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

TL;DR

This paper introduces NovArch, a novel genetically encoded voltage indicator activated by two-photon excitation, enabling high-resolution, high-contrast voltage imaging in brain tissue with improved sensitivity and temporal resolution.

Contribution

The study develops a high-throughput screening method to identify a new Archaerhodopsin-based voltage indicator with unique photophysical properties, advancing voltage imaging technology.

Findings

NovArch enables optical mapping of dendritic action potentials.

Two-photon excitation enhances fluorescence catalytically, improving imaging efficiency.

The system achieves high contrast and temporal resolution in brain tissue.

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 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.