FRET

Fluorescence Resonance Energy Transfer (FRET) is an increasingly popular microscopy technique used to measure the proximity of two fluorophores. FRET is used to image protein-protein interactions in cells and colocalization.

Resonance energy transfer occurs only over very short distances, typically within a few nanometers, and involves the direct transfer of excited state energy from the donor fluorophore to an acceptor fluorophore as an alternative to fluorescence emissive decay from the donor. Upon transfer of energy, the acceptor molecule enters an excited state from which it decays emissively (always of a longer wavelength than that of the acceptor emission). FRET decreases the donor fluorescence and increases the acceptor fluorescence. Thus, by exciting the donor and then monitoring the relative donor and acceptor emissions, either sequentially or simultaneously, one can determine when FRET has occurred and at what efficiency.

FRET can also be used to detect intramolecular conformational changes of proteins tagged with pairs of fluorophores^1,2. Combining two-photon excitation microscopy with FRET could allow the measurement of biochemical dynamics in neuronal microcompartments within intact neural networks⁴.

Since fluorophores can be employed to specifically label biomolecules and the distance condition for FRET is a few nanometers (a distance sufficiently close for molecular interactions to occur), FRET is often used to determine when and where two or more biomolecules, often proteins, interact within their physiological surroundings. A FRET signal corresponding to a particular location within a microscope image provides an additional distance accuracy surpassing the optical resolution (~0.25 μm) of the light microscope. Aside from spatial proximity, for efficient FRET to take place, the FRET fluorophore pair must also exhibit significant overlap of the donor's excitation spectrum with the acceptor's absorption spectrum. This is one of the experimental contradiction factors of FRET; the spectral profiles of the FRET pair must have a significant overlap, yet one wants to avoid "cross-talk" between the two imaging channels, i.e. ideally the donor emission filter set must collect only the light from the donor and none from the acceptor, and vice versa. In practice, this can be realized by employing short bandpass filters that collect light from only the shorter wavelength side of the donor emission and the longer wavelength side of the acceptor emission.

Currently, FRET is quantified using two approaches. The first approach measures the intensities at two or more wavelengths; this can report the quenching of the donor fluorescence and the enhancement of acceptor fluorescence due to FRET⁴. In the second approach, FRET can be quantified using measurements of the fluorescence lifetime, the average time elapsed between fluorophore excitation and photon emission⁵. Fluorescence lifetimes are on the order of nanoseconds. Fluorescence lifetime measurements (FLIM) can be easily combined with two-photon excitation microscopy in order to provide quantitative FRET imaging⁴. Using a Prairie laser light delivery system coupled to an inverted TE2000 microscope, Bal M., et al.⁶ studied the Calmodulin binding to M-type K⁺ channels. The Calmodulin binding has been assayed by TIRF/FRET in living cells.

References

1. Clegg, R.M., Murchie, A.I., Zechel, A., Carlberg, C., Diekmann, S., and Lilley, D.M. (1992). Fluorescence resonance energy transfer analysis of the structure of the four-way DNA junction. Biochemistry 31, 4846–4856.
2. Selvin, P.R. (2000). The renaissance of fluorescence resonance energy transfer. Nat. Struct. Biol. 7, 730–734.
3. Yasuda, R., Masaike, T., Adachi, K., Noji, H., Itoh, H., and Kinosita, K., Jr. (2003a). The ATP-waiting conformation of rotating F1-ATPase revealed by single-pair fluorescence resonance energy transfer. Proc. Natl. Acad. Sci. USA 100, 9314–9318.
4. Karel Svoboda and Ryohei Yasuda, Principles of Two-Photon Excitation Microscopy and Its Applications to Neuroscience, Neuron 50, 823–839, June 15, 2006 2006 Elsevier Inc.
5. Lakowicz, J.R. (1999). Principles of Fluorescence Spectroscopy, Second edition (New York: Plenum).
6. Manjot Bal, Oleg Zaika, Pamela Martin and Mark S. Shapiro, Calmodulin binding to M-type K+ channels assayed by TIRF/FRET in living cells, J Physiol 586.9 (2008) pp 2307–2320.