Video-based pooled screening yields improved far-red genetically encoded voltage indicators

Video-based screening of pooled libraries is a powerful approach for directed evolution of biosensors because it enables selection along multiple dimensions simultaneously from large libraries. Here we develop a screening platform, Photopick, which achieves precise phenotype-activated photoselection over a large field of view (2.3 × 2.3 mm, containing >10³ cells, per shot). We used the Photopick platform to evolve archaerhodopsin-derived genetically encoded voltage indicators (GEVIs) with improved signal-to-noise ratio (QuasAr6a) and kinetics (QuasAr6b). These GEVIs gave improved signals in cultured neurons and in live mouse brains. By combining targeted in vivo optogenetic stimulation with high-precision voltage imaging, we characterized inhibitory synaptic coupling between individual cortical NDNF (neuron-derived neurotrophic factor) interneurons, and excitatory electrical synapses between individual hippocampal parvalbumin neurons. The QuasAr6 GEVIs are powerful tools for all-optical electrophysiology and the Photopick approach could be adapted to evolve a broad range of biosensors.