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

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.