Don’t fret about it, just get it (a FRET primer – Part I)

Why should you know FRET? Well, FRET is used when you do a real-time qPCR, or you might be using it in assays like HTRF, or to detect biochemical reactions in single living cells. You might measure protein-protein interactions, probe cell signalling, cell metabolism or nano-meter scale conformational changes. Or what about dimerization, protein – nucleic acids interactions, checking splicing variants by FISH, or detect fast conformational changes in structural studies? This is why some of us are very fond of FRET, and many others are using it without being fully aware of it. The usefulness of FRET arises from its capability to translate molecular properties occurring at a nanometer and nanosecond scales to optical signals that can be easily detected with a microscope or a spectrofluorimeter.

Figure 1. Energy flows from a donor ‘D’ to an acceptor fluorophore by FRET. The ration of fluorescence emitted by the donor and acceptors (IDD and IDA) can be used to estimate how much energy is transferred from donor to the acceptor, quantity that is proportional to the distance of the two molecules.

What is FRET? When a fluorescent molecule is in close proximity to another that might be, in principle, capable to absorb the light emitted by the first, FRET might occur. However, FRET is not the emission and re-absorption of light, but the non-radiative transfer of energy. This is important because the molecule that will donate energy and the one that will accept it become coupled and will inform us about the distance between the two molecules only if they are within a few nanometer ranges, with sub-nanometer precision. Most of us do not use this capability directly but to engineer probes that can sense specific biochemical reactions. Ok, now you are ready. What FRET stands for? RET is Resonance Energy Transfer and it says with three simple words what I have just described. For the “F”… you would think it is simple, but the community is a bit split on the meaning of that “F”. There are two camps. One that says “F” is for Foerster, from Theodor Foerster who developed the theoretical background to describe the phenomenon. Others say that “F” is for “Fluorescence” as it is detected by means of fluorescence emission. Who prefers Foerster-type energy transfer means to distinguish it from other possible mechanisms but, most importantly, to avoid misinterpretation of the acronym. Indeed, it is not fluorescence that is transferred from donor to acceptor and the acceptor does not need to be fluorescent. Those who use Fluorescence RET often say that Foerster did not discover FRET (correct, he did a mathematical description of a known phenomenon). Does it matter? Not really, but at least now we know what FRET means. Ah, I almost forgot… FRET for me is Foerster Resonance Energy Transfer… I heard you asking.

Next. How do we measure FRET? There are many ways to measure the occurrence of FRET but today I will focus only on ratiometric FRET and Fluorescence Lifetime Imaging Microscopy (FLIM). I am going to use an analogy that is very useful, that of buckets filled with water (Fig. 1). The tap is your light source, which is filling a donor bucket with water (energy). The bucket has one hole, from which water is dripping into a plate (a detector). That stream of water highlighted in green in Figs. 1-2 is the fluorescence signal that we measure, emitted by the donor. FRET is another hole punched into the donor-bucket. Water will flow into an acceptor-bucket from where it will drip (red flow) into a second plate (detector). The ratio of the water we collect in the blue and yellow plates will tell us the fraction of water that passed through the FRET “hole”. In a real FRET experiment, this fraction, called the ‘FRET efficiency’ is proportional to the inverse of the sixth power of the distance between the buckets, er… fluorophores.

Figure 2. Cross-talks between donor and acceptor excitation. DE: direct excitation of the acceptor. SBT: spectral bleed-through of the donor emission into the acceptor channel.

Unfortunately, the excitation and emission spectra of typical fluorophores are broad and spill-over of fluorescent signals (or water!) is usually unavoidable (Fig. 2). The buckets are large compared to their distance (the excitation spectra overlap) and part of the water we wish to put into the donor bucket will fill the acceptor bucket. This is called ‘direct excitation’ of the acceptor. The water we now collect in the yellow plate flows from one hole in the acceptor-bucket, but it originates from two different flows. Direct excitation (black flow) and FRET (red flow). The latter, FRET sensitised emission, is the signal that matters. At the same time, water flowing from the donor bucket spills-over into the yellow plate (the emission spectra overlap), adding a third (green) unwanted flow into the yellow plate.

So, how do we correct cross-talks? The good news is that sometimes you do not need to. If what you need to measure is a semiquantitative measure, the detection of changes, measuring the relative quantity of water that fell into the yellow plate compared to the blue plate will suffice. This, however, will require to ensure the stoichiometry of donor-acceptor fluorophores does not change, for instance when using typical FRET-based probes for kinase activity.

In other cases, you will need to correct for these cross-talks and techniques like ‘precision FRET’ and ‘three cube FRET’ comes to the rescue (see reference section).

Figure 3. FLIM measure the time the donor-bucket needs tobe emptied, thus inferring the size of the second (FRET) hole.

Another technique that can be used to measure FRET is Fluorescence Lifetime Imaging Microscopy or FLIM. FLIM does not need measuring the flow of water from the acceptor. FLIM requires to turn the tap on and off, and measuring the time that the donor buckets requires to be emptied. When a second hole (FRET) is punched into the donor-bucket, this will empty faster. We do not measure directly any signal from the acceptor and, therefore we avoid the need to correct for spill-overs.

