Enabling high-fidelity population voltage imaging with next-generation optical platforms and indicators

The National Institute of Mental Health awarded The Rockefeller University $1,443,030 on September 20, 2025, under Mental Health Research Grants (CFDA 93.242) to develop an optimized two-photon optical imaging system for high-fidelity population voltage imaging in intact neural tissue using genetically encoded voltage indicators (GEVIs).

The recipient will design and implement a two-photon multiplexing platform adapted to the unique requirements of GEVIs, incorporating a novel spike detection algorithm. The system extends prior work on mesoscale volumetric calcium imaging to voltage imaging and is designed to achieve imaging from fields of view several hundred microns wide at 750 Hz and depths of approximately 500 microns, with smaller fields achievable at speeds up to 2 kHz. The optical platform will enable iterative development of voltage indicators with tailored kinetics and support in vivo screening and diverse biological applications to overcome current limitations restricting voltage imaging to small neuron populations, superficial cortical layers, or sparse labeling conditions.

Work will be performed in New York, NY 10065 with a period of performance through June 30, 2030. Baylor College of Medicine serves as a sub-recipient, conducting screening for two-photon-optimized GEVIs, performing in vitro characterization of identified indicators, and sharing new indicators with The Rockefeller University in the form of plasmids and adeno-associated viruses along with performance data (sub-award R01MH141913-SUB00000505, awarded December 18, 2025).

Abstract

Simultaneously monitoring neuronal activity in large populations with single-neuron resolution in vivo is critical for understanding the mechanisms underlying brain function and the generation of flexible, adaptive behavior. Genetically encoded voltage indicators (GEVIS) offer a promising approach for optically capturing electrical membrane potentials, enabling spatially resolved recordings with genetic specificity. However, both the intrinsic properties and the current limitations of GEVIS present significant challenges for the design and implementation of optical systems, particularly of two-photon (2P) imaging systems, that are maximally adapted to GEVIS. These include their millisecond response times, membrane localization, low brightness, and limited signal-to-noise ratio (SNR). Consequently, current voltage imaging techniques have been restricted to small neuron populations, superficial cortical layers, or sparse labeling conditions. In our previous work on calcium imaging, we demonstrated mesoscale volumetric imaging of up to one million neurons in the mouse cortex by implementing the many-fold axial multiplexing module (MAXIMUM). This project extends that conceptual framework to voltage imaging while incorporating a novel spike detection algorithm. The proposed optical platform is designed in a principled manner to deliver a highly optimized 2P multiplexing system that meets the unique requirements of GEVIS in terms of temporal, spatial, and energy efficiency. This system will drive iterative development of voltage indicators with tailored kinetics, while its versatility enables efficient in vivo screening and diverse biological applications. Among its capabilities, the system will allow imaging from fields of view several hundred microns wide at 750 HZ and depths of ~500 ΜM. Smaller fovs will be achievable at higher speeds (up to 2 KHZ) which will enable detection of single spikes within bursts. A high-sensitivity mode for detecting sub-threshold potentials will also be implemented. By combining this platform with an innovative excitation strategy and the CO-development of advanced GEVIS, we aim to achieve targeted multi-plane population voltage imaging of large populations of neurons in the mouse brain.