FRET as a biomolecular research tool — understanding its potential while avoiding pitfalls

Abstract

The applications of Förster resonance energy transfer (FRET) grow with each year. However, different FRET techniques are not applied consistently, nor are results uniformly presented, which makes implementing and reproducing FRET experiments challenging. We discuss important considerations for designing and evaluating ensemble FRET experiments. Alongside a primer on FRET basics, we provide guidelines for making experimental design choices such as the donor-acceptor pair, instrumentation and labeling chemistries; selecting control experiments to unambiguously demonstrate FRET and validate that the experiments provide meaningful data about the biomolecular process in question; analyzing raw data and assessing the results; and reporting data and experimental details in a manner that easily allows for reproducibility. Some considerations are also given for FRET assays and FRET imaging, especially with fluorescent proteins. Our goal is to motivate and empower all biologists to consider FRET for the powerful research tool it can be.

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Fig. 1: The range of FRET detection versus selected microscopical techniques and a general process for designing a FRET experiment.
Fig. 2: Flowchart for designing a FRET system.
Fig. 3: Designing and running control experiments.
Fig. 4: Data analysis.
Fig. 5: Principles of FRET assays and their quantification.
Fig. 6: FLIM-FRET cellular imaging.

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Acknowledgements

This Perspective originates from the International Discussion Meeting on Förster Resonance Energy Transfer (FRET) in the Life Sciences II 2016 (http://fret.uni-duesseldorf.de/cms/), held at the Max Planck Institute for Biophysical Chemistry in Göttingen, Germany — where Förster first developed his seminal FRET theory. I.L.M. acknowledges the Office of Naval Research, the NRL Nanosciences Institute and a LUCI project in support of the VBFF through the OSD. N.H. acknowledges the Institut Universitaire de France (IUF). W.R.A. acknowledges the Natural Sciences and Engineering Research Council (NSERC) of Canada, the Canada Foundation for Innovation (CFI), BCKDF, a Canada Research Chair (Tier 2), a Michael Smith Foundation for Health Research Scholar Award and an Alfred P. Sloan Foundation Research Fellowship. S.S.V. acknowledges funding by the intramural program of the National Institutes of Health, National Institute on Alcohol Abuse and Alcoholism, Bethesda, MD. The mention of commercial and other websites in this article does not constitute any endorsement by the authors.

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Correspondence to Igor L. Medintz.

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