This brings me back to the time I was a PhD student. A very smart master student entered my office and popped the question “how FLIM can detect the presence of FRET if the only photons we measure are those that do not experience energy transfer?”. Back then, I was taken aback from the question and I could not respond immediately in a satisfactory way. The bucket analogy should do the trick.

To conclude, this was just a brief overview of FRET and how we can measure it. There are plenty of great reviews out there to improve your understanding of FRET, but I hope that the analogy with buckets might provide a simple model for the non-specialist, albeit physically inaccurate for other aspects of FRET. Below, you can find a few references. Let me also refer to my new study published on Biomedical Optics Express entitled “How many photons are needed for FRET imaging?”. It is a theoretical study, but even the non-specialist might find some sections interesting and, plenty of more bucket figures there!


J. R. Lakowicz, Principles of Fluorescence Spectroscopy (Kluwer Academic/Plenum Publishers, New York, 1999).

T. Förster, “Zwischenmolekulare Energiewanderung und Fluoreszenz,” Annalen der Physik 437, 55-75 (1948).

L. Stryer and R. P. Haugland, “Energy Transfer – A Spectroscopic Ruler,” Proceedings of the National Academy of Sciences of the United States of America 58, 719-& (1967).

G. Bunt and F. S. Wouters, “Visualization of molecular activities inside living cells with fluorescent labels,” International Review of Cytology 237, 205-277 (2004).

E. A. Jares-Erijman and T. M. Jovin, “FRET imaging,” Nat. Biotechnol. 21, 1387-1395 (2003).

J. Zhang and M. D. Allen, “FRET-based biosensors for protein kinases: illuminating the kinome,” Mol Biosyst 3, 759-765 (2007).

M. Y. Berezin and S. Achilefu, “Fluorescence lifetime measurements and biological imaging,” Chem Rev 110, 2641-2684 (2010).

A. D. Elder, A. Domin, G. S. Kaminski Schierle, C. Lindon, J. Pines, A. Esposito, and C. F. Kaminski, “A quantitative protocol for dynamic measurements of protein interactions by Förster resonance energy transfer-sensitized fluorescence emission,” Journal of the Royal Society, Interface/the Royal Society (2008).

A. Hoppe, K. Christensen, and J. A. Swanson, “Fluorescence resonance energy transfer-based stoichiometry in living cells,” Biophys J 83, 3652-3664 (2002).

M. Elangovan, H. Wallrabe, Y. Chen, R. N. Day, M. Barroso, and A. Periasamy, “Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy,” Methods 29(2003).

G. W. Gordon, G. Berry, X. H. Liang, B. Levine, and B. Herman, “Quantitative fluorescence resonance energy transfer measurements using fluorescence microscopy,” Biophysical Journal 74, 2702-2713 (1998).

C. Berney and G. Danuser, “FRET or no FRET: A quantitative comparison,” Biophysical Journal 84, 3992-4010 (2003).

J. Wlodarczyk, A. Woehler, F. Kobe, E. Ponimaskin, A. Zeug, and E. Neher, “Analysis of FRET signals in the presence of free donors and acceptors,” Biophysical Journal 94, 986-1000 (2008).

A. Zeug, A. Woehler, E. Neher, and E. G. Ponimaskin, “Quantitative intensity-based FRET approaches–a comparative snapshot,” Biophys J 103, 1821-1827 (2012).

H. C. Gerritsen, A. V. Agronskaia, A. N. Bader, and A. Esposito, “Time Domain FLIM: theory, Instrumentation and data analysis,” in FRET & FLIM Imaging Techniques, T. W. Gadella, ed. (Elsevier, Amsterdam, The Netherlands, 2009).

R. A. Neher and E. Neher, “Applying spectral fingerprinting to the analysis of FRET images,” Microscopy Research and Technique 64, 185-195 (2004).

H. Wallrabe, Y. Chen, A. Periasamy, and M. Barroso, “Issues in confocal microscopy for quantitative FRET analysis,” Microscopy Research and Technique 69, 196-206 (2006).

S. Ganesan, S. M. Ameer beg, T. Ng, B. Vojnovic, and F. S. Wouters, “A YFP-based Resonance Energy Accepting Chromoprotein (REACh) for efficient FRET with GFP,” Proceedings of the National Academy of Sciences of the United States of America 103, 4089-4094 (2006).

J. Klarenbeek, J. Goedhart, A. van Batenburg, D. Groenewald, and K. Jalink, “Fourth-generation epac-based FRET sensors for cAMP feature exceptional brightness, photostability and dynamic range: characterization of dedicated sensors for FLIM, for ratiometry and with high affinity,” PLoS ONE 10, e0122513 (2015).

K. J. Martin, E. J. McGhee, J. P. Schwarz, M. Drysdale, S. M. Brachmann, V. Stucke, O. J. Sansom, and K. I. Anderson, “Accepting from the best donor; analysis of long-lifetime donor fluorescent protein pairings to optimise dynamic FLIM-based FRET experiments,” PLoS ONE 13, e0183585 (2018).

M. W. Fries, K. T. Haas, S. Ber, J. Saganty, E. K. Richardson, A. R. Venkitaraman, and A. Esposito, “Multiplexed biochemical imaging reveals caspase activation patterns underlying single cell fate,” bioRxiv, 427237 (2018).

Author: Alessandro

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