title: Prism-type total internal reflection microscopy for single-molecule FRET.
abstract: 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 the setup for prism-type TIR microscopy (excitation and emission), which has been a major imaging tool for smFRET since it was first introduced.
authors.forename: Chirlmin
authors.initials: C
authors.lastname: Joo
authors.forename: Taekjip
authors.initials: T
authors.lastname: Ha
mesh_terms: D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D008856:Microscopy, Fluorescence / Q000379:methods*; D008967:Molecular Biology / Q000379:methods*