Objective-type total internal reflection microscopy (excitation) for single-molecule FRET

Cold Spring Harb Protoc. 2012 Nov 1;2012(11):1189-91. doi: 10.1101/pdb.prot072025.


Single-molecule (sm) fluorescence detection is a powerful method for studying biological events without time and population averaging. Förster (fluorescence) resonance energy transfer (FRET) is a spectroscopic technique in which the efficiency of energy transfer from donor to acceptor molecules is used to determine distances between molecules in the 30-80 Å range. Structural changes in biological molecules or relative motion between two interacting molecules can be detected by a change in FRET. A variant of smFRET is based on total internal reflection (TIR) microscopy, which can be set up in two ways, either using an oil-immersion (objective-type) or a water-immersion (prism-type) lens. This protocol describes objective-type TIR microscopy (excitation), including setup of the laser beam and epifluorescence microscopy, and conversion of the latter into TIR.

MeSH terms

  • Fluorescence Resonance Energy Transfer / methods*
  • Microscopy, Fluorescence / methods*