Multi-layer neuronal imaging with reverberation multiphoton microscopy

摘要 大脑的许多区域，如新皮质和嗅球，都是垂直组织成 包含不同单元格类型的图层，这些单元格显示不同的活动剖面并投影到 不同的下游目标。因此，快速的体积成像对于捕获来说是必不可少的 这种神经元群体在其分层环境中的动态。而当 多光子显微镜(MPM)已成为高分辨率的黄金标准 从脑组织深处进行成像，一般仅限于2D平面成像。我们 建议开发一种执行体积MPM的技术，其中远程Z堆叠 是通过近乎瞬时的轴向扫描获得的，同时保持3D微米级 决议。我们的技术，称为混响MPM，使监测 大尺度上的神经元种群，包括深度尺度，没有速度损失 与传统的MPM相比。 混响MPM是我们最近才演示的一种新技术 并对双光子原理实验进行了证明。我们的大部分提案将集中在 进一步开发这一工具并对其性能进行表征。此外，我们建议 将我们的技术扩展到三光子显微镜，以增加深度穿透。我们的 目标是对大脑中的神经元群体进行全面的3D分辨率成像 体积高达1×1×1mm3，横跨小鼠皮质的整个厚度。 混响MPM的一个关键优势是其极其简单。它只需要 在配备了快速检测的传统MPM上增加了混响回路 电子产品。此外，它允许获得任意数量的飞机，而不需要 增加了设置的复杂性。我们的其他优势是我们的系统是轻便高效的 轻松兼容视频率扫描，使其成为体积钙扫描的理想选择 使用基因编码的钙指示剂进行成像。这些优势使 混响MPM作为一种快速、高分辨率 脑组织中的大规模体积成像。

ABSTRACT Many brain areas, such as neocortex and olfactory bulb, are vertically organized into layers containing distinct cell types that show different activity profiles and project to different downstream targets. Fast, volumetric imaging is thus indispensable to capture the dynamics of such neuronal populations within their stratified environments. While multiphoton microscopy (MPM) has become the gold standard for high resolution imaging from deep within brain tissue, it is generally restricted to 2D planar imaging. We propose to develop a technique to perform volumetric MPM where a long-range z-stack is acquired by near-instantaneous axial scanning, while maintaining 3D micron-scale resolution. Our technique, called reverberation MPM, enables the monitoring of neuronal populations over large scales, including the depth scale, with no speed penalty compared to conventional MPM. Reverberation MPM is a new technique which we have demonstrated only recently with proof of principle two-photon experiments. Much of our proposal will be focused on further developing this tool and characterizing its performance. Moreover, we propose to extend our technique to three-photon microscopy, for increased depth penetration. Our goal is to perform comprehensive 3D-resolved imaging of neuronal populations within volumes up to 1×1×1mm3, spanning the entire thickness of the mouse cortex. A key advantage of reverberation MPM is its extreme simplicity. It requires only the addition of a reverberation loop to a conventional MPM equipped with fast detection electronics. Moreover, it allows the acquisition of an arbitrary number of planes without increasing setup complexity. Other advantages are that our system is light efficient and easily compatible with video-rate scanning, making it ideal for volumetric calcium imaging using genetically encoded calcium indicators. These advantages make reverberation MPM particularly attractive as a general tool for fast, high resolution, large-scale volumetric imaging in brain tissue.