Extracellular Fluorescence Recording of BacFlash Dynamics Reveals Two Distinct Modes of Proton Extrusion

BacFlash is a recently described transient bacterial process characterized by synchronized intracellular alkalization, membrane depolarization, and reactive oxygen species (ROS) generation, driven by regulated proton efflux. This phenomenon reveals a previously unrecognized mode of rapid physiological regulation in bacteria. Current techniques for studying BacFlash dynamics rely on intracellular fluorescent labeling with organic dyes or fluorescent proteins sensitive to pH, membrane potential, or ROS. Here, we introduce an extracellular, label-free fluorescence imaging strategy for real-time monitoring of BacFlash-associated proton dynamics at the single-cell level. By confining individual E. Coli cells within femtoliter-scale microwells and minimizing extracellular buffering, we directly quantified transient proton extrusion, revealing an estimated release of ∼105-106 protons per cell per second-a parameter not previously reported. Further analysis under alkaline conditions uncovered two kinetically distinct forms of BacFlash, differing by over an order of magnitude in proton efflux rates and durations. These dynamic patterns were further modulated by ultraviolet stimulation. Our findings demonstrate that BacFlash is not a stochastic fluctuation but a tunable, metabolically linked signaling process. Moreover, our ability to quantify proton translocation at single-cell resolution opens new avenues for mechanistic studies of bacterial bioenergetics and antimicrobial strategies targeting proton-driven physiological processes.