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bioRxiv · 10.1101/211946

Two-photon photoactivated voltage imaging in tissue with an Archaerhodopsin-derived reporter

Chien, M.-P.·Brinks, D.·Adam, Y.·Bloxham, W.·Kheifets, S.·Cohen, A. E.
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Abstract

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 highresolution 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.

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Chien, M.-P., Brinks, D., Adam, Y., Bloxham, W., Kheifets, S., Cohen, A. E.. 2017-10-31. Two-photon photoactivated voltage imaging in tissue with an Archaerhodopsin-derived reporter. https://doi.org/10.1101/211946

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