Fluorogenic Probes for Real-Time Tracking of Bacterial Cell Wall Dynamics with Nanoscopy

The bacterial cell wall, an essential structure for maintaining cell morphology and protecting against environmental hazards, is predominantly composed of peptidoglycan (PG). This intricate macromolecule undergoes dynamic synthesis and remodeling throughout the cell cycle. Despite its importance, monitoring PG dynamics in live cells, particularly with detailed spatial distribution, poses significant challenges. To this end, we present a series of rhodamine-based fluorogenic probes specifically optimized for real-time and super-resolution imaging of PG synthesis. By fine-tuning the self-aggregation of the probes through the incorporation of hydrophobic linkers, we achieved a substantial reduction in background fluorescence and significant fluorogenicity after labeling. These advancements have enabled us to attain wash-free labeling across a diverse array of bacterial species. Our approach facilitates the direct visualization of PG synthesis patterns, enabling the quantification of septal PG (sPG) synthesis rates in living bacterial cells. Furthermore, it allows for simultaneous imaging of cell division machinery in living cells via both two-dimensional (2D) and three-dimensional (3D) STED microscopy. This study provides a powerful toolkit for investigating the architecture and dynamics of the bacterial cell wall, paving new paths for research on PG-related cellular processes.