Advanced Voltage Indicators Tailored for Two-Photon Resonant Scanning Applications - SUMMARY Understanding brain function requires tools capable of recording membrane voltage with high spatial and temporal resolution, cell-type specificity, and compatibility with animal models. Genetically encoded voltage indicators (GEVIs), protein-based fluorescent biosensors of membrane potential, have the potential to achieve these specifications and enable optical measurements voltage dynamics in the brain. This project focuses on developing next-generation GEVIs optimized for two-photon (2P) microscopy, the gold standard for deep-tissue imaging in scattering brain tissues. Specifically, we aim to improve GEVI sensitivity for detecting subthreshold signals and enhance response kinetics to reliably capture rapid action potentials in awake, behaving animals. A key objective is to tune indicators for maximal performance when using resonant scanning microscopy, the most widely accessible two-photon imaging method. Key innovations include refining our high-throughput screening platform to identify fast-kinetics GEVI variants, engineering ultrasensitive GEVIs tailored for synaptic activity, and designing advanced subcellular targeting strategies to minimize background fluorescence. These efforts will incorporate directed evolution, synthetic dosage compensation circuits, and clustering strategies to enhance signal-to-noise ratios. Preliminary data demonstrate substantial advancements in GEVI sensitivity and voltage response dynamics, including new mutations that improve fluorescence changes across a broad voltage range. The optimized GEVIs will undergo rigorous validation in vitro and in vivo, combining state-of-the-art electrophysiology and 2P microscopy to benchmark their performance against existing tools. By addressing current limitations in GEVI technology, this project aims to expand the utility of voltage imaging, providing neuroscience researchers with powerful tools to explore neural computations, synaptic integration, and their disruption in neurological disorders. These innovations are expected to catalyze widespread adoption of GEVIs, advancing our understanding of brain function in health and disease
