A high-throughput all-optical platform for mapping gene perturbations to electrophysiology

Electrical phenotyping remains a bottleneck in linking genes to cellular function. We developed a high-throughput all-optical electrophysiology platform combining an engineered human induced pluripotent stem cell (hiPSC) line, wide-field voltage imaging, and automated analysis. The hiPSC line integrates inducible NGN2, CRISPR interference, and degron-gated expression of Voltron2 and CheRiff, enabling neuronal differentiation, targeted gene knockdown, and optical electrophysiology. The microscope records approximately 1,500 spiking glutamatergic neurons at 800 Hz across a 4x4 mm field, with larger fields supporting approximately 3,000 neurons at 660 Hz. Automated analysis extracts single-cell voltage traces and quantifies firing patterns, action-potential waveforms, and subthreshold dynamics. Arrayed knockdown of five sodium- and potassium-channel genes produced distinct electrophysiological phenotypes. The engineered hiPSC line also supported optical electrophysiology recordings from motor neurons, astrocytes, and cardiomyocytes. These results establish a scalable platform for high-throughput electrophysiological phenotyping and functional genomics in hiPSC-derived cells.