Electrochemiluminescence microscopy for real-time, single-cell imaging of surface charge in electroactive bacteria.

Understanding how applied voltage regulates metabolism in microbial electrochemical systems is challenging due to the lack of adequate electrophysiological tools for microorganisms. Here, we present an imaging platform based on electrochemiluminescence (ECL) that allows real-time, femtocoulomb-scale quantification of surface charge in single bacterial cells. The method exploits the electrostatic enrichment of cationic luminophores at bacterial membranes to amplify ECL signals, thereby linking surface charge dynamics to intracellular electron metabolism and interfacial electron flow. Using Shewanella oneidensis MR-1 as a model, we show voltage-activated metabolic enhancement mediated by outer-membrane cytochromes. By modulating direct and indirect electron-transfer pathways, we reveal that cytochrome-dependent direct electron transfer initiates activation, while mediator-enabled indirect electron transfer prevents charge saturation and sustains elevated metabolic turnover. Furthermore, dual-parameter screening enables identification of bacterial subpopulations with high metabolic activity and efficient electron transfer capability. Thus, our work provides a framework for microbial electrophysiology at the single-cell level and opens potential avenues for engineering high-performance electroactive strains for bio-electrochemical applications.