, gives a measure of bleed through of the donor fluorescence into the acceptor channel)
- donor excitation, acceptor emission (FRET sample, estimated values for 1 and 2 can be subtracted from this measurement to yield a value for FRET)
Spectral Imaging
This approach requires that we know the spectral profile of the acceptor and donor fluorophores thus acceptor only and donor only samples are required along with the ‘FRET’ sample. This approach is a variation of the sensitized emission technique however, instead of recording data through two individual channels, the complete emission spectrum of the donor and acceptor fluorophores is collected. In doing this it is possible to determine the levels of donor and acceptor fluorescence and as such, any FRET contribution should be evident.
