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<p>Understanding how neurons integrate synaptic inputs requires imaging techniques capable of capturing rapid, three-dimensional dendritic events. These processes occur on millisecond timescales and submicron spatial scales, exceeding the speed of conventional two-photon microscopy (2PM). We developed dual-view Bessel two-photon projection microscopy (dv-B2PM), a high-speed volumetric imaging approach that achieves 100 Hz whole-volume acquisition with synaptic-level resolution. dv-B2PM simultaneously records two orthogonal projections of the same 3D volume, preserving spatial information while minimizing ambiguity from structural overlap. Combining dv-B2PM with two-photon glutamate uncaging, we visualized 3D Ca²⁺ dynamics in neurons following localized stimulation. Multi-timescale analysis revealed dendrite-to-soma Ca²⁺ signal propagation, back propagated Ca²⁺ signal from the soma, and multi-frequency (5–40 Hz) Ca²⁺ transients activated along apical dendrites at speeds from ten of microns per second to millimeters per seconds. These findings demonstrate dv-B2PM as a powerful tool for direct visualization of 3D calcium dynamics associated with dendritic integration across extended neuronal structures, bridging the gap between optical imaging and the dynamic biophysics of neuronal integration.</p>

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Two-photon 3D imaging of optically stimulated neural activity at 100 Hz

  • 期刊论文

  • 出版模式：

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  • 发表日期：

    2026年7月3日

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  • Dongli Xu,
  • Fuu-Jiun Hwang,
  • Jun B. Ding,
  • Leilei Peng

摘要

Understanding how neurons integrate synaptic inputs requires imaging techniques capable of capturing rapid, three-dimensional dendritic events. These processes occur on millisecond timescales and submicron spatial scales, exceeding the speed of conventional two-photon microscopy (2PM). We developed dual-view Bessel two-photon projection microscopy (dv-B2PM), a high-speed volumetric imaging approach that achieves 100 Hz whole-volume acquisition with synaptic-level resolution. dv-B2PM simultaneously records two orthogonal projections of the same 3D volume, preserving spatial information while minimizing ambiguity from structural overlap. Combining dv-B2PM with two-photon glutamate uncaging, we visualized 3D Ca²⁺ dynamics in neurons following localized stimulation. Multi-timescale analysis revealed dendrite-to-soma Ca²⁺ signal propagation, back propagated Ca²⁺ signal from the soma, and multi-frequency (5–40 Hz) Ca²⁺ transients activated along apical dendrites at speeds from ten of microns per second to millimeters per seconds. These findings demonstrate dv-B2PM as a powerful tool for direct visualization of 3D calcium dynamics associated with dendritic integration across extended neuronal structures, bridging the gap between optical imaging and the dynamic biophysics of neuronal integration.