Detecting rapid pan-cortical voltage dynamics in vivo with a brighter and faster voltage indicator

Widefield imaging with genetically encoded voltage indicators (GEVIs) is a promising approach for understanding the role of large cortical networks in the neural coding of behavior. However, the slow kinetics of current GEVIs limit their deployment for single-trial imaging of rapid neuronal voltage dynamics. Here, we developed a high-throughput platform to screen for GEVIs that combine fast kinetics with high brightness, sensitivity, and photostability under widefield one-photon illumination. Rounds of directed evolution produced JEDI-1P, a green-emitting fluorescent indicator whose performance is improved for all metrics. Next, we optimized a neonatal intracerebroventricular delivery method to achieve cost-effective and wide-spread JEDI-1P expression in mice. We also developed an approach to effectively correct optical measurements from hemodynamic and motion artifacts. Finally, we achieved stable brain-wide voltage imaging and successfully tracked gamma-frequency whisker and visual stimulations in awake mice in single trials, opening the door to investigating the role of high-frequency signals in brain computations.