Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

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

Nature Methods volume 16, pages 815–829 (2019)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Relevant articles

Open Access articles citing this article.

Access options

Rent or buy this article

Get just this article for as long as you need it

$39.95

Learn more

Prices may be subject to local taxes which are calculated during checkout

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.

References

  1. Web of Science (Clarivate Analytics, accessed 3 June 2019); https://apps.webofknowledge.com

  2. Medintz, I. L. & Hildebrandt, N. (eds) FRET – Förster Resonance Energy Transfer from Theory to Applications (Wiley-VCH, 2013).

  3. McNutt, M. Journals unite for reproducibility. Science 346, 679–679 (2014).

    Article  CAS  Google Scholar 

  4. Lerner, E. et al. Toward dynamic structural biology: two decades of single-molecule Forster resonance energy transfer. Science 359, eaan1133 (2018).

    Article  Google Scholar 

  5. Hellenkamp, B. et al. Precision and accuracy of single-molecule FRET measurements—a multi-laboratory benchmark study. Nat. Meth. 15, 669–676 (2018).

    Article  CAS  Google Scholar 

  6. Kalinin, S. et al. A toolkit and benchmark study for FRET-restrained high-precision structural modeling. Nat. Meth. 9, 1218–1225 (2012).

    Article  CAS  Google Scholar 

  7. Stryer, L. & Haugland, R. P. Energy transfer – a spectroscopic ruler. Proc. Natl. Acad. Sci. USA 58, 719–726 (1967).

    Article  CAS  Google Scholar 

  8. Marx, V. Probes: FRET sensor design and optimization. Nat. Methods 14, 949–953 (2017).

    Article  CAS  Google Scholar 

  9. Ai, H. W., Hazelwood, K. L., Davidson, M. W. & Campbell, R. E. Fluorescent protein FRET pairs for ratiometric imaging of dual biosensors. Nat. Methods 5, 401–403 (2008).

    Article  CAS  Google Scholar 

  10. Greenwald, E. C., Mehta, S. & Zhang, J. Genetically encoded fluorescent biosensors illuminate the spatiotemporal regulation of signaling networks. Chem. Rev. 118, 11707–11794 (2018).

    Article  CAS  Google Scholar 

  11. Miyawaki, A. & Niino, Y. Molecular spies for bioimaging-fluorescent protein-based probes. Mol. Cell 58, 632–643 (2015).

    Article  CAS  Google Scholar 

  12. Bajar, B. T. et al. Improving brightness and photostability of green and red fluorescent proteins for live cell imaging and FRET reporting. Sci. Rep. 6, 20889 (2016).

    Article  CAS  Google Scholar 

  13. Hochreiter, B., Garcia, A. P. & Schmid, J. A. Fluorescent proteins as genetically encoded FRET biosensors in life sciences. Sensors 15, 26281–26314 (2015).

    Article  CAS  Google Scholar 

  14. Sahl, S. J., Hell, S. W. & Jakobs, S. Fluorescence nanoscopy in cell biology. Nat. Rev. Mol. Cell Bio. 18, 685–701 (2017).

    Article  CAS  Google Scholar 

  15. Boeneman, K. et al. Quantum dot DNA bioconjugates: attachment chemistry strongly influences the resulting composite architecture. ACS Nano. 4, 7253–7266 (2010).

    Article  CAS  Google Scholar 

  16. Sapsford, K. E., Berti, L. & Medintz, I. L. Materials for fluorescence resonance energy transfer: beyond traditional ‘dye to dye’ combinations. Angew. Chem. Int. Ed. 45, 4562–4588 (2006).

    Article  CAS  Google Scholar 

  17. Sapsford, K. E., Tyner, K. M., Dair, B. J., Deschamps, J. R. & Medintz, I. L. Analyzing nanomaterial bioconjugates: a review of current and emerging purification and characterization techniques. Anal. Chem. 83, 4453–4488 (2011).

    Article  CAS  Google Scholar 

  18. Hermanson, G. T. (ed.) Bioconjugate Techniques 3rd edn (Acad. Press, 2013).

  19. Byrne, A. G. et al. in FRET - Förster Resonance Energy Transfer from Theory to Applications (eds Medintz, I. L. & Hildebrandt, N.) 657–756 (Wiley-VCH, 2013).

  20. Lakowicz, J. R. Principles of Fluorescence Spectroscopy (Springer, 2006).

  21. Bhuckory, S., Lefebvre, O., Qiu, X., Wegner, K. D. & Hildebrandt, N. Evaluating quantum dot performance in homogeneous FRET immunoassays for prostate specific antigen. Sensors 16, 197 (2016).

    Article  Google Scholar 

  22. Wegner, K. D. et al. Three-dimensional solution-phase Förster resonance energy transfer analysis of nanomolar quantum dot bioconjugates with subnanometer resolution. Chem. Mat. 26, 4299–4312 (2014).

