Broader contextThe direct conversion of organic wastes and biomass to electricity with microbial fuel cells offers the potential for producing high-value, carbon-neutral energy, from inexpensive source materials. However, at present, the power output of microbial fuel cells is too low for most envisioned applications. Further optimization has been stymied by a lack of understanding of the factors controlling the activity of the microorganisms that colonize the anode of microbial fuel cells and are responsible for producing the current. Here we report on a novel approach which makes it feasible to image actively metabolizing and growing cells within the anode biofilm in real time with a confocal scanning laser microscope.G. sulfurreducens, a well studied current-producing microorganism, was engineered to express the red fluorescent protein, mcherry. The growth of the fluorescent cells on the anode was monitored over time. When a fluorescent dye that is sensitive to pH was introduced it was possible to measure pH throughout different layers of the biofilm. During active current production, the pH deep within the biofilm dropped to levels shown to inhibit the activity ofG. sulfurreducens. These results suggest that strategies to facilitate proton flux out of the biofilm may increase power output.