Real-Time Monitoring of Self-Repair in Single Bacteria after Photodamage by Synchronous...

Understanding how individual bacteria recover from cellular damage is essential for clarifying the basic principles of self-repair. However, most existing single-cell approaches often rely on extrinsic reporters or invasive methods, which perturb native metabolism and preclude longitudinal kinetic tracking. Here, we introduce a label-free methodology, termed single-bacteria bioluminescence recovery after photobleaching (BRAP), for real-time monitoring of metabolic self-repair in individual luminescent bacteria following defined photodamage. By using intrinsic bacterial bioluminescence (BL) as a real-time and noninvasive reporter closely coupled to central metabolism, BRAP enabled continuous tracking of the recovery trajectory in hundreds of single bacteria within only tens of minutes. Quantitative analysis unveiled robust recovery kinetics but substantial cell-to-cell heterogeneity in both the initial metabolic activity and repair rates. To mechanistically dissect the biochemical drivers of recovery, we integrated synchronous BL and flavin mononucleotide (FMN) autofluorescence (auto-FL) imaging, allowing real-time, temporally matched visualization of both the metabolic state and flavin regeneration. Comparative analysis of BL and FMN auto-FL revealed that successful metabolic recovery primarily reflects FMN and substrate regeneration, whereas incomplete restoration arises from partial irreversible luciferase inactivation. Overall, this work establishes an endogenous, label-free single-cell framework for monitoring metabolic recovery in naturally bioluminescent bacteria, laying the methodological foundation for future extensions to engineered luminous strains and single-cell studies of stress-induced self-repair.