A versatile, positive-going voltage indicator that enables accessible two-photon recordings in vivo

Abstract Genetically encoded voltage indicators (GEVIs) enable cell-type–specific optical readout of membrane potential, but two-photon (2P) spike detection has been hampered by low signal-to-noise and ultrafast off-kinetics, restricting use to specialized microscopes. We introduce FORCE1s, a green, positive-going GEVI engineered to make robust 2P voltage imaging broadly accessible. FORCE1s brightens from a dark baseline during depolarization, reports spikes with ∼100% ΔF/F in awake mice, and displays repolarization kinetics that are tuned for reliable spike detection at sub-kilohertz frame rates. As a result, FORCE1s supports spike-resolved multi-cell recordings on standard resonant-scanning microscopes, and further scales to larger fields of view and neuron counts on advanced modalities. FORCE1s also enables multiplexed voltage–neurotransmitter imaging and extended recordings in freely moving mice using a compact, affordable MEMS-based 2P miniscope. Together, these advances establish FORCE1s as a community-ready tool that democratizes deep-tissue voltage imaging across platforms and experimental contexts. Competing Interest Statement F.S.-P. holds a US patent for a voltage sensor design (patent #US9606100 B2) that encompasses the GEVIs reported here. BM is a co-founder, employee, and shareholder of Karthala System, the commercial provider of microscopes implementing ULoVE. The remaining authors declare that they have no competing interests.