Here, we present a protocol for labeling and tracking individual molecules, particularly cell division proteins in live bacterial cells. The protocol encompasses strain construction, single-molecule imaging, trajectory segmentation, and motion property analysis. The protocol enables the identification of distinctive motion states associated with different cell division proteins. Subsequent assessments of the dynamic behaviors of these proteins provide insights into their activities and interactions at the septum during cell division. For complete details on the use and execution of this protocol, please refer to Yang et al. (2021),1 Lyu et al. (2022),2 and Mahone et al. (2024).3.

Intracellular transport plays an important role in maintaining the physiological functions of cells. Here, we describe a protocol for 3D single-particle tracking within living cells. We detail the use of a two-focal imaging system and the analytical steps for quantifying 3D transport dynamics. This protocol can be used to characterize the intracellular diffusion and trafficking of macromolecules, nanoparticles, and endocytic vesicles in adherent cells. For complete details on the use and execution of this protocol, please refer to Jiang et al. (2022).