Tracking Single Molecules in Biomembranes: Is Seeing Always Believing?

The spatial organization of molecules in cell membranes and their dynamic interactions play a central role in regulating cell functions. Single-particle tracking (SPT), a technique in which single molecules are imaged and tracked in real time, has led to breakthrough discoveries regarding these spatiotemporal complexities of cell membranes. There are, however, emerging concerns about factors that might produce misleading interpretations of SPT results. Here, we briefly review the application of SPT to understanding the nanoscale heterogeneities of plasma membranes, with a focus on the unique challenges, pitfalls, and limitations that confront the use of nanoparticles as imaging probes for tracking the dynamics of single molecules in cell membranes.