A Brief Introduction to Single-Molecule Fluorescence Methods

One of the more popular single-molecule approaches in biological science is single-molecule fluorescence microscopy, which is the subject of the following section of this volume. Fluorescence methods provide the sensitivity required to study biology on the single-molecule level, but they also allow access to useful measurable parameters on time and length scales relevant for the biomolecular world. Before several detailed experimental approaches are addressed, we first give a general overview of single-molecule fluorescence microscopy. We start with discussing the phenomenon of fluorescence in general and the history of single-molecule fluorescence microscopy. Next, we review fluorescent probes in more detail and the equipment required to visualize them on the single-molecule level. We end with a description of parameters measurable with such approaches, ranging from protein counting and tracking, to distance measurements with F�rster Resonance Energy Transfer and orientation measurements with fluorescence polarization.