    Article  CAS  Google Scholar 

  23. Gorris, H. H. & Resch-Genger, U. Perspectives and challenges of photon-upconversion nanoparticles - Part II: bioanalytical applications. Anal. Bioanal. Chem. 409, 5875–5890 (2017).

    Article  CAS  Google Scholar 

  24. Wurth, C., Grabolle, M., Pauli, J., Spieles, M. & Resch-Genger, U. Relative and absolute determination of fluorescence quantum yields of transparent samples. Nat. Protoc. 8, 1535–1550 (2013).

    Article  Google Scholar 

  25. Stennett, E. M. S., Ma, N., van der Vaart, A. & Levitus, M. Photophysical and dynamical properties of doubly linked Cy3-DNA constructs. J. Phys. Chem. B 118, 152–163 (2014).

    Article  CAS  Google Scholar 

  26. Melinger, J. S. et al. FRET from multiple pathways in fluorophore-labeled DNA. ACS Photonics 3, 659–669 (2016).

    Article  CAS  Google Scholar 

  27. Seidel, C. A. M., Schulz, A. & Sauer, M. H. M. Nucleobase-specific quenching of fluorescent dyes. 1. Nucleobase one-electron redox potentials and their correlation with static and dynamic quenching efficiencies. J. Phys. Chem. 100, 5541–5553 (1996).

    Article  CAS  Google Scholar 

  28. Wozniak, A. K., Schroder, G. F., Grubmuller, H., Seidel, C. A. M. & Oesterhelt, F. Single-molecule FRET measures bends and kinks in DNA. Proc. Natl. Acad. Sci. USA 105, 18337–18342 (2008).

    Article  CAS  Google Scholar 

  29. Van der Meer, B. W., Van der Meer, D. M. & Vogel, S. S. in FRET - Förster Resonance Energy Transfer: From Theory to Applications (eds Medintz, I. L. & Hildebrandt, N.) Ch. 4 (Wiley-VCH, 2013).

  30. Dos Santos, M. C. & Hildebrandt, N. Recent developments in lanthanide-to-quantum dot FRET using time-gated fluorescence detection and photon upconversion. Trac. Trends Anal. Chem. 84, 60–71 (2016).

    Article  Google Scholar 

  31. Hildebrandt, N., Wegner, K. D. & Algar, W. R. Luminescent terbium complexes: superior Forster resonance energy transfer donors for flexible and sensitive multiplexed biosensing. Coord. Chem. Rev. 273, 125–138 (2014).

    Article  Google Scholar 

  32. Wild, D. The Immunoassay Handbook 3rd edn (Elsevier, 2013).

  33. Wegner, K. D., Jin, Z. W., Linden, S., Jennings, T. L. & Hildebrandt, N. Quantum-dot-based Forster resonance energy transfer immunoassay for sensitive clinical diagnostics of low-volume serum samples. ACS Nano 7, 7411–7419 (2013).

    Article  CAS  Google Scholar 

  34. Jalink, K. & Rheenen, J.v. in Laboratory Techniques in Biochemistry and Molecular Biology Vol. 33. (ed. Gadella, T. W. J.) 289–349 (Elsevier, 2009).

  35. Chen, H., Puhl, H. L., Koushik, S. V., Vogel, S. S. & Ikeda, S. R. Measurement of FRET efficiency and ratio of donor to acceptor concentration in living cells. Biophys. J. 91, L39–L41 (2006).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  37. Zal, T. & Gascoigne, N. R. J. Photobleaching-corrected FRET efficiency imaging of live cells. Biophys. J. 86, 3923–3939 (2004).

    Article  CAS  Google Scholar 

  38. Vogel, S. S., Blank, P. S., Koushik, S. V. & Thaler, C. in Laboratory Techniques in Biochemistry and Molecular Biology (ed. Gadella, T. W. J.) (Elsevier, 2009).

  39. Neher, R. A. & Neher, E. Applying spectral fingerprinting to the analysis of FRET images. Microsc. Res. Tech. 64, 185–195 (2004).

    Article  Google Scholar 

  40. Thaler, C., Koushik, S. V., Blank, P. S. & Vogel, S. S. Quantitative multiphoton spectral imaging and its use for measuring resonance energy transfer. Biophys. J. 89, 2736–2749 (2005).

    Article  CAS  Google Scholar 

  41. Wlodarczyk, J. et al. Analysis of FRET signals in the presence of free donors and acceptors. Biophys. J. 94, 986–1000 (2008).

    Article  CAS  Google Scholar 

  42. Zimmermann, T., in Advances in Biochemical Engineering-Biotechnology (ed. Rietdorf, J.) 245–265 (Springer Verlag, 2005).

