Abstract
Recent developments in single-molecule localization techniques using photoactivatable fluorescent proteins have allowed the probing of single-molecule motion in a living cell with high specificity, millisecond time resolution, and nanometer spatial resolution. Analyzing the dynamics of individual molecules at high densities in this manner promises to provide new insights into the mechanisms of many biological processes, including protein heterogeneity in the plasma membrane, the dynamics of cytoskeletal flow, and clustering of receptor complexes in response to signaling cues. Here we describe the method of single-molecule tracking photoactivated localization microscopy (sptPALM) and discuss how its use can contribute to a quantitative understanding of fundamental cellular processes.