Imaging single molecules using total internal reflection fluorescence microscopy (TIRFM).

Total internal reflection fluorescence microscopy (TIRFM) allows fluorescent molecules to be visualized with an unparalleled signal-to-noise ratio. This is achieved by illuminating only the molecules that are within a thin volume near the coverslip surface but not those that are deeper in solution. Using this technique, fluorescent molecules within approximately 100 nm of the coverslip can be visualized, and single molecules that are separated by a distance greater than the diffraction limit (approximately 200 nm) can be individually resolved. The application of centroid-tracking methods allows subdiffraction-limited localization precision as low as 1 nm. Additionally, by combining centroid-tracking methods with recent advances in fluorophore technology and imaging methods, even those molecules that are present at high concentrations and closer to one another than the diffraction limit can be individually imaged. TIRF is ideally suited for studying protein dynamics on or near the plasma membrane. Although TIRFM was pioneered in the 1980s, it was not until the mid-1990s that single biological molecules were imaged directly. The explosion of new fluorescent proteins, new organic dyes, and quantum dots (Qdots), along with commercially available TIRFMs, has made this technique increasingly useful and accessible to biologists. In this review, we first describe the theory of TIRFM. We then give a detailed description of important considerations for setting up a TIRFM, based on commercially available systems, and review considerations for purification and labeling of proteins. Finally, we discuss new techniques that allow single molecules to be imaged at cellular concentrations and with super-resolution localization.