  43. Bastiaens, P. I. H. & Squire, A. Fluorescence lifetime imaging microscopy: spatial resolution of biochemical processes in the cell. Trends Cell Bio. 9, 48–52 (1999).

    Article  CAS  Google Scholar 

  44. Chen, Y. E. & Periasamy, A. Characterization of two-photon excitation fluorescence lifetime imaging microscopy for protein localization. Microsc. Res. Tech. 63, 72–80 (2004).

    Article  CAS  Google Scholar 

  45. Wallrabe, H. & Periasamy, A. Imaging protein molecules using FRET and FLIM microscopy. Curr. Opin. Biotech. 16, 19–27 (2005).

    Article  CAS  Google Scholar 

  46. Wouters, F. S. & Bastiaens, P. I. H. Fluorescence lifetime imaging of receptor tyrosine kinase activity in cells. Curr. Bio. 9, 1127–1130 (1999).

    Article  CAS  Google Scholar 

  47. Kenworthy, A. K. Imaging protein-protein interactions using fluorescence resonance energy transfer microscopy. Methods 24, 289–296 (2001).

    Article  CAS  Google Scholar 

  48. Bader, A. N., Hofman, E. G., Voortman, J., Henegouwen, P. & Gerritsen, H. C. Homo-FRET imaging enables quantification of protein cluster sizes with subcellular resolution. Biophys. J. 97, 2613–2622 (2009).

    Article  CAS  Google Scholar 

  49. Clayton, A. H. A., Hanley, Q. S., Arndt-Jovin, D. J., Subramaniam, V. & Jovin, T. M. Dynamic fluorescence anisotropy imaging microscopy in the frequency domain (rFLIM). Biophys. J. 83, 1631–1649 (2002).

    Article  CAS  Google Scholar 

  50. Lidke, D. S. et al. Imaging molecular interactions in cells by dynamic and static fluorescence anisotropy (rFLIM and emFRET). Biochem. Soc. Trans. 31, 1020–1027 (2003).

    Article  CAS  Google Scholar 

  51. Tramier, M. & Coppey-Moisan, M. Fluorescence anisotropy imaging microscopy for homo-FRET in living cells. Meth. Cell Bio. 85, 395–414 (2008).

    Article  CAS  Google Scholar 

  52. Yeow, E. K. L. & Clayton, A. H. A. Enumeration of oligomerization states of membrane proteins in living cells by homo-FRET spectroscopy and microscopy: theory and application. Biophys. J. 92, 3098–3104 (2007).

    Article  CAS  Google Scholar 

  53. Gadella, T. W. J. (ed.) FRET and FLIM Techniques 1st edn (Elsevier Science, 2008).

  54. Periasamy, A., Mazumder, N., Sun, Y., Christopher, K. G. & Day, R. N. in Advanced Time-Correlated Single Photon Counting Applications (ed. W. Becker) 249–276 (Springer, 2015).

  55. Kim, Y. et al. Venus(A206) dimers behave coherently at room temperature. Biophys. J. 116, 1918–1930 (2019).

    Article  CAS  Google Scholar 

  56. Vogel, S. S., Nguyen, T. A., van der Meer, B. W. & Blank, P. S. The impact of heterogeneity and dark acceptor states on FRET: Implications for using fluorescent protein donors and acceptors. PLoS ONE 7, e49593 (2012).

    Article  CAS  Google Scholar 

  57. Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

    Article  CAS  Google Scholar 

  58. Chandrasekhar, S. Stochastic problems in physics and astronomy. Rev. Mod. Phys. 15, 1–89 (1943).

    Article  Google Scholar 

  59. Anikovsky, M., Dale, L., Ferguson, S. & Petersen, N. Resonance energy transfer in cells: a new look at fixation effect and receptor aggregation on cell membrane. Biophys. J. 95, 1349–1359 (2008).

    Article  CAS  Google Scholar 

  60. King, C., Sarabipour, S., Byrne, P., Leahy, D. J. & Hristova, K. The FRET signatures of noninteracting proteins in membranes: Simulations and experiments. Biophys. J. 106, 1309–1317 (2014).

    Article  CAS  Google Scholar 

  61. Loura, L. M. S. & Prieto, M. FRET in membrane biophysics: an overview. Front. Physiol. 2, 82 (2011).

    Article  CAS  Google Scholar 

  62. Ma, Y. Q. et al. An intermolecular FRET sensor detects the dynamics of T cell receptor clustering. Nat. Comm. 8, 15100 (2017).

    Article  Google Scholar 

  63. Humpolickova, J. et al. Inhibition of the precursor and mature forms of HIV-1 protease as a tool for drug evaluation. Sci. Rep. 8, 10438 (2018).

