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.

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

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:

The FRET efficiency depends on many parameters that can be grouped as follows:

The FRET efficiency relates to the quantum yield and the fluorescence lifetime of the donor molecule as follows:

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

There are several ways of measuring the FRET efficiency by monitoring changes in the fluorescence emitted by the donor or the acceptor.

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

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:

* Browser-based calculator to find the critical distance and FRET efficiency with known spectral overlap

* FRET Fluorescence resonance energy transfer is used to study protein interactions, detect specific nucleic acid sequences and used as biosensors, while fluorescence lifetime ( FLIM ) can give an additional layer of information.

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 )

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.

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

FRET efficiencies can also be inferred from the photobleaching rates of the donor in the presence and absence of an acceptor.

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.

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

FRET can also detect interaction between proteins.

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

Thus, FRET measurements using FLIM can provide a method to discriminate between the states / environments of the fluorophore.

In contrast to intensity-based FRET measurements, the FLIM-based FRET measurements are also insensitive to the concentration of fluorophores and can thus filter out artifacts introduced by variations in the concentration and emission intensity across the sample.

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

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

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.

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 ).

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

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

Fluorescence-lifetime imaging yields images with the intensity of each pixel determined by, which allows one to view contrast between materials with different fluorescence decay rates ( even if those materials fluoresce at exactly the same wavelength ), and also produces images which show changes in other decay pathways, such as in FRET imaging.

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.

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.

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 ).

When the donor and acceptor are in proximity ( 1 – 10 nm ) due to the interaction of the two molecules, the acceptor emission will increase because of the intermolecular FRET from the donor to the acceptor.

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.

FRET will respond to internal conformational changes result from reorientation of the fluorophore with respect to the other.

In addition, there has been a flurry of activity extending FCS in various ways, for instance to laser scanning and spinning-disk confocal microscopy ( from a stationary, single point measurement ), in using cross-correlation ( FCCS ) between two fluorescent channels instead of autocorrelation, and in using Förster Resonance Energy Transfer ( FRET ) instead of fluorescence.

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.

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.

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

In general, " FRET " refers to situations where the donor and acceptor proteins ( or " fluorophores ") are of two different types.