Single-molecule tracking in living microbial cells.

Some microbes are referred to as model organisms because they are easy to study in the laboratory and hold the ability to retain their characteristics during DNA replication, DNA transcription, and other fundamental processes. Studying these microbes in living cells via single-molecule imaging allows us to better understand these processes at highly improved spatiotemporal resolution. Single particle tracking photoactivated localization microscopy (sptPALM) is a robust tool for detecting the positions and motions of individual molecules with tens of nanometers of spatial and millisecond temporal resolution in vivo, providing insights into intricate intracellular environments that traditional ensemble methods cannot. With this approach, the fluorophores are photoactivated stochastically, a series of images are recorded, and the positions of fluorophores are identified in these images, and ultimately the locations are linked together to yield trajectories of individual molecules. Quantitative kinetic and spatial information, such as reaction rates, diffusion coefficients, and localization maps, can be obtained by further analysis. Here, we present a single-molecule tracking protocol that includes sample preparation, data acquisition and brief data processing. This protocol will enable researchers to directly unveil molecular and cellular mechanisms underlying the essential biological processes.