Opt Express. 2023 Sep 25;31(20):32925-32934. doi: 10.1364/OE.492674.
Development of miniature two-photon microscopy (m2PM) has made it possible to observe fine structure and activity of neurons in the brain of freely moving animals. However, the imaging field-of-view of existing m2PM is still significantly smaller than that of miniature single-photon microscopy. Here we report that, through the design of low-magnification objective, large field-of-view scan lens and small tilt angle microscanner, a 2.5-g m2PM achieved a field-of-view of 1000 × 788 µm, comparable to that of a typical single-photon miniscope. We demonstrated its capability by imaging neurons, dendrites and spines in the millimeter field-of-view, and simultaneous recording calcium activities, through a gradient-index lens, of approximately 400 neurons in the dorsal hippocampal CA1 in a freely moving mouse. Integrated with a detachable 1.2-g fast z-scanning module, it enables a 1000 × 788 × 500 µm volumetric neuronal imaging in the cerebral cortex. Thus, millimeter FOV m2PM provides a powerful tool for deciphering neuronal population dynamics in experimental paradigms allowing for animal's free movement.
微型双光子显微镜（m2PM）的发展使得在自由活动的动物的大脑中观察神经元的精细结构和活动成为可能。然而，现有的 m2PM 的成像视场仍然明显小于微型单光子显微镜。在这里，我们报告说，通过设计低倍物镜、大视场扫描透镜和小倾斜角度微扫描仪，一个 2.5 克重的 m2PM 实现了 1000×788μm 的视场，与典型的单光子微型显微镜相当。我们通过在毫米视场中成像神经元、树突和棘突，并通过梯度折射率透镜同时记录约 400 个在自由移动的小鼠背侧海马 CA1 中的神经元的钙活动，证明了它的能力。与可分离的 1.2 克快速 z 扫描模块集成，它可以在大脑皮层中进行 1000×788×500μm 的体积神经元成像。因此，毫米视场 m2PM 为在允许动物自由移动的实验范式中解析神经元群体动力学提供了强大的工具。
