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Cellular membrane potential plays a key role in the formation and retrieval of memories in the metazoan brain, but it remains unclear whether such memory can also be encoded in simpler organisms like bacteria. Here, we show that single-cell-level memory patterns can be imprinted in bacterial biofilms by light-induced changes in the membrane potential. We demonstrate that transient optical perturbations generate a persistent and robust potassium-channel-mediated change in the membrane potential of bacteria within the biofilm. The light-exposed cells respond in an anti-phase manner, relative to unexposed cells, to both natural and induced oscillations in extracellular ion concentrations. This anti-phase response, which persists for hours following the transient optical stimulus, enables a direct single-cell resolution visualization of spatial memory patterns within the biofilm. The ability to encode robust and …