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All cells maintain ionic gradients across their plasma membranes, producing transmembrane potentials (V mem ). Mounting evidence suggests a relationship between resting V mem  and the physiology of non-excitable cells with implications in diverse areas, including cancer, cellular differentiation, and body patterning. A lack of non-invasive methods to record absolute V mem  limits our understanding of this fundamental signal. To address this need, we developed a fluorescence lifetime-based approach (VF-FLIM) to visualize and optically quantify V mem  with single-cell resolution in mammalian cell culture. Using VF-FLIM, we report V mem  distributions over thousands of cells, a 100-fold improvement relative to electrophysiological approaches. In human carcinoma cells, we visualize the voltage response to growth factor stimulation, stably recording a 10–15 mV hyperpolarization over minutes. Using pharmacological inhibitors, we identify the source of the hyperpolarization as the Ca 2+ -activated K +  channel K Ca 3.1. The ability to optically quantify absolute V mem  with cellular resolution will allow a re-examination of its signaling roles.

Optical estimation of absolute membrane potential using fluorescence lifetime imaging