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Genetically encoded voltage indicators (GEVIs) have emerged as a technology to optically record neural activity with genetic specificity and millisecond-scale temporal resolution using fluorescence microscopy. GEVIs have demonstrated ultra-fast kinetics and high spike detection fidelity  in vivo , but existing red-fluorescent voltage indicators fall short of the response and brightness achieved by green fluorescent protein-based sensors. Furthermore, red-fluorescent GEVIs suffer from incomplete spectral separation from green sensors and blue-light-activated optogenetic actuators. We have developed Ace-mScarlet, a red fluorescent GEVI that fuses Ace2N, a voltage-sensitive inhibitory rhodopsin, with mScarlet, a bright red fluorescent protein (FP). Through fluorescence resonance energy transfer (FRET), our sensor detects changes in membrane voltage with high sensitivity and brightness and has kinetics comparable to the fastest green fluorescent sensors. Ace-mScarlet’s red-shifted absorption and emission spectra facilitate virtually complete spectral separation when used in combination with green-fluorescent sensors or with blue-light-sensitive sensors and rhodopsins. This spectral separation enables both simultaneous imaging in two separate wavelength channels and high-fidelity voltage recordings during simultaneous optogenetic perturbation.

A high-speed, bright, red fluorescent voltage sensor to detect neural activity