FRET Microscopy
The ‘Förster (or Fluorescence) Resonance Energy Transfer Microscopy’ (FRET) is a specialized application of fluorescence microscopy. The effect of the energy transfer was predicted by Theodor Förster (1910 – 1974) after whom the effect is named.
By Dr. Jakob Bierwagen, AHF analysentechnik AG
The 'FRET Effect'
In the FRET effect, energy is transferred from a donor molecule to an acceptor molecule. The efficiency of this energy transfer decreases with the sixths power of the distance and also depends on the orientation of the molecules. Due to this strong distance dependency, one can optically measure distances from 0 to approx. 10 nm.
To have a FRET effect, the emission spectrum of the donor must be superimposed to the excitation spectrum of the acceptor. The closer the molecules are and the more parallel they are to each other, it is more likely the excitation energy of the donor will be transferred to the acceptor. In practice, the donor is excited with a laser or a LED light source.
Then, there are several ways to observe the FRET effect: e.g. by measuring the increase of the fluorescence of the acceptor or the decrease of the fluorescence of the donor and also by the (reduced) fluorescence lifetime of the donor. This method is usually the most accurate, as bleach effects play only a minor role here. Through reference measurement, the Förster radius of the FRET pair can be determined which then allows to calculate the distance between the molecules
Fields of Application
The effect is used in the mechanism elucidation when proteins or other molecular machines are moving, either through protein-protein interaction or conformational changes. However, the effect is also important in OLEDs and occurs in nature in the photosynthesis where the captured photon is directed to the reactive center of the photo-complex by the FRET effect. Especially in biochemistry and microbiology for the observation of processes in cells, the effect is combined with microscopy to enable localization in organelles.
Optical Filters for FRET
AHF offers matching optical filters for FRET applications and can also build individual filter sets.