Single-molecule fluorescence imaging and tracking in live bacteria: observing proteins in action

SUMMARY Live-cell study at the molecular level is essential for understanding the complexities of bacterial systems and protein functions in vivo . While ensemble biochemical approaches have been widely used to characterize protein functions, they suffer from heterogeneities arising from variations in individual cells’ different physiological states and microenvironments. Single-molecule techniques have enhanced our abilities to resolve these heterogeneities by enabling the observation of individual cells and molecules, one at a time. Particularly, fluorescence single-molecule localization microscopy (SMLM) and single-molecule tracking (SMT) have emerged as powerful tools for real-time, in vivo analysis of biomolecular dynamics. SMT can reveal the heterogeneity and dynamic behaviors of individual molecules within their native cellular contexts, providing insights that are often obscured in ensemble studies. This review highlights recent progress on imaging probes and modalities and focuses on several key scientific discoveries in microbial systems, including metal regulation, electron transfer, cell division, and DNA repair. By showcasing novel insights gained from single-molecule imaging techniques, this review underscores their vital role in advancing our understanding of cellular processes at the single-molecule level and discusses future challenges and opportunities in the field.