Förster resonance energy transfer#CFP-YFP pairs

Image:Concept of FRET.png

Förster resonance energy transfer is named after the German scientist Theodor Förster.{{cite journal |doi=10.1002/andp.19484370105 |title=Zwischenmolekulare Energiewanderung und Fluoreszenz |trans-title=Intermolecular energy migration and fluorescence |date=1948 |last1=Förster |first1=Theodor | name-list-style = vanc |journal=Annalen der Physik |volume=437 |issue=1–2 |bibcode=1948AnP...437...55F |pages=55–75 |language=de|doi-access=free }} When both chromophores are fluorescent, the term "fluorescence resonance energy transfer" is often used instead, although the energy is not actually transferred by fluorescence.{{cite book |first1=Bernard |last1=Valeur |first2=Mario |last2=Berberan-Santos | name-list-style = vanc |chapter=Excitation Energy Transfer|title=Molecular Fluorescence: Principles and Applications, 2nd ed. |date=2012 |publisher=Wiley-VCH |location=Weinheim |isbn=978-3-527-32837-6 |pages=213–261 |doi=10.1002/9783527650002.ch8}}{{Cite web|url=https://www.olympusfluoview.com/%e3%83%90%e3%83%b3%e3%82%b3%e3%82%af%e3%81%ae%e3%82%b5%e3%83%bc%e3%83%93%e3%82%b9%e3%82%a2%e3%83%91%e3%83%bc%e3%83%88%e3%81%af%e5%9f%ba%e6%9c%ac%e7%9a%84%e3%81%ab%e3%81%af%ef%bc%92%e7%a8%ae%e9%a1%9e/|archiveurl=https://archive.today/20120629220328/http://www.olympusfluoview.com/applications/fretintro.html|url-status=dead|title=バンコクのサービスアパートは基本的には２種類に分かれる|first=タイ不動産マガジン|last=長嶋|date=November 15, 2019|archivedate=June 29, 2012}} In order to avoid an erroneous interpretation of the phenomenon that is always a nonradiative transfer of energy (even when occurring between two fluorescent chromophores), the name "Förster resonance energy transfer" is preferred to "fluorescence resonance energy transfer"; however, the latter enjoys common usage in scientific literature.{{cite book |title=Glossary of Terms Used in Photochemistry |edition=3rd |date=2007 |publisher=IUPAC |page=340}} FRET is not restricted to fluorescence and occurs in connection with phosphorescence as well.

The FRET efficiency ($E$) is the quantum yield of the energy-transfer transition, i.e. the probability of energy-transfer event occurring per donor excitation event:{{cite web |last=Moens |first=Pierre |name-list-style=vanc |title=Fluorescence Resonance Energy Transfer spectroscopy |url=http://www.anatomy.usyd.edu.au/mru/fret/abot.html#frete |access-date=July 14, 2012 |archive-date=July 26, 2016 |archive-url=https://web.archive.org/web/20160726180607/http://www.anatomy.usyd.edu.au/mru/fret/abot.html#frete }}

: $E\; =\; \backslash frac\{k\_\backslash text\{ET\}\}\{k\_f\; +\; k\_\backslash text\{ET\}\; +\; \backslash sum\{k\_i\}\},$

where $k\_f$ the radiative decay rate of the donor, $k\_\backslash text\{ET\}$ is the rate of energy transfer, and $k\_i$ the rates of any other de-excitation pathways excluding energy transfers to other acceptors.{{cite book |title=Molecular Imaging: FRET Microscopy and Spectroscopy |last1=Schaufele |first1=Fred |last2=Demarco |first2=Ignacio |last3=Day |first3=Richard N. | name-list-style = vanc |date=2005 |publisher=Oxford University Press |isbn=978-0-19-517720-6 |editor1-last=Periasamy |editor1-first=Ammasi |location=Oxford |pages=72–94 |chapter=FRET Imaging in the Wide-Field Microscope |doi=10.1016/B978-019517720-6.50013-4 |editor2-last=Day |editor2-first=Richard |chapter-url=https://books.google.com/books?id=K0aawJ6sX-sC&pg=PA72}}{{cite journal | vauthors = Lee S, Lee J, Hohng S | title = Single-molecule three-color FRET with both negligible spectral overlap and long observation time | journal = PLOS ONE | volume = 5 | issue = 8 | pages = e12270 | date = August 2010 | pmid = 20808851 | pmc = 2924373 | doi = 10.1371/journal.pone.0012270 | bibcode = 2010PLoSO...512270L | doi-access = free }}

The FRET efficiency depends on many physical parameters{{cite journal|doi= 10.1103/PhysRevB.85.125106|title=Exactly soluble model of resonant energy transfer between molecules|author=C. King|author2=B. Barbiellini|author3=D. Moser|author4=V. Renugopalakrishnan|name-list-style=amp|date=2012|journal=Physical Review B|volume=85|issue=12|page=125106| arxiv = 1108.0935 |bibcode=2012PhRvB..85l5106K|s2cid=16938353}} that can be grouped as: 1) the distance between the donor and the acceptor (typically in the range of 1–10 nm), 2) the spectral overlap of the donor emission spectrum and the acceptor absorption spectrum, and 3) the relative orientation of the donor emission dipole moment and the acceptor absorption dipole moment.

