The recent explosion in the availability and variety of fluorescent proteins, new organic dyes and quantum dots has been a driving force in the growing use of Total Internal Reflection Fluorescence Microscopy (TIRFM). TIRFM only illuminates molecules that are within a thin volume near the coverslip surface of a specimen and not those deeper in solution. This allows for an unparalleled signal-to-noise ratio and tremendous resolution. In the March issue of Cold Spring Harbor Protocols, Samara Reck-Peterson, Nathan Derr and Nico Stuurman present Imaging Single Molecules Using Total Internal Reflection Fluorescence Microscopy (TIRFM), which includes an overview of the theory behind TIRFM, considerations for TIRFM setup and purification/labeling of proteins, and a discussion of new techniques for imaging single molecules with super-resolution localization. In addition, the group offers step-by-step protocols for Determining Single-Molecule Intensity as a Function of Power Density and Imaging Single Molecular Motor Motility with TIRFM. An example of TIRFM imaging of single dynein molecules labeled with TMR (green) moving along axonemal microtubules labeled with Cy5 (red) can be seen here.