Improving positively tuned voltage indicators for brightness and kinetics
The recent positively tuned ASAP4-family voltage indicators feature superior photostability compared to the negatively tuned ASAP3, but signal-to-noise ratios for spike detection were not significantly different. To improve spike detection by positively tuned ASAP indicators, we performed multiple rounds of structure-guided saturation mutagenesis of an ASAP4 template while screening directly for faster responses. Our resulting variants, ASAP6.1 and ASAP6b, demonstrated sufficient dynamic range and kinetics, and reported action potentials in vivo by one-photon and two-photon microscopy with high SNR and temporal resolution.