$E$ depends on the donor-to-acceptor separation distance $r$ with an inverse 6th-power law due to the dipole–dipole coupling mechanism:

: $E\; =\; \backslash frac\{1\}\{1\; +\; (r/R\_0)^6\}$

with $R\_0$ being the Förster distance of this pair of donor and acceptor, i.e. the distance at which the energy transfer efficiency is 50%. The Förster distance depends on the overlap integral of the donor emission spectrum with the acceptor absorption spectrum and their mutual molecular orientation as expressed by the following equation all in SI units:{{cite book | last1 = Förster | first1 = Th. | name-list-style = vanc | title = Modern Quantum Chemistry. Istanbul Lectures. Part III: Action of Light and Organic Crystals | chapter = Delocalized Excitation and Excitation Transfer | volume = 3 | editor1-first = Oktay | editor1-last = Sinanoglu | publisher = Academic Press | date = 1965 | location = New York and London | pages = 93–137 | chapter-url = http://www.quantum-chemistry-history.com/Sina_Dat/BOOKIstaLec/IstaLec1.htm | access-date = 2011-06-22}}{{cite book |last=Clegg |first=Robert | name-list-style = vanc |title=FRET and FLIM Techniques |series=Laboratory Techniques in Biochemistry and Molecular Biology |volume=33 |date=2009 |publisher=Elsevier |isbn=978-0-08-054958-3 |editor1-first=Theodorus W. J. |editor1-last=Gadella |chapter=Förster resonance energy transfer—FRET: what is it, why do it, and how it's done |chapter-url=https://books.google.com/books?id=uHvqu4hLhH8C&pg=PA1 |pages=1–57 |doi=10.1016/S0075-7535(08)00001-6}}{{Cite book |last=Andrews |first=David L. |url=https://spiedigitallibrary.org/ebooks/PM/Tutorials-in-Complex-Photonic-Media/eISBN-9780819480934/10.1117/3.832717 |title=Tutorials in Complex Photonic Media |date=2009-12-29 |publisher=SPIE |isbn=978-0-8194-7773-6 |editor-last=Noginov |editor-first=Mikhail A. |chapter=Resonance Energy Transfer: Theoretical Foundations and Developing Applications |doi=10.1117/3.832717 |editor-last2=Dewar |editor-first2=Graeme |editor-last3=McCall |editor-first3=Martin W. |editor-last4=Zheludev |editor-first4=Nikolay I. |chapter-url=https://spie.org/samples/PM194.pdf}}

: $R\_0^6\; =\; \backslash frac\{9\; \backslash ,\; \backslash log(10)\; \backslash ,\; \backslash kappa^2\; \backslash ,\; Q\_D\; \backslash ,\; J\}\{128\; \backslash ,\; \backslash pi^5\; \backslash ,\; N\_A\; \backslash ,\; n^4\}$

where $Q\_\backslash text\{D\}$ is the fluorescence quantum yield of the donor in the absence of the acceptor, $\backslash kappa^2$ is the dipole orientation factor, $n$ is the refractive index of the medium, $N\_\backslash text\{A\}$ is the Avogadro constant, and $J$ is the spectral overlap integral calculated as

: $J\; =\; \backslash frac\{\backslash int\; f\_\backslash text\{D\}(\backslash lambda)\; \backslash epsilon\_\backslash text\{A\}(\backslash lambda)\; \backslash lambda^4\; \backslash ,\; d\backslash lambda\}\{\backslash int\; f\_\backslash text\{D\}(\backslash lambda)\; \backslash ,\; d\backslash lambda\}\; =\; \backslash int\; \backslash overline\{f\_\backslash text\{D\}\}(\backslash lambda)\; \backslash epsilon\_\backslash text\{A\}(\backslash lambda)\; \backslash lambda^4\; \backslash ,\; d\backslash lambda,$

where $f\_\backslash text\{D\}$ is the donor emission spectrum, $\backslash overline\{f\_\backslash text\{D\}\}$ is the donor emission spectrum normalized to an area of 1, and $\backslash epsilon\_\backslash text\{A\}$ is the acceptor molar extinction coefficient, normally obtained from an absorption spectrum.{{cite book |last=Demchenko |first=Alexander P. | name-list-style = vanc |chapter=Fluorescence Detection Techniques |chapter-url=https://books.google.com/books?id=wMARxPxkE7EC&pg=PA65 |title=Introduction to Fluorescence Sensing |date=2008 |publisher=Springer |location=Dordrecht |isbn=978-1-4020-9002-8 |pages=65–118 |doi=10.1007/978-1-4020-9003-5_3}} The orientation factor {{mvar|κ}} is given by

: $\backslash kappa\; =\; \backslash hat\backslash mu\_\backslash text\{A\}\; \backslash cdot\; \backslash hat\backslash mu\_\backslash text\{D\}\; -\; 3\; (\backslash hat\backslash mu\_\backslash text\{D\}\; \backslash cdot\; \backslash hat\; R)\; (\backslash hat\backslash mu\_\backslash text\{A\}\; \backslash cdot\; \backslash hat\; R),$

where $\backslash hat\backslash mu\_i$ denotes the normalized transition dipole moment of the respective fluorophore, and $\backslash hat\; R$ denotes the normalized inter-fluorophore displacement.{{Cite journal|last=VanDerMeer|first=B. Wieb|date=2020|title=Kappaphobia is the elephant in the fret room|journal=Methods and Applications in Fluorescence|language=en|volume=8|issue=3|page=030401|doi=10.1088/2050-6120/ab8f87|pmid=32362590|bibcode=2020MApFl...8c0401V|issn=2050-6120|doi-access=free}} $\backslash kappa^2$ = 2/3 is often assumed. This value is obtained when both dyes are freely rotating and can be considered to be isotropically oriented during the excited-state lifetime. If either dye is fixed or not free to rotate, then $\backslash kappa^2$ = 2/3 will not be a valid assumption. In most cases, however, even modest reorientation of the dyes results in enough orientational averaging that $\backslash kappa^2$ = 2/3 does not result in a large error in the estimated energy-transfer distance due to the sixth-power dependence of $R\_0$ on $\backslash kappa^2$. Even when $\backslash kappa^2$ is quite different from 2/3, the error can be associated with a shift in $R\_0$, and thus determinations of changes in relative distance for a particular system are still valid. Fluorescent proteins do not reorient on a timescale that is faster than their fluorescence lifetime. In this case 0 ≤ $\backslash kappa^2$ ≤ 4.

