A practical guide to single-molecule FRET

Nat Methods. 2008 Jun;5(6):507-16. doi: 10.1038/nmeth.1208.


Single-molecule fluorescence resonance energy transfer (smFRET) is one of the most general and adaptable single-molecule techniques. Despite the explosive growth in the application of smFRET to answer biological questions in the last decade, the technique has been practiced mostly by biophysicists. We provide a practical guide to using smFRET, focusing on the study of immobilized molecules that allow measurements of single-molecule reaction trajectories from 1 ms to many minutes. We discuss issues a biologist must consider to conduct successful smFRET experiments, including experimental design, sample preparation, single-molecule detection and data analysis. We also describe how a smFRET-capable instrument can be built at a reasonable cost with off-the-shelf components and operated reliably using well-established protocols and freely available software.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.
  • Review

MeSH terms

  • Algorithms
  • Biophysics / instrumentation
  • Biophysics / methods*
  • Biotechnology / methods
  • Biotin
  • Biotinylation
  • Calibration
  • Fluorescence Resonance Energy Transfer / instrumentation*
  • Fluorescence Resonance Energy Transfer / methods*
  • Fluorescent Dyes / pharmacology
  • Microchemistry / instrumentation
  • Microchemistry / methods
  • Oxygen / chemistry
  • Quantum Theory
  • Software
  • Spectrophotometry / methods


  • Fluorescent Dyes
  • Biotin
  • Oxygen