Multi-layer neuronal imaging with reverberation multiphoton microscopy

摘要 许多大脑区域，如新皮层和嗅球，是垂直组织成 包含不同像元类型的图层，显示不同的活动配置文件，并投影到 不同的下游目标因此，快速的体积成像对于捕获 这些神经元群体在其分层环境中的动态。而 多光子显微镜（MPM）已成为高分辨率的黄金标准 从脑组织深处成像，它通常限于2D平面成像。我们 我建议开发一种技术来执行体积MPM，其中长距离z叠加 通过近瞬时轴向扫描获得，同时保持3D微米尺度 分辨率我们的技术，称为混响MPM，使监测 大尺度上的神经元群体，包括深度尺度，没有速度惩罚 与传统的MPM相比。 混响MPM是一种新的技术，我们最近才证明 双光子实验的原理证明。我们的大部分建议将集中在 进一步发展这一工具并说明其性能。此外，我们建议 将我们的技术扩展到三光子显微镜，以增加穿透深度。我们 目标是对神经元群体进行全面的3D分辨成像， 体积高达1×1×1mm3，跨越小鼠皮层的整个厚度。 混响MPM的一个关键优势是它的极端简单。它只需要 在配备有快速检测的常规MPM上增加混响回路 electronics.此外，它允许获取任意数量的平面，而不需要 增加了设置复杂性。其他优点是我们的系统是光效率高的， 易于与视频速率扫描兼容，使其成为体积钙的理想选择 使用基因编码的钙指示剂成像。这些优点使得 混响MPM作为快速、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.