Two-Photon Photoactivated Voltage Imaging in Tissue with an Archaerhodopsin-Derived Reporter
September 20th, 2018 DAAN BRINKS Delft University of Technology

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.

Seminar, September 20, 2018, 17:00. ICFO’s Seminar Room

Hosted by Prof. Niek van Hulst