Single-molecule gold-nanoparticle tracking

Gold nanoparticles, like single fluorophores, can be used to locate single molecules with nanometer accuracy. Unlike an optical trap, the gold particle label does not exert an external load, which is important for studying diffusive processes. Thus, a gold particle can be used analogously to a single fluorophore, providing similar information but with submillisecond time resolution. The features of gold-nanoparticle tracking (high temporal resolution, small label size, and lack of applied force) facilitate the characterization of structural properties of short-lived intermediates, as shown by our work with myosin V. This protocol provides details for gold-nanoparticle-tracking experiments, including flow cell construction, microscopy, and data analysis, along with a brief outline of actin and myosin preparation. Although details particular to our experiment are given, the approach should be generally applicable.