  Robust voltage imaging in tissue remains a technical challenge. Existing
combinations of genetically encoded voltage indicators (GEVIs) and microscopy
techniques cannot simultaneously achieve sufficiently high voltage sensitivity,
background rejection, and time resolution for high-resolution mapping of
sub-cellular voltage dynamics in intact brain tissue. We developed a pooled
high-throughput screening approach to identify Archaerhodopsin mutants with
unusual photophysical properties. After screening ~105 cells, we identified a
novel GEVI, NovArch, whose 1-photon near infrared fluorescence is reversibly
enhanced by weak 2-photon excitation. Because the 2-photon excitation acts
catalytically rather than stoichiometrically, high fluorescence signals,
optical sectioning, and high time resolution are achieved simultaneously, at
modest 2- photon laser power. We developed a microscopy system optimized for
NovArch imaging in tissue. The combination of protein and optical engineering
enhanced signal contrast sufficiently to enable optical mapping of
back-propagating action potentials in dendrites in acute mouse brain slice.
