Förster resonance energy transfer
FRET is analogous to near-field communication, in that the radius of interaction is much smaller than the wavelength of light emitted.
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.
= 2/3 does not result in a large error in the estimated energy-transfer distance due to the sixth-power dependence of
The inverse sixth-power distance dependence of Förster resonance energy transfer was experimentally confirmed by Wilchek, Edelhoch and Brand[26] using tryptophyl peptides.
Stryer, Haugland and Yguerabide[27][citation needed][28] also experimentally demonstrated the theoretical dependence of Förster resonance energy transfer on the overlap integral by using a fused indolosteroid as a donor and a ketone as an acceptor.
[22][24] However, a lot of contradictions of special experiments with the theory was observed under complicated environment when the orientations and quantum yields of the molecules are difficult to estimate.
There are several ways of measuring the FRET efficiency by monitoring changes in the fluorescence emitted by the donor or the acceptor.
[32] Its use of an entire curve of points to extract the time constants can give it accuracy advantages over the other methods.
It is, however, important to keep the illumination the same for the with- and without-acceptor measurements, as photobleaching increases markedly with more intense incident light.
smFRET is a group of methods using various microscopic techniques to measure a pair of donor and acceptor fluorophores that are excited and detected at the single molecule level.
The variation of the smFRET signal is useful to reveal kinetic information that an ensemble measurement cannot provide, especially when the system is under equilibrium.
Labeling with organic fluorescent dyes requires purification, chemical modification, and intracellular injection of a host protein.
[34] A limitation of FRET performed with fluorophore donors 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.
BRET has also been implemented using a different luciferase enzyme, engineered from the deep-sea shrimp Oplophorus gracilirostris.
[42][44] A split-protein version of NanoLuc developed by Promega[45] has also been used as a BRET donor in experiments measuring protein-protein interactions.
[46] In general, "FRET" refers to situations where the donor and acceptor proteins (or "fluorophores") are of two different types.
[49] 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.
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.
[50] In the field of nano-photonics, FRET can be detrimental if it funnels excitonic energy to defect sites, but it is also essential to charge collection in organic and quantum-dot-sensitized solar cells, and various FRET-enabled strategies have been proposed for different opto-electronic devices.
[51] Fluorescence microscopy study of such single chains demonstrated that energy transfer by FRET between neighbor platelets causes energy to diffuse over a typical 500-nm length (about 80 nano emitters), and the transfer time between platelets is on the order of 1 ps.
[53] The applications of fluorescence resonance energy transfer (FRET) have expanded tremendously in the last 25 years, and the technique has become a staple in many biological and biophysical fields.
FRET can be used as a spectroscopic ruler to measure distance and detect molecular interactions in a number of systems and has applications in biology and biochemistry.
[61] Applied in vivo, FRET has been used to detect the location and interactions of cellular structures including integrins and membrane proteins.
Similarly, FRET systems have been designed to detect changes in the cellular environment due to such factors as pH, hypoxia, or mitochondrial membrane potential.
[67] For example, FRET and BRET have been used in various experiments to characterize G-protein coupled receptor activation and consequent signaling mechanisms.
[68] Other examples include the use of FRET to analyze such diverse processes as bacterial chemotaxis[69] and caspase activity in apoptosis.
In addition to common uses previously mentioned, FRET and BRET are also effective in the study of biochemical reaction kinetics.
[71] FRET is increasingly used for monitoring pH dependent assembly and disassembly and is valuable in the analysis of nucleic acids encapsulation.
[72][73][74][75] This technique can be used to determine factors affecting various types of nanoparticle formation[76][77] as well as the mechanisms and effects of nanomedicines.
