Encyclopedia of Biophysics

Living Edition
| Editors: Gordon Roberts, Anthony Watts, European Biophysical Societies

Single-Molecule Fluorescence Resonance Energy Transfer

  • Alexander E. Johnson-BuckEmail author
  • Mario R. Blanco
  • Nils G. Walter
Living reference work entry
DOI: https://doi.org/10.1007/978-3-642-35943-9_492-1



Single-molecule fluorescence resonance energy transfer (smFRET) is a technique used to measure nanometer-scale distances between specific sites on an individual molecule, usually as a function of time.


In this entry, we review smFRET, a powerful technique for measuring distances and monitoring dynamics at the molecular scale. In this technique, the researcher monitors distances between two or more individual fluorescent labels by measuring how efficiently electronic energy is transferred between them, a phenomenon known as Förster resonance energy transfer (FRET). Its power lies in its ability to detect distance changes as small as ~0.3 nm in individual molecules (or pairs of molecules) within a heterogeneous population (Roy et al. 2008). Although it requires a specialized fluorescence microscope, smFRET...

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  1. Benítez JJ, Keller AM, Chen P (2010) Nanovesicle trapping for studying weak protein interactions by single-molecule FRET. Methods Enzymol 472:41–60CrossRefPubMedPubMedCentralGoogle Scholar
  2. Blanco M, Walter NG (2010) Analysis of complex single molecule FRET time trajectories. Methods Enzymol 472:153–178CrossRefPubMedPubMedCentralGoogle Scholar
  3. Ha T, Enderle T, Ogletree DF, Chemla DS, Selvin PR, Weiss PR (1996) Probing the interaction between two single molecules: fluorescence resonance energy transfer between a single donor and a single acceptor. Proc Natl Acad Sci U S A 93:6264–6268CrossRefPubMedPubMedCentralGoogle Scholar
  4. Hwang LC, Hohlbein J, Holden SJ, Kapanidis AN (2009) Single-molecule FRET: methods and biological applications. In: Hinterdorfer P, van Oijen A (eds) Handbook of single-molecule biophysics, vol 1. Springer, Dordrecht, pp 129–163CrossRefGoogle Scholar
  5. Krishnan R, Blanco MR, Kahlscheuer ML, Abelson J, Guthrie C, Walter NG (2013) Biased Brownian ratcheting leads to pre-mRNA remodeling and capture prior to first-step splicing. Nat Struct Mol Biol 20:1450–1457CrossRefPubMedPubMedCentralGoogle Scholar
  6. Roy R, Hohng S, Ha T (2008) A practical guide to single-molecule FRET. Nat Methods 5:507–516CrossRefPubMedPubMedCentralGoogle Scholar
  7. Sakmann BA, Neher E (eds) (2009) Single channel recording, 2nd edn. Springer, New YorkGoogle Scholar
  8. Stryer L (1978) Fluorescence energy transfer as a spectroscopic ruler. Annu Rev Biochem 47:819–846CrossRefPubMedGoogle Scholar
  9. Uphoff S, Holden SJ, Le Reste L, Periz J, van de Linde S, Heilemann M, Kapanidis AN (2010) Monitoring multiple distances within a single molecule using switchable FRET. Nat Methods 7:831–836CrossRefPubMedGoogle Scholar
  10. Walter NG, Huang C-Y, Manzo AJ, Sobhy MA (2008) Do-it-yourself guide: how to use the modern single-molecule toolkit. Nat Methods 5:475–489CrossRefPubMedPubMedCentralGoogle Scholar
  11. Weiss S (1999) Fluorescence spectroscopy of single biomolecules. Science 283:1676–1683CrossRefPubMedGoogle Scholar

Copyright information

© European Biophysical Societies' Association (EBSA) 2018

Authors and Affiliations

  • Alexander E. Johnson-Buck
    • 1
    Email author
  • Mario R. Blanco
    • 2
  • Nils G. Walter
    • 1
  1. 1.Department of ChemistryThe University of MichiganAnn ArborUSA
  2. 2.Department of Cellular and Molecular BiologyThe University of MichiganAnn ArborUSA

Section editors and affiliations

  • Nils G. Walter
    • 1
  1. 1.Department of ChemistryThe University of MichiganAnn ArborUSA