Enabling Extended Widefield Voltage Imaging with Low Phototoxicity

/ABSTRACT Membrane voltage regulation is fundamental to neuronal signaling, and its disruption is implicated in a wide range of neurological diseases. Because neurons encode and transmit information through rapid voltage changes, there is a critical need for tools that can faithfully capture these electrical events. Existing approaches – such as patch-clamp recordings, voltage-sensitive dyes, and genetically encoded calcium indicators – each have limitations, often lacking the necessary combination of speed, resolution, and cell-type specificity to fully resolve fast and localized voltage dynamics in genetically defined groups of neurons. Genetically encoded voltage indicators (GEVIs) offer a promising solution. These protein-based sensors convert changes in membrane potential into optical signals, allowing real-time, cell-type-specific recordings of neuronal activity with subcellular spatial precision and submillisecond temporal resolution. Unlike calcium indicators, GEVIs can resolve single spikes, subthreshold events, and fast dynamics across populations. Despite this potential, most current GEVIs require high- intensity illumination to achieve usable signals under widefield one-photon (1P) imaging – introducing phototoxicity. 1P widefield setups can image populations of cells, are relatively affordable, and easily adoptable by most neuroscience labs, especially with the increasing availability of fast scientific CMOS cameras. To fully harness these advantages, GEVIs must be optimized specifically for low-light, high-SNR 1P performance. This project aims to enhance GEVI performance through a multi-faceted approach utilizing advanced imaging technologies, optimizing codon usage and trafficking signals, and exploring alternative voltage-sensitive domains. Three specific aims will guide this work: (1) utilize high-throughput screening to rapidly optimize GEVI characteristics, (2) improve the signal- to-noise ratio (SNR) by optimizing GEVI expression levels and enhancing their trafficking to the plasma membrane, and (3) develop alternative GEVI architectures to enhance voltage sensitivity and photostability. By achieving the proposed aims, this research will produce next-generation voltage indicators that meet the growing need for precise, noninvasive tools to monitor neuronal activity in real-time. These improved sensors will have wide-reaching applications, from elucidating the basic mechanisms of neural computation to enabling new insights into the pathophysiology of brain disorders. This work will bridge the gap between fundamental discovery and translational research to improve neurological health. Project Number: 1F31NS147837-01 | Fiscal Year: 2026 | NIH Institute/Center: National Institute of Neurological Disorders and Stroke (NINDS) | Principal Investigator: Adrian Lankenau Ahumada | Institution: RICE UNIVERSITY, HOUSTON, TX | Award Amount: $53,114 | Activity Code: F31 | Study Section: Special Emphasis Panel[ZRG1 F03B-W (20)] View on NIH RePORTER: https://reporter.nih.gov/project-details/11317782