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

TL;DR

This paper reviews recent advances in high-speed optical microscopy techniques for neuronal voltage imaging, highlighting methods, trade-offs, and future opportunities for capturing rapid neural activity.

Contribution

It provides a comprehensive overview of recent technological developments enabling millisecond-scale voltage imaging in neuroscience.

Findings

Advanced microscopy methods now enable kilohertz-level neuronal imaging.

Trade-offs among speed, resolution, and signal-to-noise remain a key challenge.

Alternative scanning techniques improve the capture of rapid neural events.

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.