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bioRxiv · 10.1101/2025.07.02.662755

A Compact Two-Photon Module for Simultaneous Dual-Depth, Multi-Region in vivo Brain Imaging

Zhao, Y.·Gu, X.·Kong, C.·Liu, S.·Zhu, Y.·Su, J.·Wu, J.·Xu, F.·Chen, L.·Mao, Y.·Li, B.
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Abstract

Understanding how distributed brain regions interact across cortical depths is crucial for uncovering the neural basis of behavior and cognition. Two-photon microscopy (2PM) excels at imaging the superficial layer, while three-photon microscopy (3PM) penetrates deep tissues but lacks sufficient speed for capturing fast superficial dynamics. Here, we present a compact two-photon imaging module (2PIM) that seamlessly integrates with existing 3PM to achieve simultaneous, high-speed imaging of superficial and deep brain regions, while preserving the deep tissue penetration of 3PM. This dual-modality system allows for remote and independent control of imaging planes in both axial and lateral dimensions. Leveraging this technology, we discovered context-dependent modulation of hippocampal CA1 and visual cortex responses to identical tactile stimuli, layer-specific temporal dynamics in sensory cortex during air puff stimulation, and depth-divergent neuronal activity patterns in motor cortex across acute-to-chronic pain. These results highlight the systems potential for dissecting the spatiotemporal coordination of distributed brain circuits.

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Zhao, Y., Gu, X., Kong, C., Liu, S., Zhu, Y., Su, J., Wu, J., Xu, F., Chen, L., Mao, Y., Li, B.. 2025-07-05. A Compact Two-Photon Module for Simultaneous Dual-Depth, Multi-Region in vivo Brain Imaging. https://doi.org/10.1101/2025.07.02.662755

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