Optical estimation of absolute membrane potential using fluorescence lifetime imaging

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.