Single-Molecule Fluorescence Resonance Energy Transfer

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 is employed in many laboratories worldwide and has been used to address questions about topics ranging from intermolecular interactions to macromolecular folding and catalysis. Although smFRET has only been technically possible since the mid-1990s, its theoretical foundations were laid much earlier through the pioneering work of Theodor Förster in the 1940s and through others who elucidated the distance dependence of FRET and its use as a molecular ruler (Stryer 1978). A separate branch of inquiry, the first single-molecule measurements of ion channels using the patch clamp technique, already established some of the core aspects of analyzing time-lapsed recordings from individual molecules in the 1970s and early 1980s (Sakmann and Neher 2009). By the mid-1990s, improvements in fluorescence detection brought about by total internal reflection fluorescence microscopy made it possible to measure the weak emission of single organic fluorophores under ambient conditions and, soon thereafter, smFRETwas realized (Ha et al. 1996).