The units of the data are usually not in SI units. Using the original units to calculate the Förster distance is often more convenient. For example, the wavelength is often in unit nm and the extinction coefficient is often in unit $M^\{-1\}\; cm^\{-1\}$, where $M$ is concentration $mol/L$. $J$ obtained from these units will have unit $M^\{-1\}\; cm^\{-1\}\; nm^4$. To use unit Å ($10^\{-10\}m$) for the $R\_0$, the equation is adjusted to{{Cite web|url=https://www.fpbase.org/fret/|title=FPbase FRET Calculator|first=Talley|last=Lambert|website=FPbase}}{{Cite journal |vauthors=Chan YH, Chen J, Wark SE, Skiles SL, Son DH, Batteas JD |date=2009|title=Using patterned arrays of metal nanoparticles to probe plasmon enhanced luminescence of CdSe quantum dots (supporting information)|journal=ACS Nano|language=en|volume=3|issue=7|pages=1735–1744|doi=10.1021/nn900317n|pmid=19499906|url = https://pubs.acs.org/doi/10.1021/nn900317n|url-access=subscription}}{{Cite journal |vauthors=Wu PG, Brand L |date=1994|title=Resonance Energy Transfer: Methods and Applications|journal=Analytical Biochemistry|language=en|volume=218|issue=1|pages=1–13|doi=10.1006/abio.1994.1134|pmid=8053542 |doi-access=free}}

: $\{R\_0\}^6\; =\; 8.785\; \backslash times\; 10^\{-5\}\; \backslash frac\{\backslash kappa^2\; \backslash ,Q\_D\}\{n^4\}\; J$ (Å$^6$)

For time-dependent analyses of FRET, the rate of energy transfer ($k\_\backslash text\{ET\}$) can be used directly instead:

: $k\_\backslash text\{ET\}\; =\; \backslash left(\backslash frac\{R\_0\}\{r\}\backslash right)^6\; \backslash ,\; \backslash frac\{1\}\{\backslash tau\_D\}$

where $\backslash tau\_D$ is the donor's fluorescence lifetime in the absence of the acceptor.

The FRET efficiency relates to the quantum yield and the fluorescence lifetime of the donor molecule as follows:{{cite book |first1=Irina |last1=Majoul |first2=Yiwei |last2=Jia |first3=Rainer |last3=Duden |title=Handbook of Biological Confocal Microscopy | name-list-style = vanc |chapter=Practical Fluorescence Resonance Energy Transfer or Molecular Nanobioscopy of Living Cells |pages=[https://archive.org/details/handbookbiologic00pawl/page/n813 788]–808 |editor-last=Pawley |editor1-first=James B. |url=https://archive.org/details/handbookbiologic00pawl |url-access=limited |date=2006 |publisher=Springer |location=New York, NY |isbn=978-0-387-25921-5 |edition=3rd |doi=10.1007/978-0-387-45524-2_45}}

: $E\; =\; 1\; -\; \backslash tau\text{'}\_\backslash text\{D\}/\backslash tau\_\backslash text\{D\},$

where $\backslash tau\_\backslash text\{D\}\text{'}$ and $\backslash tau\_\backslash text\{D\}$ are the donor fluorescence lifetimes in the presence and absence of an acceptor respectively, or as

: $E\; =\; 1\; -\; F\_\backslash text\{D\}\text{'}/F\_\backslash text\{D\},$

where $F\_\backslash text\{D\}\text{'}$ and $F\_\backslash text\{D\}$ are the donor fluorescence intensities with and without an acceptor respectively.

The inverse sixth-power distance dependence of Förster resonance energy transfer was experimentally confirmed by Wilchek, Edelhoch and Brand{{cite journal | vauthors = Edelhoch H, Brand L, Wilchek M | title = Fluorescence studies with tryptophyl peptides | journal = Biochemistry | volume = 6 | issue = 2 | pages = 547–59 | date = February 1967 | pmid = 6047638 | doi = 10.1021/bi00854a024 }} using tryptophyl peptides. Stryer, Haugland and Yguerabide{{cite book |editor1-last=Lakowicz |editor1-first=Joseph R. | name-list-style = vanc |title=Principles |date=1991 |publisher=Plenum Press |location=New York |isbn=978-0-306-43875-2 |page=172 }}{{citation needed|date=August 2019}} 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. Calculations on FRET distances of some example dye-pairs can be found here. 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.{{cite book | vauthors = Vekshin NL | chapter = Energy Transfer in Macromolecules, SPIE | date = 1997 | veditors = Vekshin NL | title = Photonics of Biopolymers | publisher = Springer }}

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, DNA-DNA interactions,{{cite journal |last1=Kowalski |first1=Adam |title=Effective screening of Coulomb repulsions in water accelerates reactions of like-charged compounds by orders of magnitude |journal=Nature Communications |date=2022 |volume=13 |issue=1 |page=6451 |doi=10.1038/s41467-022-34182-z |pmid=36307412 |pmc=9616817 |url=https://doi.org/10.1038/s41467-022-34182-z}} and protein conformational changes. For monitoring the complex formation between two molecules, one of them is labeled with a donor and the other with an acceptor. 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.{{cite web|title=Fluorescence Resonance Energy Transfer Protocol |url=http://coil.bio.ed.ac.uk/Protocols/FRET.htm |publisher=Wellcome Trust |access-date=24 June 2012 |archive-url=https://web.archive.org/web/20130717083912/http://coil.bio.ed.ac.uk/Protocols/FRET.htm |archive-date=July 17, 2013 }}

One method of measuring FRET efficiency is to measure the variation in acceptor emission intensity. 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. For monitoring protein conformational changes, the target protein is labeled with a donor and an acceptor at two loci. 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.

FRET efficiencies can also be inferred from the photobleaching rates of the donor in the presence and absence of an acceptor. This method can be performed on most fluorescence microscopes; one simply shines the excitation light (of a frequency that will excite the donor but not the acceptor significantly) on specimens with and without the acceptor fluorophore and monitors the donor fluorescence (typically separated from acceptor fluorescence using a bandpass filter) over time. The timescale is that of photobleaching, which is seconds to minutes, with fluorescence in each curve being given by

:$\backslash text\{background\}\; +\; \backslash text\{constant\}\; \backslash cdot\; e^\{-\backslash text\{time\}/\backslash tau\_\backslash text\{pb\}\},$

where $\backslash tau\_\backslash text\{pb\}$ is the photobleaching decay time constant and depends on whether the acceptor is present or not. 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:

:$E\; =\; 1\; -\; \backslash tau\_\backslash text\{pb\}/\backslash tau\_\backslash text\{pb\}\text{'},$

where $\backslash tau\_\backslash text\{pb\}\text{'}$ and $\backslash tau\_\backslash text\{pb\}$ are the photobleaching decay time constants of the donor in the presence and in the absence of the acceptor respectively. (Notice that the fraction is the reciprocal of that used for lifetime measurements).

