Protocol for single-molecule labeling and tracking of bacterial cell division proteins

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.