Two-photon laser scanning microscopy has been extensively applied to study in vivo neuronal activity at cellular and subcellular resolutions in mammalian brains. However, the extent of such studies is typically confined to a single functional region of the brain. Here, we demonstrate a novel technique, termed the multiarea two-photon real-time in vivo explorer (MATRIEX), that allows the user to target multiple functional brain regions distributed within a zone of up to 12 mm in diameter, each with a field of view (FOV) of ~200 μm in diameter, thus performing two-photon Ca²⁺ imaging with single-cell resolution in all of the regions simultaneously. For example, we demonstrate real-time functional imaging of single-neuron activities in the primary visual cortex, primary motor cortex and hippocampal CA1 region of mice in both anesthetized and awake states. A unique advantage of the MATRIEX technique is the configuration of multiple microscopic FOVs that are distributed in three-dimensional space over macroscopic distances (>1 mm) both laterally and axially but that are imaged by a single conventional laser scanning device. In particular, the MATRIEX technique can be effectively implemented as an add-on optical module for an existing conventional single-beam-scanning two-photon microscope without requiring any modification to the microscope itself. Thus, the MATRIEX technique can be readily applied to substantially facilitate the exploration of multiarea neuronal activity in vivo for studies of brain-wide neural circuit function with single-cell resolution.

两光子激光扫描显微镜已广泛应用于研究哺乳动物大脑中细胞和亚细胞分辨率下的体内神经元活性。但是，此类研究的范围通常仅限于大脑的单个功能区域。在这里，我们演示了一种称为多区域两光子实时体内浏览器（MATRIEX）的新颖技术，该技术使用户可以定位在直径最大12毫米的区域内分布的多个功能性大脑区域，每个区域都有一个视野直径（FOV）约为200μm，从而产生了两个光子Ca ²⁺同时在所有区域中以单细胞分辨率成像。例如，我们展示了处于麻醉状态和清醒状态的小鼠初级视觉皮层，初级运动皮层和海马CA1区中单个神经元活动的实时功能成像。MATRIEX技术的独特优势是多个微观FOV的配置，这些FOV在三维空间内横向和轴向分布在宏观距离（> 1毫米）上，但由单个常规激光扫描设备成像。特别地，可以将MATRIEX技术有效地实现为现有的传统单光束扫描双光子显微镜的附加光学模块，而无需对显微镜本身进行任何修改。因此，