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

Project Summary 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.