National Biomedical Imaging Center, State Key Laboratory of Membrane Biology, Institute of Molecular Medicine, Peking-Tsinghua Center for Life Sciences, College of Future Technology, Peking University, Beijing, China.
Research Unit of Mitochondria in Brain Diseases, Chinese Academy of Medical Sciences, PKU-Nanjing Institute of Translational Medicine, Nanjing Raygen Health, Nanjing, China.
Nat Methods. 2023 Apr;20(4):617-622. doi: 10.1038/s41592-023-01777-3. Epub 2023 Feb 23.
In deep-tissue multiphoton microscopy, diffusion and scattering of fluorescent photons, rather than ballistic emanation from the focal point, have been a confounding factor. Here we report on a 2.17-g miniature three-photon microscope (m3PM) with a configuration that maximizes fluorescence collection when imaging in highly scattering regimes. We demonstrate its capability by imaging calcium activity throughout the entire cortex and dorsal hippocampal CA1, up to 1.2 mm depth, at a safe laser power. It also enables the detection of sensorimotor behavior-correlated activities of layer 6 neurons in the posterior parietal cortex in freely moving mice during single-pellet reaching tasks. Thus, m3PM-empowered imaging allows the study of neural mechanisms in deep cortex and subcortical structures, like the dorsal hippocampus and dorsal striatum, in freely behaving animals.
在深层组织多光子显微镜中，荧光光子的扩散和散射，而不是从焦点处的弹道发射，一直是一个令人困惑的因素。在这里，我们报告了一种 2.17 克的微型三光子显微镜（m3PM），其配置在高散射区域成像时最大限度地提高了荧光收集效率。我们通过在安全的激光功率下对整个皮层和背侧海马 CA1 进行成像，直至 1.2 毫米的深度，证明了其功能。它还可以在自由移动的小鼠执行单个颗粒抓取任务时，检测到后顶叶皮层中与感觉运动行为相关的第 6 层神经元的活动。因此，m3PM 增强的成像允许在自由活动的动物中研究深层皮层和皮质下结构（如背侧海马体和背侧纹状体）中的神经机制。
