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arXiv · 2512.15587

High-speed optical microscopy for neural voltage imaging: Methods, trade-offs, and opportunities

Zhaoqiang Wang·Ruth R. Sims·Sheng Xiao·Ruixuan Zhao·Ohr Benshlomo·Zihan Zang·Jiamin Wu·Valentina Emiliani·Liang Gao
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Abstract

High-speed optical imaging of dynamic neuronal activity is essential yet challenging in neuroscience. While calcium imaging has been firmly established as a workhorse technique for monitoring neuronal activity, its limited temporal resolution and indirect measurement restrict its ability to capture rapid inhibitory and excitatory events and subthreshold voltage oscillations. In contrast, voltage imaging directly measures membrane potential fluctuations, providing a comprehensive and precise representation of neuronal circuit dynamics. Recent advancements in voltage-sensitive dyes and, particularly, genetically encoded voltage indicators have significantly enhanced the feasibility of voltage imaging, prompting the development of advanced fluorescence microscopy methods optimized for high-speed acquisition. However, achieving millisecond-scale temporal resolution remains challenging due to inherent trade-offs among imaging speed, spatial resolution, and signal-to-noise ratio. Conventional raster-scanning approaches, including confocal microscopy, are fundamentally limited by their slow frame rates, precluding the capture of rapid neuronal events from multiple neurons simultaneously. Alternative techniques such as random-access scanning, spatiotemporal multiplexing, and computational optical imaging have successfully addressed these constraints, enabling kilohertz-level imaging of neuronal activity in both two-dimensional and three-dimensional contexts. This review summarizes recent progress in high-speed optical microscopy for voltage imaging and discusses its transformative potential for neuroscience research.

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Zhaoqiang Wang, Ruth R. Sims, Sheng Xiao, Ruixuan Zhao, Ohr Benshlomo, Zihan Zang, Jiamin Wu, Valentina Emiliani, Liang Gao. 2025-12-17. High-speed optical microscopy for neural voltage imaging: Methods, trade-offs, and opportunities. https://doi.org/10.1038/s41566-026-01965-5

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