TWO-PHOTON VOLTAGE IMAGING WITH RHODOPSIN-BASED GEVIS

Recording neuronal membrane potential with optical approaches overcomes the limitations of traditional electrical methods, promising minimally-invasive, real-time observation of population activity with unprecedented single-cell and single-spike resolution. Building on early work with voltage-sensitive dyes, advances in protein engineering have led to the development and continuous improvement of genetically-encoded voltage indicators (GEVIs). GEVIs used to detect action potentials, fall into two categories: ASAP-type indicators and those utilizing microbial rhodopsins as voltage sensing domain. For applications demanding two-photon (2P) illumination predominantly ASAP-type sensors have been used, while rhodopsin-based GEVIs have been reported to lose voltage sensitivity under 2P illumination and were mainly used under one-photon (1P) illumination. Here, we describe the rational design of a novel FRET-opsin GEVI, Jarvis, which combines Acetabularia rhodopsin with the brightest known fluorescent protein, AaFP1. The bright Jarvis expresses well in neurons, has comparable sensitivity and kinetics to pAce, and shows extraordinary photostability under 1P and 2P illumination. Both Jarvis and pAce are highly compatible with scanless 2P illumination, allowing to detect action potentials at kilohertz rates with high fidelity, while SNR was significantly reduced for 2P scanning approaches at similar conditions. Using Jarvis and pAce under scanless 2P illumination, we successfully recorded single action potentials in vitro and in vivo with unprecedented contrast (SNR > 5).