How to Measure FRET

In my previous article on FRET, I gave you some background on FRET – its mechanism and its applications. Here, I will expand, including what to measure when doing FRET.

There are a number of approaches to FRET quantification:

  1. Sensitized Emission – This two-channel imaging technique uses an algorithm that corrects for excitation and emission crosstalk.
  2. Acceptor Photobleaching – Sometimes called donor dequenching, this technique measures increased donor emission when the acceptor is photobleached.
  3. Fluorescence Lifetime Imaging Microscopy FRET (FLIM FRET) – This technique detects fluorescence lifetime changes of donor.
  4. Fluorophore donor Spectral Imaging – This technique involves exciting at one or two wavelengths and measuring the spectral profiles of both donor and acceptor.
  5. Homo-FRET and polarization Anisotropy Imaging – This technique uses identical donor and acceptor fluorophores and detects FRET through measurements of polarization.

1. Sensitized Emission

As I mentioned in my first article on FRET, sensitized emission is perhaps the simplest method of FRET. In this technique the donor fluorophore is excited and the signal is collected using emission filters chosen for both the donor fluorescence and the acceptor fluorescence. The acceptor fluorescence increases in the presence of donor, whereas the donor fluorescence decreases in the presence of the acceptor. The ratiometric change of the fluorescence intensity can then be used to measure FRET.

If there was no crosstalk between the two fluorophores, this method would be ideal – however, crosstalk between fluorescent proteins does exist. Thus, it is difficult to obtain quantitatively accurate FRET data with this approach. Additionally, you still need appropriate control experiments to establish the presence or absence of FRET. But despite these limitations, sensitized emission measurements can be useful for detecting rapid dynamic changes, and is especially useful when examining fluorescent protein biosensors where the FRET dynamic range is large and the stoichiometry of the donor and acceptor is fixed at a 1:1 ratio.

2. Acceptor Photobleaching

Acceptor photobleaching is a technique based on the fact that donor fluorescence is quenched during FRET when some of the donor fluorescence energy is transferred to the acceptor. But photobleaching the acceptor fluorophore stops it from fluorescing and thus from using some of the donor’s energy, resulting in an increase of donor fluorescence. This phenomenon can be used to calculate FRET efficiency by subtracting the donor intensity in the presence of the acceptor from its intensity after photobleaching the acceptor, and dividing the result by the donor intensity after bleaching.

Acceptor photobleaching is very straightforward technique and is, perhaps, the most widely used method of FRET. Its main drawback is that it can be used only once per cell. Also, it is a relatively slow process. An important thing to keep in mind when design acceptor photobleaching experiments is that you need to ensure that donor fluorescence is not affected (this involves careful selection of bleaching wavelength and intensity) and that the acceptor is photobleached to approximately 10% of its initial value.


All fluorescent molecules exhibit a decay pattern in their fluorescence emission on a nanosecond scale, and the rate of this decay is sensitive to environmental variables. Fluorescence-lifetime imaging microscopy (FLIM) is the technique that calculates this decay pattern and, through it, gives information about the state of the protein and about factors in the cellular microenvoronment. In FLIM FRET, the donor fluorescence is quenched by the FRET interaction, and the amount of quenching can be calculated by measuring the decrease in fluorescence decay time of the donor molecule.

A significant advantage to the FLIM-FRET technique of measurements is its insensitivity to direct acceptor excitation artifacts. The downside to this technique is that it requires highly specialized and expensive instrumentation. Also, just like Acceptor Photobleaching, FLIM-FRET is relatively slow, and that limits its applications. Finally, because FLIM is sensitive to local microenvironment factors, when interpreting FLIM-FRET you need to be careful to exclude artifacts.

4. Spectral Imaging

In spectral imaging, the entire emission spectrum containing both donor and acceptor fluorescence is recorded upon excitation of the donor. This is, in a way, spectroscopy for the microscope, and is based on the principle that overlapping spectra can be separated not just by their emission peaks but also their distinct overall shapes.

This technique also requires specialized equipment, but the cost is not as high as for FLIM-FRET. The main disadvantage of this approach is the reduced signal-to-noise ratio associated with acquiring the complete emission spectra of both fluorophores, opposed to collecting two channels at the emission peak.

5. Homo-FRET and Polarization Anisotropy Imaging

Just like regular FRET, homo-FRET involves the transfer of excited-state energy between fluorophores that are located within ~10 nm of each other, but with the difference that these fluorophores are identical. There are no changes in lifetime or emission spectrum in this case. How do we make any sense of it, then?

When a randomly oriented population of fluorescent molecules is excited with linearly polarized light, there is preferential excitation of only those fluorophores whose absorption dipole vector happens to be parallel to the polarization azimuth. Most of the emission light remains polarized after the excitation, and this light can be observed using an analyzer oriented parallel to the excitation light polarization vector. The signal level after the analyzer will decrease either if the fluorophore rotates in the timescale of the experiment or if it transfers excitation energy due to FRET to a neighboring protein having a different orientation. However, as energy transfer can occur far more rapidly than molecular rotation for molecules of this size, depolarization due to FRET can be recognized. This way, we are able to image clustering of identical molecules in cells. This technique is well-suited for high-content screening applications because the data can be acquired rapidly.

Which Fluorophores to Use?

The fluorophore absorption spectra become broader as we move to longer wavelengths, making such fluorophores less appropriate for this FRET. And although significant advances have been made in expanding the useable area for FRET into the orange, red, and far-red regions of the spectrum, the optimized versions of CFP and YFP remain the most useful and most popular FRET pair. In any case, there is no such thing as a perfect FRET pair. Likewise, there is no perfect technique with which to measure FRET. The user has to decide which pair and which technique to use, depending on the type of experiment in question, and based on the available literature.

Although the issue is vast, I can give you some one-liners about how one can go about optimizing FRET in its most common form, acceptor photobleaching:

  • Select the appropriate donor and acceptor fluorophores.
  • Select an appropriate labeling scheme.
  • When using antibodies, optimize antibody labeling ratios and conditions, and make sure antibodies do not react with each other.
  • Identify relevant positive and negative controls.
  • Identify optimal photobleaching conditions for the acceptor.
  • Only apply quantitative comparisons in experiments performed at the same time.
  • Be aware of artifactual reasons for changes in donor fluorescence (e.g. chemical conversion).
  • Optimize sample size for statistical analysis.

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