Robinson, Jacob T.St-Pierre, François2023-09-012023-082023-08-09August 202Lu, Helen. "Developing Genetically Encoded Voltage Indicators for in vivo Neuroimaging." (2023) Diss., Rice University. https://hdl.handle.net/1911/115262.https://hdl.handle.net/1911/115262EMBARGO NOTE: This item is embargoed until 2025-08-01A fundamental goal of neuroscience is to decipher the neural activities underlying behaviors in vivo. Among existing tools for neural recording, genetically encoded voltage indicators (GEVIs) — protein-based fluorescent indicators whose brightness is directly modulated by membrane potential — hold the most promise for large-scale measuring of neural activities with cell-type specificity, subcellular spatial resolution, and sub-millisecond temporal resolution. However, current GEVIs have limited utility in vivo due to their suboptimal performance, especially under two-photon microscopy (2PM), a desired method for deep-tissue imaging. To address these limitations, we started from building a high-throughput screening platform that can evaluate all key metrics of a GEVI under one-photon illumination. Directed evolution on this platform led to JEDI-1P, a green-emitting GEVI optimized for widefield voltage imaging. With improved brightness, kinetics, sensitivity and photostability, JEDI-1P empowered chronic pan-cortical voltage imaging and robust detection of rapid voltage signals in behaving mice. Next, we sought to optimize GEVI for deep tissue imaging by extending the screening platform with a two-photon resonant scanning system. Using this 2P screening platform, we identified JEDI-2P, whose brightness, sensitivity and photostability under 2PM were all significantly improved over its parental indicator. We showed that JEDI-2P can capture voltage responses to visual stimuli in the amacrine cells of isolated mouse retina and the axonal projections of Drosophila interneurons. With excellent 2P photostability, JEDI-2P enabled prolonged continuous recording from individual cortical neurons in awake behaving mice with both resonant-scanning 2PM and ULoVE random-access microscopy. In particular, we highlighted that the improved sensitivity and brightness of JEDI-2P allowed the first high-fidelity voltage recording from mice Layer 5 cortical neurons as well as robust recordings of pairwise voltage correlations during behavior. Taken together, JEDI-2P fills the vacancy of a GEVI optimized for 2P applications and paves the way for long-term studying of deep brain neural activities. Finally, to enable all-optical electrophysiology and multi-spectral imaging under two-photon microscopy, we designed a red-emitting voltage indicator from scratch. After rationally engineering the interface between the chromophore and the voltage-sensing domain, we identified VADER1, the first red-emitting GEVI that has demonstrated the capability to resolve single action potentials in mice under two-photon illumination. We anticipate the expanded GEVI toolbox will enable high-throughput and real-time recording of action potentials from the genetically specified group of neurons in live animals, thereby helping interpret the computation mechanism of neural circuits with unprecedented spatiotemporal resolution.application/pdfengCopyright is held by the author, unless otherwise indicated. Permission to reuse, publish, or reproduce the work beyond the bounds of fair use or other exemptions to copyright law must be obtained from the copyright holder.Genetically encoded voltage indicatorGEVIVoltage imagingProtein engineeringWidefield imagingTwo-photon microscopyThesis2023-09-01