Imaging single molecular motor motility with total internal reflection fluorescence microscopy (TIRFM)

Total internal reflection fluorescence microscopy (TIRFM) allows fluorescent molecules on or near the plasma membrane to be visualized with a very high signal-to-noise ratio. This strategy has been very successful for imaging molecular machines as they move and do work. We provide here a general protocol for imaging single molecular motors as they move along microtubule tracks. Our protocol is designed for the study of cytoplasmic dynein purified from Saccharomyces cerevisiae, but it represents a general framework for any in vitro single-molecule assay.