This technique was introduced by Jovin in 1989.{{cite book |first1=János |last1=Szöllősi |first2=Denis R. |last2=Alexander |chapter=The Application of Fluorescence Resonance Energy Transfer to the Investigation of Phosphatases |pages=203–24 |editor1-first=Susanne |editor1-last=Klumpp |editor2-first=Josef |editor2-last=Krieglstein | name-list-style = vanc |title=Protein Phosphatases |volume=366 |series=Methods in Enzymology |date=2007 |publisher=Elsevier |location=Amsterdam |isbn=978-0-12-182269-9 |doi=10.1016/S0076-6879(03)66017-9|pmid=14674251 }} Its use of an entire curve of points to extract the time constants can give it accuracy advantages over the other methods. Also, the fact that time measurements are over seconds rather than nanoseconds makes it easier than fluorescence lifetime measurements, and because photobleaching decay rates do not generally depend on donor concentration (unless acceptor saturation is an issue), the careful control of concentrations needed for intensity measurements is not needed. 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.

FRET efficiency can also be determined from the change in the fluorescence lifetime of the donor. The lifetime of the donor will decrease in the presence of the acceptor. Lifetime measurements of the FRET-donor are used in fluorescence-lifetime imaging microscopy (FLIM).

{{Main article|Single-molecule FRET}}

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. In contrast to "ensemble FRET" or "bulk FRET" which provides the FRET signal of a high number of molecules, single-molecule FRET is able to resolve the FRET signal of each individual molecule. 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. Heterogeneity among different molecules can also be observed. This method has been applied in many measurements of biomolecular dynamics such as DNA/RNA/protein folding/unfolding and other conformational changes, and intermolecular dynamics such as reaction, binding, adsorption, and desorption that are particularly useful in chemical sensing, bioassays, and biosensing.

File:Proteolytic cleavage of a Dual-GFP fusion FRET-pair.png

One common pair fluorophores for biological use is a cyan fluorescent protein (CFP) – yellow fluorescent protein (YFP) pair.{{cite journal | vauthors = Periasamy A | title = Fluorescence resonance energy transfer microscopy: a mini review | journal = Journal of Biomedical Optics | volume = 6 | issue = 3 | pages = 287–91 | date = July 2001 | pmid = 11516318 | doi = 10.1117/1.1383063 | url = https://pdfs.semanticscholar.org/3b4d/4d92c44059eabee448451f83bc7ac71f9630.pdf | archive-url = https://web.archive.org/web/20200210040345/https://pdfs.semanticscholar.org/3b4d/4d92c44059eabee448451f83bc7ac71f9630.pdf | archive-date = 2020-02-10 | s2cid = 39759478 | bibcode = 2001JBO.....6..287P }} Both are color variants of green fluorescent protein (GFP). Labeling with organic fluorescent dyes requires purification, chemical modification, and intracellular injection of a host protein. GFP variants can be attached to a host protein by genetic engineering which can be more convenient. Additionally, a fusion of CFP and YFP ("tandem-dimer") linked by a protease cleavage sequence can be used as a cleavage assay.{{cite journal | vauthors = Nguyen AW, Daugherty PS | title = Evolutionary optimization of fluorescent proteins for intracellular FRET | journal = Nature Biotechnology | volume = 23 | issue = 3 | pages = 355–60 | date = March 2005 | pmid = 15696158 | doi = 10.1038/nbt1066 | s2cid = 24202205 }}

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. To avoid this drawback, bioluminescence resonance energy transfer (or BRET) has been developed.{{cite book |first1=Nicola |last1=Bevan |first2=Stephen |last2=Rees | name-list-style = vanc |chapter=Pharmaceutical Applications of GFP and RCFP |chapter-url=https://books.google.com/books?id=v8Y4zrEofpIC&pg=PA361 |pages=361–90 |editor1-first=Martin |editor1-last=Chalfie |editor2-first=Steven R. |editor2-last=Kain |series=Methods of Biochemical Analysis |volume=47 |title=Green Fluorescent Protein: Properties, Applications and Protocols |date=2006 |publisher=John Wiley & Sons |location=Hoboken, NJ |isbn=978-0-471-73682-0 |edition=2nd |doi=10.1002/0471739499.ch16|pmid=16335721 }}{{cite journal | vauthors = Pfleger KD, Eidne KA | title = Illuminating insights into protein-protein interactions using bioluminescence resonance energy transfer (BRET) | journal = Nature Methods | volume = 3 | issue = 3 | pages = 165–74 | date = March 2006 | pmid = 16489332 | doi = 10.1038/nmeth841 | s2cid = 9759741 }} This technique uses a bioluminescent luciferase (typically the luciferase from Renilla reniformis) rather than CFP to produce an initial photon emission compatible with YFP.

