@article {Lazzari-Dean519736, author = {Julia R. Lazzari-Dean and Anneliese M.M. Gest and Evan W. Miller}, title = {Optical determination of absolute membrane potential}, elocation-id = {519736}, year = {2019}, doi = {10.1101/519736}, publisher = {Cold Spring Harbor Laboratory}, abstract = {All cells maintain ionic gradients across their plasma membranes, producing transmembrane potentials (Vmem). Mounting evidence suggests a relationship between resting Vmem 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 Vmem 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 Vmem with single-cell resolution. Using VF-FLIM, we report Vmem distributions over thousands of cells, a 100-fold improvement relative to electrophysiological approaches. In human carcinoma cells, we visualize the voltage response to epidermal growth factor stimulation, stably recording a 10-15 mV hyperpolarization over minutes. Using pharmacological inhibitors, we identify the source of the hyperpolarization as the Ca2+-activated K+ channel Kca3.1. The ability to optically quantify absolute Vmem with cellular resolution will allow a re-examination of its roles as a cellular signal.}, URL = {https://www.biorxiv.org/content/early/2019/01/14/519736}, eprint = {https://www.biorxiv.org/content/early/2019/01/14/519736.full.pdf}, journal = {bioRxiv} }