Kobayashi, A.; Park, J.; Mine-Hattab, J.; Fernandez, F. G.

Live-cell Single-Particle Tracking (SPT) is a powerful super resolution microscopy approach. It allows us to observe live recordings of individual molecules in living cells at high temporal and spatial resolution (100 Hz, 20 nm). Here, we discuss the implementation and application of super-resolution microscopy to quantify the mobility of single molecules in living human cells. We first describe the human cell sample preparation for SPT with an experimental time line overview, then super-resolution microscope setting and procedure to acquire SPT films for further SPT video analyses. We provide a practical guide to determine the quality of acquired SPT films in particular, number of photons and fluorescent background. Furthermore, we introduce several SPT video analysis software comparing their advantages and limitations. Expectantly, this SPT protocol will serve as a foundation for a better SPT performance.