Kilohertz-rate two-photon voltage imaging of population dynamics in vivo

Abstract

Understanding how neural circuits compute requires capturing voltage dynamics across large neuronal populations with millisecond resolution in vivo. However, two-photon voltage imaging remains fundamentally limited by a trade-off among imaging speed, field of view, and excitation efficiency. We introduce HS2PM, a hybrid scanning two-photon microscope that overcomes this bottleneck, enabling kilohertz-rate imaging over a 650 x 524 m2 field of view at single-cell resolution while preserving the photon efficiency of single-point excitation. HS2PM stably records deep-layer membrane potentials from over 160 neurons simultaneously, with minimal photobleaching and phototoxicity. It resolves both spikes and subthreshold voltage dynamics in vivo, revealing how these jointly shape sensory adaptation and population coding. Beyond voltage imaging, HS2PM supports high-speed fluorescence lifetime and vascular flow imaging, establishing a multimodal platform for dissecting fast circuit dynamics with precision previously inaccessible to optical methods.