BRET has also been implemented using a different luciferase enzyme, engineered from the deep-sea shrimp Oplophorus gracilirostris. This luciferase is smaller (19 kD) and brighter than the more commonly used luciferase from Renilla reniformis,{{cite journal | vauthors = Mo XL, Luo Y, Ivanov AA, Su R, Havel JJ, Li Z, Khuri FR, Du Y, Fu H | display-authors = 6 | title = Enabling systematic interrogation of protein-protein interactions in live cells with a versatile ultra-high-throughput biosensor platform | journal = Journal of Molecular Cell Biology | volume = 8 | issue = 3 | pages = 271–81 | date = June 2016 | pmid = 26578655 | pmc = 4937889 | doi = 10.1093/jmcb/mjv064 }}{{cite journal | vauthors = Robers MB, Dart ML, Woodroofe CC, Zimprich CA, Kirkland TA, Machleidt T, Kupcho KR, Levin S, Hartnett JR, Zimmerman K, Niles AL, Ohana RF, Daniels DL, Slater M, Wood MG, Cong M, Cheng YQ, Wood KV | display-authors = 6 | title = Target engagement and drug residence time can be observed in living cells with BRET | journal = Nature Communications | volume = 6 | page = 10091 | date = December 2015 | pmid = 26631872 | pmc = 4686764 | doi = 10.1038/ncomms10091 | bibcode = 2015NatCo...610091R }}{{cite journal | vauthors = Stoddart LA, Johnstone EK, Wheal AJ, Goulding J, Robers MB, Machleidt T, Wood KV, Hill SJ, Pfleger KD | display-authors = 6 | title = Application of BRET to monitor ligand binding to GPCRs | journal = Nature Methods | volume = 12 | issue = 7 | pages = 661–663 | date = July 2015 | pmid = 26030448 | pmc = 4488387 | doi = 10.1038/nmeth.3398 }}{{cite journal | vauthors = Machleidt T, Woodroofe CC, Schwinn MK, Méndez J, Robers MB, Zimmerman K, Otto P, Daniels DL, Kirkland TA, Wood KV | display-authors = 6 | title = NanoBRET--A Novel BRET Platform for the Analysis of Protein-Protein Interactions | journal = ACS Chemical Biology | volume = 10 | issue = 8 | pages = 1797–804 | date = August 2015 | pmid = 26006698 | doi = 10.1021/acschembio.5b00143 | doi-access = free }} and has been named NanoLuc{{cite journal | vauthors = Hall MP, Unch J, Binkowski BF, Valley MP, Butler BL, Wood MG, Otto P, Zimmerman K, Vidugiris G, Machleidt T, Robers MB, Benink HA, Eggers CT, Slater MR, Meisenheimer PL, Klaubert DH, Fan F, Encell LP, Wood KV | display-authors = 6 | title = Engineered luciferase reporter from a deep sea shrimp utilizing a novel imidazopyrazinone substrate | journal = ACS Chemical Biology | volume = 7 | issue = 11 | pages = 1848–57 | date = November 2012 | pmid = 22894855 | pmc = 3501149 | doi = 10.1021/cb3002478 }} or NanoKAZ.{{cite journal | vauthors = Inouye S, Sato J, Sahara-Miura Y, Yoshida S, Kurakata H, Hosoya T | title = C6-Deoxy coelenterazine analogues as an efficient substrate for glow luminescence reaction of nanoKAZ: the mutated catalytic 19 kDa component of Oplophorus luciferase | journal = Biochemical and Biophysical Research Communications | volume = 437 | issue = 1 | pages = 23–8 | date = July 2013 | pmid = 23792095 | doi = 10.1016/j.bbrc.2013.06.026 }} Promega has developed a patented substrate for NanoLuc called furimazine,{{Cite web|url=https://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/nanoluc-luciferase-redefining-reporter-assays/|title=NanoLuc product page|access-date=2016-10-25|archive-date=2016-12-25|archive-url=https://web.archive.org/web/20161225070250/http://www.promega.com/products/reporter-assays-and-transfection/reporter-assays/nanoluc-luciferase-redefining-reporter-assays/}} though other valuables coelenterazine substrates for NanoLuc have also been published.{{cite journal | vauthors = Coutant EP, Gagnot G, Hervin V, Baatallah R, Goyard S, Jacob Y, Rose T, Janin YL | display-authors = 6 | title = Bioluminescence Profiling of NanoKAZ/NanoLuc Luciferase Using a Chemical Library of Coelenterazine Analogues | journal = Chemistry: A European Journal | volume = 26 | issue = 4 | pages = 948–958 | date = January 2020 | pmid = 31765054 | doi = 10.1002/chem.201904844 | s2cid = 208276133 | url = https://hal-pasteur.archives-ouvertes.fr/pasteur-02988525/file/Coutant_et-al_Chemistry2020-26.pdf }} A split-protein version of NanoLuc developed by Promega{{cite journal | vauthors = Dixon AS, Schwinn MK, Hall MP, Zimmerman K, Otto P, Lubben TH, Butler BL, Binkowski BF, Machleidt T, Kirkland TA, Wood MG, Eggers CT, Encell LP, Wood KV | display-authors = 6 | title = NanoLuc Complementation Reporter Optimized for Accurate Measurement of Protein Interactions in Cells | journal = ACS Chemical Biology | volume = 11 | issue = 2 | pages = 400–8 | date = February 2016 | pmid = 26569370 | doi = 10.1021/acschembio.5b00753 | doi-access = free }} has also been used as a BRET donor in experiments measuring protein-protein interactions.{{cite journal | vauthors = Hoare BL, Kocan M, Bruell S, Scott DJ, Bathgate RA | title = Using the novel HiBiT tag to label cell surface relaxin receptors for BRET proximity analysis | journal = Pharmacology Research & Perspectives | volume = 7 | issue = 4 | pages = e00513 | date = August 2019 | pmid = 31384473 | pmc = 6667744 | doi = 10.1002/prp2.513 }}

In general, "FRET" refers to situations where the donor and acceptor proteins (or "fluorophores") are of two different types. In many biological situations, however, researchers might need to examine the interactions between two, or more, proteins of the same type—or indeed the same protein with itself, for example if the protein folds or forms part of a polymer chain of proteins{{cite journal | vauthors = Gautier I, Tramier M, Durieux C, Coppey J, Pansu RB, Nicolas JC, Kemnitz K, Coppey-Moisan M | display-authors = 6 | title = Homo-FRET microscopy in living cells to measure monomer-dimer transition of GFP-tagged proteins | journal = Biophysical Journal | volume = 80 | issue = 6 | pages = 3000–8 | date = June 2001 | pmid = 11371472 | pmc = 1301483 | doi = 10.1016/S0006-3495(01)76265-0 | bibcode = 2001BpJ....80.3000G }} or for other questions of quantification in biological cells{{cite journal | vauthors = Bader AN, Hofman EG, Voortman J, en Henegouwen PM, Gerritsen HC | title = Homo-FRET imaging enables quantification of protein cluster sizes with subcellular resolution | journal = Biophysical Journal | volume = 97 | issue = 9 | pages = 2613–22 | date = November 2009 | pmid = 19883605 | pmc = 2770629 | doi = 10.1016/j.bpj.2009.07.059 | bibcode = 2009BpJ....97.2613B }} or in vitro experiments.{{cite journal |last1=Heckmeier |first1=Philipp J. |last2=Agam |first2=Ganesh |last3=Teese |first3=Mark G. |last4=Hoyer |first4=Maria |last5=Stehle |first5=Ralf |last6=Lamb |first6=Don C. |last7=Langosch |first7=Dieter |title=Determining the Stoichiometry of Small Protein Oligomers Using Steady-State Fluorescence Anisotropy |journal=Biophysical Journal |date=July 2020 |volume=119 |issue=1 |pages=99–114 |doi=10.1016/j.bpj.2020.05.025|doi-access=free |pmid=32553128 |pmc=7335908 |bibcode=2020BpJ...119...99H }}

Obviously, differences in conventional UV-vis spectra will not be the tool used to detect and measure homogeneous FRET, as both the acceptor and donor emit light with the same wavelengths. However, there is evidence that certain nonlinear spectroscopies may provide signatures of homogeneous FRET that would be invisible in linear spectra. {{cite journal | vauthors = Petkov BK, Gellen TA, Farfan CA, Carbery WP, Hetzler BE, Trauner D, Li X, Glover WJ, Ulness DJ, Turner DB | title = Two-Dimensional Electronic Spectroscopy Reveals the Spectral Dynamics of Förster Resonance Energy Transfer | journal = Cell Chem | volume = 5 | issue = 8 | pages = 2111-2125 | date = August 2019 | doi = 10.1016/j.chempr.2019.05.005 }} 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.{{cite journal | vauthors = Gradinaru CC, Marushchak DO, Samim M, Krull UJ | title = Fluorescence anisotropy: from single molecules to live cells | journal = The Analyst | volume = 135 | issue = 3 | pages = 452–9 | date = March 2010 | pmid = 20174695 | doi = 10.1039/b920242k | url = https://zenodo.org/record/896379 | bibcode = 2010Ana...135..452G }}

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. It is then essential to understand how isolated nano-emitters behave when they are stacked in a dense layer. Nanoplatelets are especially promising candidates for strong homo-FRET exciton diffusion because of their strong in-plane dipole coupling and low Stokes shift.{{cite journal |last1=Liu |first1=Jiawen |last2=Guillemeney |first2=Lilian |last3=Choux |first3=Arnaud |last4=Maître |first4=Agnès |last5=Abécassis |first5=Benjamin |last6=Coolen |first6=Laurent |title=Fourier-Imaging of Single Self-Assembled CdSe Nanoplatelet Chains and Clusters Reveals out-of-Plane Dipole Contribution |journal=ACS Photonics |date=21 October 2020 |volume=7 |issue=10 |pages=2825–2833 |doi=10.1021/acsphotonics.0c01066|arxiv=2008.07610 |s2cid=221150436 }} 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.{{cite journal |last1=Liu |first1=Jiawen |title=Long Range Energy Transfer in Self-Assembled Stacks of Semiconducting Nanoplatelets |journal=Nano Letters |date=April 21, 2020 |volume=20 |issue=5 |pages=3465–3470 |doi=10.1021/acs.nanolett.0c00376|pmid=32315197 |bibcode=2020NanoL..20.3465L |s2cid=216075288 |url=https://hal.archives-ouvertes.fr/hal-02565031/file/Article%20FRET%20revised%20v1%20SOUMIS%20LE%2017%20AVRIL.pdf }}

Various compounds beside fluorescent proteins.{{cite journal | vauthors = Wu P, Brand L | title = Resonance energy transfer: methods and applications | journal = Analytical Biochemistry | volume = 218 | issue = 1 | pages = 1–13 | date = April 1994 | pmid = 8053542 | doi = 10.1006/abio.1994.1134 | doi-access = free }}

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.{{cite book|last=Lakowicz|first=Joseph R.| name-list-style = vanc |title=Principles of fluorescence spectroscopy|url=https://archive.org/details/principlesfluore00lako|url-access=limited|date=1999|publisher=Kluwer Acad./Plenum Publ.|location=New York, NY |isbn=978-0-306-46093-7|pages=[https://archive.org/details/principlesfluore00lako/page/n397 374]–443|edition=2nd}}{{Cite journal|last1=Szabó|first1=Ágnes|last2=Szendi-Szatmári|first2=Tímea|last3=Szöllősi|first3=János|last4=Nagy|first4=Peter|date=2020-07-07|title=Quo vadis FRET? Förster's method in the era of superresolution|journal=Methods and Applications in Fluorescence|volume=8|issue=3|page=032003|doi=10.1088/2050-6120/ab9b72|pmid=32521530|bibcode=2020MApFl...8c2003S|s2cid=219588720|issn=2050-6120}}

FRET is often used to detect and track interactions between proteins.{{cite journal | vauthors = Pollok BA, Heim R | title = Using GFP in FRET-based applications | journal = Trends in Cell Biology | volume = 9 | issue = 2 | pages = 57–60 | date = February 1999 | pmid = 10087619 | doi = 10.1016/S0962-8924(98)01434-2 }}{{cite journal | vauthors = Shi Y, Stouten PF, Pillalamarri N, Barile L, Rosal RV, Teichberg S, Bu Z, Callaway DJ | display-authors = 6 | title = Quantitative determination of the topological propensities of amyloidogenic peptides | journal = Biophysical Chemistry | volume = 120 | issue = 1 | pages = 55–61 | date = March 2006 | pmid = 16288953 | doi = 10.1016/j.bpc.2005.09.015 }}{{cite journal | vauthors = Matsumoto S, Hammes GG | title = Fluorescence energy transfer between ligand binding sites on aspartate transcarbamylase | journal = Biochemistry | volume = 14 | issue = 2 | pages = 214–24 | date = January 1975 | pmid = 1091284 | doi = 10.1021/bi00673a004 }}{{cite journal | vauthors = Martin SF, Tatham MH, Hay RT, Samuel ID | title = Quantitative analysis of multi-protein interactions using FRET: application to the SUMO pathway | journal = Protein Science | volume = 17 | issue = 4 | pages = 777–84 | date = April 2008 | pmid = 18359863 | pmc = 2271167 | doi = 10.1110/ps.073369608 }} Additionally, FRET can be used to measure distances between domains in a single protein by tagging different regions of the protein with fluorophores and measuring emission to determine distance. This provides information about protein conformation, including secondary structures and protein folding.{{cite journal | vauthors = Truong K, Ikura M | title = The use of FRET imaging microscopy to detect protein-protein interactions and protein conformational changes in vivo | journal = Current Opinion in Structural Biology | volume = 11 | issue = 5 | pages = 573–8 | date = October 2001 | pmid = 11785758 | doi = 10.1016/S0959-440X(00)00249-9 }}{{cite journal | vauthors = Chan FK, Siegel RM, Zacharias D, Swofford R, Holmes KL, Tsien RY, Lenardo MJ | title = Fluorescence resonance energy transfer analysis of cell surface receptor interactions and signaling using spectral variants of the green fluorescent protein | journal = Cytometry | volume = 44 | issue = 4 | pages = 361–8 | date = August 2001 | pmid = 11500853 | doi = 10.1002/1097-0320(20010801)44:4<361::AID-CYTO1128>3.0.CO;2-3 | doi-access = free }} This extends to tracking functional changes in protein structure, such as conformational changes associated with myosin activity.{{cite journal | vauthors = Shih WM, Gryczynski Z, Lakowicz JR, Spudich JA | title = A FRET-based sensor reveals large ATP hydrolysis-induced conformational changes and three distinct states of the molecular motor myosin | journal = Cell | volume = 102 | issue = 5 | pages = 683–94 | date = September 2000 | pmid = 11007486 | doi = 10.1016/S0092-8674(00)00090-8 | doi-access = free }} Applied in vivo, FRET has been used to detect the location and interactions of cellular structures including integrins and membrane proteins.{{cite journal | vauthors = Sekar RB, Periasamy A | title = Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein localizations | journal = The Journal of Cell Biology | volume = 160 | issue = 5 | pages = 629–33 | date = March 2003 | pmid = 12615908 | pmc = 2173363 | doi = 10.1083/jcb.200210140 }}

FRET can be used to observe membrane fluidity, movement and dispersal of membrane proteins, membrane lipid-protein and protein-protein interactions, and successful mixing of different membranes.{{cite journal | vauthors = Loura LM, Prieto M | title = FRET in Membrane Biophysics: An Overview | journal = Frontiers in Physiology | volume = 2 | page = 82 | date = 2011-11-15 | pmid = 22110442 | pmc = 3216123 | doi = 10.3389/fphys.2011.00082 | doi-access = free }} FRET is also used to study formation and properties of membrane domains and lipid rafts in cell membranes{{cite journal | vauthors = Silvius JR, Nabi IR | title = Fluorescence-quenching and resonance energy transfer studies of lipid microdomains in model and biological membranes | journal = Molecular Membrane Biology | volume = 23 | issue = 1 | pages = 5–16 | date = 2006 | pmid = 16611577 | doi = 10.1080/09687860500473002 | s2cid = 34651742 | doi-access = free }} and to determine surface density in membranes.{{cite journal | vauthors = Fung BK, Stryer L | title = Surface density determination in membranes by fluorescence energy transfer | journal = Biochemistry | volume = 17 | issue = 24 | pages = 5241–8 | date = November 1978 | pmid = 728398 | doi = 10.1021/bi00617a025 }}

File:FRET probe for the detection of Cd2+.gif

FRET-based probes can detect the presence of various molecules: the probe's structure is affected by small molecule binding or activity, which can turn the FRET system on or off. This is often used to detect anions, cations, small uncharged molecules, and some larger biomacromolecules as well. Similarly, FRET systems have been designed to detect changes in the cellular environment due to such factors as pH, hypoxia, or mitochondrial membrane potential.{{cite journal | vauthors = Wu L, Huang C, Emery BP, Sedgwick AC, Bull SD, He XP, Tian H, Yoon J, Sessler JL, James TD | display-authors = 6 | title = Förster resonance energy transfer (FRET)-based small-molecule sensors and imaging agents | journal = Chemical Society Reviews | volume = 49 | issue = 15 | pages = 5110–5139 | date = August 2020 | pmid = 32697225 | pmc = 7408345 | doi = 10.1039/C9CS00318E | url = http://xlink.rsc.org/?DOI=C9CS00318E }}

Another use for FRET is in the study of metabolic or signaling pathways.{{cite book | vauthors = Ni Q, Zhang J | title = Nano/Micro Biotechnology | chapter = Dynamic visualization of cellular signaling | journal = Advances in Biochemical Engineering/Biotechnology | pages = 79–97 | date = 2010 | volume = 119 | pmid = 19499207 | doi = 10.1007/10_2008_48 | publisher = Springer | bibcode = 2010nmb..book...79N | isbn = 978-3-642-14946-7 | veditors = Endo I, Nagamune T | chapter-url = https://books.google.com/books?id=qrGsL_wYdHMC&pg=PA79 }} For example, FRET and BRET have been used in various experiments to characterize G-protein coupled receptor activation and consequent signaling mechanisms.{{cite journal | vauthors = Lohse MJ, Nuber S, Hoffmann C | title = Fluorescence/bioluminescence resonance energy transfer techniques to study G-protein-coupled receptor activation and signaling | journal = Pharmacological Reviews | volume = 64 | issue = 2 | pages = 299–336 | date = April 2012 | pmid = 22407612 | doi = 10.1124/pr.110.004309 | s2cid = 2042851 }} Other examples include the use of FRET to analyze such diverse processes as bacterial chemotaxis{{cite book | vauthors = Sourjik V, Vaknin A, Shimizu TS, Berg HC | title = Two-Component Signaling Systems, Part B | chapter = In vivo measurement by FRET of pathway activity in bacterial chemotaxis | series = Methods in Enzymology | volume = 423 | pages = 365–91 | date = 2007-01-01 | pmid = 17609141 | doi = 10.1016/S0076-6879(07)23017-4 | publisher = Academic Press | isbn = 978-0-12-373852-3 | veditors = Simon MI, Crane BR, Crane A }} and caspase activity in apoptosis.{{cite journal | vauthors = Wu Y, Xing D, Luo S, Tang Y, Chen Q | title = Detection of caspase-3 activation in single cells by fluorescence resonance energy transfer during photodynamic therapy induced apoptosis | journal = Cancer Letters | volume = 235 | issue = 2 | pages = 239–47 | date = April 2006 | pmid = 15958279 | doi = 10.1016/j.canlet.2005.04.036 }}

Proteins, DNAs, RNAs, and other polymer folding dynamics have been measured using FRET. Usually, these systems are under equilibrium whose kinetics is hidden. However, they can be measured by measuring single-molecule FRET with proper placement of the acceptor and donor dyes on the molecules. See single-molecule FRET for a more detailed description.

