If you think FRET stands for Fluorescence Resonance Energy Transfer, you are wrong…in good company but wrong. FRET actually stands for Förster Resonance Energy Transfer. Find out why and more about FRET in this article.
In 2011, it was the centenary of the birth of eminent German physical chemist Theodor Förster. Förster was a very significant figure in his field. His work provided important insights to the nature of excited-state photochemical reactions and the understanding of organic dyes. His most famous contribution was his work on the electrostatically mediated energy transfer between sensitiser and acceptor molecules. And while it is quite possible that you have never heard Förster name before – I beat you HAVE heard of the technique that bears his name: Förster Resonance Energy Transfer or FRET. One of the reasons for this discrepancy maybe because you always thought these initials stood for “Fluorescence Resonance Energy Transfer” – which is OK, but strictly speaking not entirely correct, as we will see below.
FRET is key to a wide range of natural phenomena, including photosynthesis. However, its application in microscopy and its ability to uncover molecular processes in biological systems at the microscopical level is what I will focus on in this article.
Investigating the interactions between different proteins in a cell is paramount in understanding protein function. Certainly, biochemical approaches such as immunoprecipitation, pull-down assays, etc., are indispensable for identifying protein-protein interactions. However, these techniques do not provide spatial resolution or even temporal information in the context of a living cell. On the other hand in living cells, immunofluorescence can provide spatial resolution; and fluorescent tags, such as GFP, can provide spatial and temporal information in living cells. But these approaches tell us little about protein-protein interactions.
It’s All About Scale
It is only logical: To have an interaction, there must be colocalization. You could detect colocalization by 1) employing fluorescent antibodies or tags, such as one green tag and a second red tag and 2) scanning the sample with a confocal microscope, or using deconvolution, or both to identify colocalization or yellow. But yellow may or may not equal colocalization, depending on your definition of colocalization, specifically what scale you are interested in.
If for example, you want to know if a certain non-pyramidal neuron expresses both GABA and Parvalbumin, and you stain for both (GABA in red and Parvalbumin in green) and the neuron looks yellow, then you CAN say that these proteins colocalize in that neuron. But things are not so definitive the closer you look.
When using a diffraction-limited microscope, we need to be aware that we cannot decipher distances that are (at best) less than 200nm. This means that with these microscopes any two signals that originate from points closer to each other than 200nm are considered “colocalized” -not because they are but because of limitations in the technology. Second, we must remember that, if we are looking for an interaction, this kind of “colocalization” (vicinity, to be more realistic), is not enough. It would be better to know that the two proteins are actually close enough to interact, enter FRET.
Principles of FRET
The use of FRET in optical microscopy makes it possible to detect two molecules approaching within the range of a few nanometers. To perform FRET you need two fluorescent molecules, a donor and an acceptor. When these molecules are close and the donor fluorophore is excited, part of its energy is transferred to the acceptor fluorophore. This transfer causes the acceptor fluorophore to fluoresce, despite the fact that it has not been directly excited by light.
In FRET the transfer of energy between the donor and the acceptor happens in non-radiative fashion through long-range dipole-dipole interactions. In short the excited fluorophore acts as an oscillating dipole that can undergo an energy exchange with a second dipole having a similar resonance frequency. This is why calling FRET “Fluorescence Resonance Energy Transfer” is not entirely correct, as it implies that it is through fluorescence that the energy is transferred, which is not the case. The range over which this energy transfer can occur is limited to about 10nm. And as the efficiency of transfer is highly sensitive to the distance between fluorophores, FRET measurements prove very useful for assessing close molecular interactions.
The efficiency of the energy transfer also depends on the spectral overlap of the two fluorophores. That is, the overlap between the donor fluorophore’s emission spectrum and the acceptor fluorophore’s excitation spectrum. Therefore you need to take care when choosing your fluorophore pairs for your FRET experiment. For example, CFP and YFP are a very popular FRET pair, and one can easily understand why when you look at their excitation and emission spectra (they overlap well). But this is not the only thing to consider when designing your FRET experiment. I will go into more detail about FRET in my next article.