Principle of FRET
Occurrence of FRET
Jablonsky diagram showing the energy levels of the fluorescent dyes and the rates and states involved in FRET.
FRET is a phenomenon that occurs between a fluorescent dye (donor) and a dye (acceptor) when they are close (< 10nm) together. In that case the fluorescent energy is transferred without the emission of light form the donor to the acceptor.
Therefore FRET can be used to estimate the distance between two dyes. FRET was characterized in the 1940ies by T. Förster.
Spectral requirements
Excitation and emission spectra of the FRET pair, CFP and YFP, illustrating the overlap of the CFP emission and the YFP excitation spectrum.
The emission specturm of the donor and the absorbtion spectrum of the acceptor are required to exhibit a significant overlap (approx. 30%) for FRET.
Applications of FRET in Bioscience
There are various assays used in Bioscience that use FRET. With the dawn of the Fluorescent Proteins (FP) the popularity of FRET in Bioscience is increased. As now it is possible to tag proteins of interest directly via cloning a FP to it.
Two kinds of assays emerge from that possibility. First the construction of Biosensors by tagging a protein that undergoes a conformational change under circumstances of interest (e.g. Ca2+ concentration) on each side with FPs which belong to a FRET pair. An example for a FRET based biosensore is Cameleon. This biosensore is a reporter for changes of intracellular calcium concantration. Second the interaction between two proteins can be monitored by tagging them with appropriate FPs.
One of the most popular FRET pairs consists of CFP and YFP, the cyan and the yellow variant of GFP. Originally GFP was discovered in the jelly fish Aequorea Victoria. The family of FPs is grown, now there are members derived from other species aswell.