Single-Molecule FRET with Total Internal Reflection Microscopy

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. This article focuses primarily on smFRET based on total internal reflection (TIR) microscopy. It begins with discussions of dye choice and labeling of nucleic acids and proteins. These are followed by information on surface preparation and data acquisition. Various methods of data analysis are then presented, as is information on setting up TIR microscopy, both the objective and the prism types.