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Screening fluorescent voltage probes with spontaneously spiking HEK cells (779.5)

A major difficulty for imaging brain activity comes from the lack of voltage‐sensitive probes that have fast, sensitive and bright characteristics. Unlike other probe development challenges, screening fluorescent voltage probes has been hampered by the low throughput of patch‐clamp characterization. We introduce a line of non‐fluorescent HEK cells that stably express NaV 1.3 and KIR 2.1 and generate spontaneous electrical action potentials. These cells enable rapid, electrode‐free screening of speed and sensitivity of voltage sensitive dyes or fluorescent proteins on a standard fluorescence microscope. We screened a small library of mutants of Archaerhodopsin 3 (Arch) in spiking HEK cells and identified two mutants with greater voltage‐sensitivity than found in previously published Arch voltage indicators.