Enabling extended widefield voltage imaging with low phototoxicity

The National Institute of Neurological Disorders and Stroke awarded William Marsh Rice University $105,652 on September 10, 2026, under the Extramural Research Programs in the Neurosciences and Neurological Disorders program (CFDA 93.853) to develop genetically encoded voltage indicators (GEVIs) optimized for low-phototoxicity widefield one-photon imaging of neuronal activity.

The project addresses a critical need for tools that capture rapid voltage changes underlying neuronal signaling without the phototoxicity limitations of current GEVI approaches. Existing voltage-recording methods—patch-clamp recordings, voltage-sensitive dyes, and genetically encoded calcium indicators—lack the necessary combination of speed, spatial resolution, and cell-type specificity to resolve fast, localized voltage dynamics in defined neuronal populations. The work will enhance GEVI performance through high-throughput screening to optimize protein characteristics, codon usage, and trafficking signals, while exploring alternative voltage-sensitive protein domains. The resulting sensors will convert membrane potential changes into optical signals, enabling real-time, cell-type-specific recordings with subcellular spatial precision and submillisecond temporal resolution. One-photon widefield imaging platforms offer relative affordability and broad adoptability across neuroscience laboratories, particularly with increasingly available fast scientific CMOS cameras.

Performance occurs in Houston, Texas. The period of performance runs through March 31, 2029.

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.