Multi-Layer Neuronal Imaging with Reverberation Multiphoton Microscopy

使用混响多光子显微镜进行多层神经元成像

• 批准号: 10543772
• 负责人: Jerome Mertz
• 金额: $ 40.05万
• 依托单位: BOSTON UNIVERSITY (CHARLES RIVER CAMPUS)
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-03-15 至 2024-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10543772
• 关键词: 3-Dimensional; Area; Biology; Brain; Brain imaging; Calcium; Color; Communities; Detection; Development; Effectiveness; Electronics; Environment; Goals; Image; Lasers; Letters; Light; Lighting; Microscope; Microscopy; Monitor; Mus; Neocortex; Neurons; Neurosciences Research; Penetration; Performance; Physiologic pulse; Population; Reporting; Resolution; Sampling; Scanning; Series; Source; Speed; Surface; System; Techniques; Technology; Thick; brain tissue; calcium indicator; cell type; experimental study; fluorescence imaging; high resolution imaging; improved; in vivo calcium imaging; millimeter; multiphoton microscopy; neocortical; neuroimaging; new technology; olfactory bulb; optical imaging; photonics; preservation; prototype; three photon microscopy; tool; tool development; two-photon

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.

抽象的 许多大脑区域，例如新皮层和嗅球，垂直组织到 包含不同细胞类型的层显示不同的活动概况和项目 不同的下游目标。因此，快速，体积成像是必不可少的 这种神经元种群在其分层环境中的动力学。尽管 多光子显微镜（MPM）已成为高分辨率的金标准 从大脑组织内部的成像，通常仅限于2D平面成像。我们 提议开发一种执行体积MPM的技术，其中远程Z-stack 在维持3D微米尺度的同时，通过近乎实用的轴向扫描获得 解决。我们的技术称为Reverberation MPM，可以监视 在包括深度量表在内的大尺度上的神经元种群，没有速度惩罚 与常规MPM相比。 混响MPM是一种新技术，我们才最近展示 带有原理两光子实验的证明。我们的大部分建议将集中在 进一步开发此工具并表征其性能。而且，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 目标是对内部神经元种群进行全面的3D分辨成像 量高达1×1×1mm3，涵盖了小鼠皮质的整个厚度。 Reverberation MPM的关键优势是其极端简单性。它只需要 在配备快速检测的常规MPM中添加了重复循环 电子产品。此外，它允许在没有的情况下获取任意数量的飞机 增加设置复杂性。其他优点是我们的系统效率很轻， 易于与视频率扫描兼容，使其非常适合体积钙 使用一般编码的钙指标进行成像。这些优势 回响mpm特别吸引人，作为快速，高分辨率的一般工具， 脑组织中的大规模体积成像。