Far-red Microscopy for Deep Voltage Imaging of Large Neuronal Populations

This proposal aims to overcome the current limitations in voltage imaging by developing high- speed microscopy techniques and new genetically engineered voltage indicators (GEVIs) optimized for deep-tissue imaging. The project brings together experts in microscopy, sensor development, and neuroscience to enable kHz-rate voltage imaging of hundreds of neurons down to cortical layer V in the mouse brain. Our first aim focuses on advancing microscope technology through two complementary approaches: a large-field-of-view (FOV) imaging system using targeted illumination and confocal line-scanning (TICO2) and a prism-based multiplane imaging system (Multi-P) that employs inter-plane deconvolution for gapless volumetric imaging. Both systems will be designed for high signal-to-background ratio (SBR) and fast imaging rates, addressing key challenges in capturing weak fluorescence signals from large neuronal populations in thick tissue. In parallel, our second aim will focus on advancing indicator technology. Specifically, we will develop a new class of fully genetically encoded, far-red GEVIs (FAR-OUT) based on novel rhodopsin proteins. Through site-directed mutagenesis, these GEVIs will be designed to exhibit high brightness and a far-red spectrum that will enable significantly deeper tissue penetration and improved signal detection. The culmination of these efforts will be in-vivo mouse imaging experiments that benchmark and validate the performance of our newly developed microscopes and GEVIs. These experiments will demonstrate the ability to achieve high-SNR voltage imaging of hundreds of neurons across cortical layers and perform dense volumetric imaging of dendritic arbors. Ultimately, our project aims to provide the neurophysiology community with powerful new tools for studying neural circuits at unprecedented scales and depths, significantly advancing our understanding of brain function. Project Number: 1R01NS147518-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Jerome Mertz (+2 co-PIs) | Institution: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS), BOSTON, MA | Award Amount: $595,774 | Activity Code: R01 | Study Section: Special Emphasis Panel[ZRG1 NV-N (96)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11320517