Fluorescence Resonance Energy Transfer, also known as Foerster Resonance Energy Transfer ( FRET in both cases ) is the term given to the process where two excited " fluorophores " pass energy one to the other non-radiatively ( i. e., without exchanging a photon ).

Other optical techniques are also used: Fluorescence Correlation and Cross-Correlation Spectroscopy ( FCS / FCCS ) can be used to gain information of fluorophore mobility in the membrane, Fluorescence Resonance Energy Transfer ( FRET ) can detect when fluorophores are in close proximity and optical tweezer techniques can give information on membrane viscosity.

Förster ( Fluorescence ) resonance energy transfer ( FRET ), resonance energy transfer ( RET ) or electronic energy transfer ( EET ), is a mechanism describing energy transfer between two chromophores.

One of the most useful techniques to study kinase action is Fluorescence Resonance Energy Transfer ( FRET ).

FRET has also been used in tandem with Fluorescence Lifetime Imaging Microscopy ( FLIM ) or fluorescently conjugated antibodies and flow cytometry to provide a detailed, specific, quantitative results with excellent temporal and spatial resolution.

Since photobleaching consists in the permanent inactivation of excited fluorophores, resonance energy transfer from an excited donor to an acceptor fluorophore prevents the photobleaching of that donor fluorophore, and thus high FRET efficiency leads to a longer photobleaching decay time constant:

FRET ( fluorescent resonance energy transfer ) / FRET quenching

* Fluorescent methods: fluorescence resonance energy transfer ( FRET ), fluorescence correlation spectroscopy ( FCS ), total internal reflection fluorescence ( TIRF )

The efficiency of this energy transfer is inversely proportional to the sixth power of the distance between donor and acceptor making FRET extremely sensitive to small distances.

These virtual photons are undetectable, since their existence violates the conservation of energy and momentum, and hence FRET is known as a radiationless mechanism.

Quantum electrodynamical calculations have been used to determine that radiationless ( FRET ) and radiative energy transfer are the short-and long-range asymptotes of a single unified mechanism.

The FRET efficiency () is the quantum yield of the energy transfer transition, i. e. the fraction of energy transfer event occurring per donor excitation event:

Since the fluorescence lifetime of a fluorophore depends on both radiative ( i. e. fluorescence ) and non-radiative ( i. e. quenching, FRET ) processes, energy transfer from the donor molecule to the acceptor molecule will decrease the lifetime of the donor.

A limitation of FRET is the requirement for external illumination to initiate the fluorescence transfer, which can lead to background noise in the results from direct excitation of the acceptor or to photobleaching.

Measurements of these rates using time-resolved FRET revealed that DNA within the nucleosome remains fully wrapped for only 250 ms before it is unwrapped for 10-50 ms and then rapidly rewrapped.

FRET is analogous to near field communication, in that the radius of interaction is much smaller than the wavelength of light emitted.

In fluorescence microscopy, fluorescence confocal laser scanning microscopy, as well as in molecular biology, FRET is a useful tool to quantify molecular dynamics in biophysics and biochemistry, such as protein-protein interactions, protein – DNA interactions, and protein conformational changes.

The FRET efficiency is measured and used to identify interactions between the labeled complexes.

One method of measuring FRET efficiency is to measure the in acceptor emission intensity.

When a twist or bend of the protein brings the change in the distance or relative orientation of the donor and acceptor, FRET change is observed.

If a molecular interaction or a protein conformational change is dependent on ligand binding, this FRET technique is applicable to fluorescent indicators for the ligand detection.

Yet researchers can detect differences in the polarisation between the light which excites the fluorophores and the light which is emitted, in a technique called FRET anisotropy imaging ; the level of quantified anisotropy ( difference in polarisation between the excitation and emission beams ) then becomes an indicative guide to how many FRET events have happened.

FRET is also used to study lipid rafts in cell membranes.

FLIM has primarily been used in biology as a method to detect photosensitizers in cells and tumors as well as FRET in instances where ratiometric imaging is difficult.

One very simple system showing chemical relaxation would be a stationary binding site in the measurement volume, where particles only produce signal when bound ( e. g. by FRET, or if the diffusion time is much faster than the sampling interval ).

By carefully selecting the excitation of these flurophores and detecting the emission, FRET has become one of the most widely used techniques in the field of Biophotonics, giving scientists the chance to investigate sub-cellular environments.

Fluorophores can also be used to quench the fluorescence of other fluorescent dyes ( see article Quenching ( fluorescence )) or to relay their fluorescence at even longer wavelength ( see article FRET )

NMR and FRET methods can be used to determine complementary information including relative distances,

Measurements of FRET efficiency can be used to determine if two fluorophores are within a certain distance of each other.

Lifetime measurements of FRET are used in Fluorescence-lifetime imaging microscopy.

Obviously, spectral differences will not be the tool used to detect and measure FRET, as both the acceptor and donor protein emit light with the same wavelengths.

FRET has been used to measure distance and detect molecular interactions in a number systems and has applications in biology and chemistry.

FRET can be used to measure distances between domains a single protein and therefore to provide information about protein conformation.

Applied in vivio in living cells, FRET has been used to detect the location and interactions of genes and cellular structures including intergrins and membrane proteins.

FRET can be used to obtain information about metabolic or signaling pathways.

FRET and BRET are also the common tools in the study of biochemical reaction kinetics and molecular motors.

There are also several quantitative protein phosphorylation methods, including fluorescence immunoassays, Microscale Thermophoresis, FRET, TRF, fluorescence polarization, fluorescence-quenching, mobility shift, bead-based detection, and cell-based formats.

Example of FRET between CFP and YFP ( Wavelength vs. Absorption ): a Genetic engineering | fusion protein containing CFP and YFP excited at 440nm wavelength.

FRET can also detect interaction between proteins.

FRET can detect dynamic protein-protein interaction in live cells providing the fluorophores get close enough.

To utilize FRET for phosphorylation studies, fluorescent proteins are coupled to both a phosphoamino acid binding domain and a peptide that can by phosphorylated.