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，例如新皮质和嗅球，垂直组织成 包含不同细胞类型的层显示不同的活动概况并投射到 因此，快速的体积成像对于捕捉不同的下游目标是必不可少的。 这些神经群体在其分层环境中的动态。 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，通常仅限于二维平面成像。 建议开发一种技术来执行体积 MPM，其中远程 z 堆栈 通过近乎瞬时的轴向扫描获得，同时保持 3D 微米级 我们的技术称为混响 MPM，可以监控 大尺度（包括深度尺度）的神经群体，没有速度损失 与传统的 MPM 相比。 混响 MPM 是我们最近才展示的一项新技术 我们的大部分提案将集中于双光子实验的原理证明。 此外，我们建议进一步开发该工具并表征其性能。 将我们的技术扩展到三光子显微镜，以增加我们的深度穿透力。 目标是对神经群体进行全面的 3D 分辨率成像 体积高达 1×1×1mm3，跨越小鼠皮层的整个厚度。 混响 MPM 的一个关键优势是其极其简单，它只需要 在配备快速检测功能的传统 MPM 中添加混响环路 此外，它允许获取任意数量的平面，而无需使用电子设备。 增加设置复杂性是我们的系统光效率高且 与视频速率扫描轻松兼容，使其成为容量钙的理想选择 这些优点使得使用基因编码的钙指示剂进行成像。 混响 MPM 作为快速、高分辨率、 脑组织的大规模体积成像。