Total Internal Reflection Fluorescence Microscopy (TIRFM) is a common method to study structural and dynamic processes at the single-molecule level. The principle is based on an optical phenomenon where electromagnetic radiation is not refracted but totally reflected on the boundary surface of two different media. An evanescent field is generated at the boundary surface and penetrates the medium with lower optical density where fluorescent molecules can be excited. This evanescent wave attains its maximum intensity at the surface and then exponentially decays with increasing penetration depth. Therefore, only molecules located near the surface are excited, as opposed to molecules situated at distances exceeding approx. 100 nm, and thus TIRF efficiently decreases background signals of the bulk solution and allows sensitive detection down to single molecules.