    Article  Google Scholar 

  64. Dou, J. Y. et al. De novo design of a fluorescence-activating beta-barrel. Nature 561, 485–491 (2018).

    Article  CAS  Google Scholar 

  65. Mastop, M. et al. Characterization of a spectrally diverse set of fluorescent proteins as FRET acceptors for mTurquoise2. Sci. Rep. 7, 11999 (2017).

    Article  Google Scholar 

  66. Murakoshi, H. & Shibata, A. C. E. ShadowY: a dark yellow fluorescent protein for FLIM-based FRET measurement. Sci. Rep. 7, 6791 (2017).

    Article  Google Scholar 

  67. Shaner, N. C., Steinbach, P. A. & Tsien, R. Y. A guide to choosing fluorescent proteins. Nat. Methods 2, 905–909 (2005).

    Article  CAS  Google Scholar 

  68. Piatkevich, K. D. & Verkhusha, V. V. in Recent Advances in Cytometry, Part A: Instrumentation, Methods 5th edn (eds Darzynkiewicz, Z. et al.) 431–461 (2011).

  69. Lam, A. J. et al. Improving FRET dynamic range with bright green and red fluorescent proteins. Nat. Methods 9, 1005–1012 (2012).

    Article  CAS  Google Scholar 

  70. Abraham, B. G. et al. Fluorescent protein based FRET pairs with improved dynamic range for fluorescence lifetime measurements. PLoS ONE 10, e0134436 (2015).

    Article  Google Scholar 

  71. Bajar, B. T., Wang, E. S., Zhang, S., Lin, M. Z. & Chu, J. A guide to fluorescent protein FRET pairs. Sensors 16, E1488 (2016).

    Article  Google Scholar 

  72. Zipfel, W. R., Williams, R. M. & Webb, W. W. Nonlinear magic: multiphoton microscopy in the biosciences. Nat. Biotechnol. 21, 1368–1376 (2003).

    Article  Google Scholar 

  73. Schermelleh, L., Heintzmann, R. & Leonhardt, H. A guide to super-resolution fluorescence microscopy. J. Cell Bio. 190, 165–175 (2010).

    Article  CAS  Google Scholar 

  74. Valeur, B. & Berberan-Santos, M. N. (eds) Molecular Fluorescence: Principles and Applications (Wiley-VCH, 2012).

  75. Clegg, R. M. & Sener, M., Govindjee. From Forster resonance energy transfer to coherent resonance energy transfer and back. Opt. Biopsy 7561, 75610C (2010).

    Google Scholar 

  76. Clegg, R. M. in FRET and FLIM Techniques (ed. Gadella, T. W. J.) Ch. 1 (Elsevier, 2009).

  77. Sapsford, K. E., Wildt, B., Mariani, A., Yeatts, A. B. & Medintz, I. L. in FRET – Förster Resonance Energy Transfer From Theory to Applications (eds Medintz, I. L. & Hildebrandt, N.) 165–268 (Wiley-VCH, 2013).

  78. Jameson, D. M. Introduction to Fluorescence (CRC Press, 2014).

  79. Cranfill, P. J. et al. Quantitative assessment of fluorescent proteins. Nat. Methods 13, 557–562 (2016).

    Article  CAS  Google Scholar 

  80. Fritz, R. D. et al. A versatile toolkit to produce sensitive FRET biosensors to visualize signaling in time and space. Sci. Signal. 6, rs12 (2013).

    Article  Google Scholar 

Download references

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.

Author information

Authors and Affiliations

  1. Department of Chemistry, University of British Columbia, Vancouver, British Columbia, Canada

    W. Russ Algar

  2. NanoBioPhotonics, Institute for Integrative Biology of the Cell, Université Paris-Saclay, Université Paris-Sud, CNRS, CEA, Orsay, France

    Niko Hildebrandt

  3. National Institute on Alcohol Abuse and Alcoholism, National Institutes of Health, Bethesda, MD, USA

    Steven S. Vogel

  4. Center for Bio/Molecular Science and Engineering, US Naval Research Laboratory, Washington, USA

    Igor L. Medintz

Authors
  1. W. Russ Algar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Niko Hildebrandt
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Steven S. Vogel
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Igor L. Medintz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Igor L. Medintz.

Ethics declarations

Competing interests

The authors declare no competing interests

Additional information

Peer review information: Rita Strack was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Figures 1–4, Supplementary Tables 1–4 and Supplementary Notes 1–33

Rights and permissions

Reprints and Permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Algar, W.R., Hildebrandt, N., Vogel, S.S. et al. FRET as a biomolecular research tool — understanding its potential while avoiding pitfalls. Nat Methods 16, 815–829 (2019). https://doi.org/10.1038/s41592-019-0530-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41592-019-0530-8

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing