This section on FRET is an excerpt from George McNamara's Multi-Probe Microscopy document, available from his web site. Dr. McNamara has given permission for FRETImaging.org to display and edit the contents.

Author: George McNamara, Ph.D.
Imaging Scientist
Address: Childrens Hospital Los Angeles
CHLA Research Institute - Image Core Cellular Imaging Facility
Room 1016 Smith Research Tower
4650 Sunset Blvd., MS 84
Los Angeles 90027
Contact: gmcnamara@chla.usc.edu
geomcnamara@earthlink.net
Internet: Personal Web Site

© George McNamara 1997-2003

Fluorescence Resonance Energy Transfer (FRET) Imaging

FRET is used in microscopy to measure how close two fluorophores are together. Resonance energy transfer is a mechanism by which energy is transferred directly from one molecule to another. This only occurs over a very small distance, usually less than 10 nm, which is on the order of the size of a typical protein. Lubert Stryer, of biochemistry textbook fame, pioneered the use of FRET as a "spectroscopic ruler" (Stryer and Haugland, 1967; Stryer 1978). Roger Tsien, Atsucshi Miyawaki, Richard (Dick) Pagano, Keller, Robert Blumenthal, Donna Arndt-Jovin and Thomas Jovin, Brian Herman, Ammasi Periasamy, Richard Day, Joel Swanson and many others have used FRET microscopy in many interesting ways. See the discussion of BFP-GFP FRET in the GFP section and the discussion of FlCRhR in the Ratiometric Fluorophore section.

See Clegg (1996) for an excellent review on FRET. Selvin (1995, 2000) also has a review. Wu and Brand (1994) for a large list of fluorophores examined for FRET. P. Wu and L. Brand (use medline for references) have used resonance energy transfer in several papers on the orientation of bio-molecules. For example, in Wu and Brand (1992) they discuss the issue of "When static orientational disorder exists, the (three dimensional) orientation factor κ² can vary from 0 to 4, leading to considerable uncertainty in estimation of distances." In almost all papers κ² is assumed to be 2/3 (random orientation of the dipole moments).

Several of Paul Selvin's FRET articles are available online at his web site (P.R. Selcin, UIUC).

An excellent overview of FRET including the conditions for FRET and the FRET equation is available online from the Molecular Probes Handbook of Fluorescent Probes and Research Products. See also the equations and explanations in the Atto-Tec 2001 catalog (PDF)

Primary Conditions for FRET (from the Molecular Probes Handbook)

Förster Radius

The distance at which energy transfer is 50% efficient (i.e. 50% of excited donors are deactivated by FRET) is defined by the Förster Radius (R0). The magnitude of R0 is dependent on the spectral properties of the donor and acceptor dyes. With respect to the dipole moments condition, in many situations the donor and acceptor dipole moments will successfully be approximated by assuming random orientation (κ² = 2/3). If both fluorophores are in a membrane or are locked together with a rigid linker, the random approximation may be poor.

FRET Imaging Examples

A few words on standard FRET and a potential new method, Spectral FRET Imaging (SFRETI?):

In standard FRET imaging one excites the donor fluorophore with excitation light, and collects sequentially the fluorescence emission of the donor and acceptor. For the donor-acceptor pair of fluorescein-rhodamine one might use a 470-490 nm excitation filter, and a 500-520 nm emission filter for collecting the light from the fluorescein donor. One might then use a 600-650 nm emission filter for collecting the light from the rhodamine acceptor. These emission filters need to collect just the shorter wavelength range of the donor, and just the long emission tail of the acceptor, to avoid cross-talk between the two image channels. That is, one would want to avoid collecting fluorescein emission in the rhodamine channel and vica versa. The problem is that for FRET to work, the donor emission and acceptor excitation spectra must overlap (high overlap is good), but for good signal-to-noise ratio imaging one must avoid collecting the "wrong" photons through a filter.

A potential alternative collection method is to use a spectral imaging device that would collect all the photons of both the donor and acceptor simultaneously and somehow separate out the two on the basis of their spectra. One would still excite fluorescein from 470-490 nm, but one would collect all the emission photons from 500-650 nm simultaneously. A device that should be able to do this is a fluorescence microscope with the suggested 470-490nm/500-650 nm filter cube and an Applied Spectral Imaging, Inc., SpectraCube® spectral imaging device. The SpectraCube® is a Fourier transform interferometer spectral imaging device that attaches to a standard microscope (i.e. Axioplan-2, Axioskop-2, Eclipse E800, or their inverted microscope equivalents) by a C-mount adapter. The device collects all the photons all the time, and uses clever mathematical analyses to quantify the amount of each fluorophore by their spectra. Using an interferometer allows the device to "collect all the photons all the time" and compute the spectrum later (offline).

Note that by 2003 the sales of spectral confocal microscopes is such that more users are likely to have access to a Zeiss 510 Meta or Leica SP1 or SP2 or SP2 AOBS or Lightform PARISS (standard PARISS + slit at the excitation field aperture focus) spectral confocal microscope than they are to a SKY™ system, or SKY + spinning disk confocal or CRI LCTF + spinning disk confocal.

A. Miyawaki, R.Y. Tsien (2000) Monitoring protein conformations and interactions by fluorescence resonance energy transfer between mutants of green fluorescent protein. Methods Enzymol. 327: 472-500. PMID: 11045004 (Excellent review in a terrific volume of MoE).

Energy Transfer Primers (molecular beacons or Sunrise primers)

Hung et al. (1996) describe a clever FRET donor for DNA sequencing machines that could also have uses in multicolor light microscopy. They discuss, “Energy transfer (ET) fluorescent primers … in which a donor chromophore with a large absorption cross section but a low fluorescence quantum yield is exploited to increase the Stokes-shifted fluorescence emission of acceptor dyes” (quoting from their abstract). The donor chromophore (fluorophore) absorbs light well but does not itself emit much light (low quantum yield for fluorescence). The donor does however transfer energy by FRET to each of the four acceptor dyes. The donor is excited by 488 nm light and the acceptors emit in the green and red. This could be an interesting set of dyes to use with the SpectraCube® and a long-pass emission filter that spans the emission range of the four acceptors.

"Sunrise™ amplification detection system" primers, described at http://www.oncor.com/pp2-cr.htm, were originally developed at Oncor and are now available from Intergen Company (http://www.intergenco.com, 800-431-4505. Incidentally, Intergen Co. also has the Cryptofluors® line of reagents … now at http://www.serologicals.com/products/int_prod/index.html).

A similar approach was adopted for multiplex PCR by L.G. Lee et al (1999). The fluorescent dyes used were:

Dye Name Ex/Em Wavelength
6FAM 497/517
dR110 519/539
dR6G 547/567
dTMR 575/595
dROX 601/621
JAX 622/642
AlPcTS 665/685

Notes: The Ex/Em value was the wavelength used in dual scan monochromator (em set to ex + 20 nm). These may not be the excitation or emission maxima of the dyes. AlPcTS was used as a reference dye for evaporation)

Used nitrothiozole blue (NYB) was the quencher in the Taqman assay. Epoch Biosciences has made a newer “dark quenchers” called Eclipse, that they claim is better than older generation quenchers.

L.G. Lee, K.J. Livak, R. Mullah, R.J. Graham, R.S. Vinayak, T.M. Woudenberg (1999) Seven-color, homogeneous detection of six PCR products. Biotechniques 27(2):342-349. Please note that the authors are from PE Biosystems, a manufacturer of PCR machines.

Joel Swanson New FRET Imaging Calculation

Joel Swanson (Hoppe et al 2002) published a new method for FRET imaging corrections, and compares his to many of the older methods.

Ammasi Periasamy FRET Imaging Calculation and Reviews

Ammasi Periasamy has published his own new imagingcorrection method, and fluorescence lifetime and multiphoton FRET methods and several reviews (Day et al. 2001; Elangovan et al. 2002, 2003; Periasamy et al. 2002; Periasamy 2001; Sekar and Periasamy 2003). See also chapters in the book Periasamy edited (2001).