In addition to common uses previously mentioned, FRET and BRET are also effective in the study of biochemical reaction kinetics.{{cite journal | vauthors = Liu Y, Liao J | title = Quantitative FRET (Förster Resonance Energy Transfer) analysis for SENP1 protease kinetics determination | journal = Journal of Visualized Experiments | issue = 72 | pages = e4430 | date = February 2013 | pmid = 23463095 | pmc = 3605757 | doi = 10.3791/4430 }} FRET is increasingly used for monitoring pH dependent assembly and disassembly and is valuable in the analysis of nucleic acids encapsulation.{{cite journal | vauthors = Sapkota K, Kaur A, Megalathan A, Donkoh-Moore C, Dhakal S | title = Single-Step FRET-Based Detection of Femtomoles DNA | journal = Sensors | volume = 19 | issue = 16 | page = 3495 | date = August 2019 | pmid = 31405068 | pmc = 6719117 | doi = 10.3390/s19163495 | bibcode = 2019Senso..19.3495S | doi-access = free }}{{cite journal | vauthors = Lu KY, Lin CW, Hsu CH, Ho YC, Chuang EY, Sung HW, Mi FL | title = FRET-based dual-emission and pH-responsive nanocarriers for enhanced delivery of protein across intestinal epithelial cell barrier | journal = ACS Applied Materials & Interfaces | volume = 6 | issue = 20 | pages = 18275–89 | date = October 2014 | pmid = 25260022 | doi = 10.1021/am505441p }}{{cite journal | vauthors = Yang L, Cui C, Wang L, Lei J, Zhang J | title = Dual-Shell Fluorescent Nanoparticles for Self-Monitoring of pH-Responsive Molecule-Releasing in a Visualized Way | journal = ACS Applied Materials & Interfaces | volume = 8 | issue = 29 | pages = 19084–91 | date = July 2016 | pmid = 27377369 | doi = 10.1021/acsami.6b05872 }}{{cite journal | vauthors = Heitz M, Zamolo S, Javor S, Reymond JL | title = Fluorescent Peptide Dendrimers for siRNA Transfection: Tracking pH Responsive Aggregation, siRNA Binding, and Cell Penetration | journal = Bioconjugate Chemistry | volume = 31 | issue = 6 | pages = 1671–1684 | date = June 2020 | pmid = 32421327 | doi = 10.1021/acs.bioconjchem.0c00231 | s2cid = 218689921 | url = https://boris.unibe.ch/148853/1/acs.bioconjchem.0c00231.pdf }} This technique can be used to determine factors affecting various types of nanoparticle formation{{cite journal | vauthors = Sanchez-Gaytan BL, Fay F, Hak S, Alaarg A, Fayad ZA, Pérez-Medina C, Mulder WJ, Zhao Y | title = Real-Time Monitoring of Nanoparticle Formation by FRET Imaging | journal = Angewandte Chemie International Edition in English | volume = 56 | issue = 11 | pages = 2923–2926 | date = March 2017 | pmid = 28112478 | pmc = 5589959 | doi = 10.1002/anie.201611288 }}{{cite journal | vauthors = Alabi CA, Love KT, Sahay G, Stutzman T, Young WT, Langer R, Anderson DG | title = FRET-labeled siRNA probes for tracking assembly and disassembly of siRNA nanocomplexes | journal = ACS Nano | volume = 6 | issue = 7 | pages = 6133–41 | date = July 2012 | pmid = 22693946 | pmc = 3404193 | doi = 10.1021/nn3013838 }} as well as the mechanisms and effects of nanomedicines.{{cite journal | vauthors = Chen T, He B, Tao J, He Y, Deng H, Wang X, Zheng Y | title = Application of Förster Resonance Energy Transfer (FRET) technique to elucidate intracellular and In Vivo biofate of nanomedicines | journal = Advanced Drug Delivery Reviews | volume = 143 | pages = 177–205 | date = March 2019 | pmid = 31201837 | doi = 10.1016/j.addr.2019.04.009 | series = Unraveling the In Vivo Fate and Cellular Pharmacokinetics of Drug Nanocarriers | s2cid = 189898459 }}

A different, but related, mechanism is Dexter electron transfer.

An alternative method to detecting protein–protein proximity is the bimolecular fluorescence complementation (BiFC), where two parts of a fluorescent protein are each fused to other proteins. When these two parts meet, they form a fluorophore on a timescale of minutes or hours.{{cite journal | vauthors = Hu CD, Chinenov Y, Kerppola TK | title = Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation | journal = Molecular Cell | volume = 9 | issue = 4 | pages = 789–98 | date = April 2002 | pmid = 11983170 | doi = 10.1016/S1097-2765(02)00496-3 | doi-access = free }}

• Dexter electron transfer
• Förster coupling
• Surface energy transfer
• Time-resolved fluorescence energy transfer

{{reflist|30em}}

• {{YouTube|id=3bmb_oDl6ws|title=FRET effect in a thin film}}
• [https://www.becker-hickl.com/applications/fret-imaging/ FRET Imaging] (Tutorial of Becker & Hickl, website)

{{DEFAULTSORT:Forster Resonance Energy Transfer}}

Category:Imaging

Category:Fluorescence

Category:Biochemistry methods

Category:Biophysics

Category:Cell imaging

Category:Optical phenomena

Category:Protein–protein interaction assays

Category:Fluorescence techniques

Category:Cell biology

Category:Laboratory techniques

Category:Molecular biology techniques

Category:Energy transfer