High Speed Multiphoton Multifocal Whole Brain Imaging

描述（由申请人提供）：在本申请中，我们描述了一种超高速串行双光子（STP）断层扫描显微镜的开发，该显微镜可以在不到一天的时间内以微米分辨率成像整个器官。它将提供最高的成像速度和灵敏度，可用于荧光亚细胞全器官成像。我们将利用TissueVision创始团队在MMM系统方面的丰富经验，在现有STP系统的基础上，实现一种新型的多焦点多光子显微镜（MMM）版本。它将解决现有mm的两个主要问题：由于中间光学引起的像差，视场受限。失扫描路径中发射光子的损失。我们将详细介绍一种避免像差和视场受限的新激励方案，并描述一种非去扫描检测方案，该方案采用高效率光纤耦合检测和原始的隔行扫描策略，以最大限度地减少焦点串扰。该提案克服了深层组织成像的MMM系统长期存在的几个技术问题，采用了一种新颖的MMM设计，非常适合体外全器官成像。在不到一天的时间内，在1微米XY采样和2微米Z采样下对整个器官进行3D荧光成像的能力将对3D组织学产生变革性影响，并为研究人员在神经科学和其他领域的大量应用提供重要工具。

DESCRIPTION (provided by applicant): In this application we describe the development of an ultra-high speed Serial Two-Photon (STP) Tomography microscope that can image entire organs with micron resolution in less than a day. It will offer the highest imaging speed and sensitivity available for fluorescent subcellular whole organ imaging. We will accomplish this by implementing a novel multi-foci multiphoton microscope (MMM) version of our current STP system that builds on TissueVision's founding team's extensive experience with MMM systems. It will address the two major problems with existing MMMs: 1. Limited field of view due to aberrations induced by the intermediate optics 2. Loss of emission photons in the descanned path. We will present details of a new excitation scheme that avoids aberrations and restricted field of views, and describe a non-descanned detection scheme that employs a high efficiency fiber coupled detection with an original interlaced scanning strategy that minimizes foci crosstalk. This proposal overcomes several long standing technical problems with MMM systems for deep tissue imaging, with a novel MMM design ideally suited for ex vivo whole organ imaging. The ability to fluorescently image a whole organ in 3D at 1 micron XY sampling and 2 micron Z sampling in less than a day will have a transformative effect on 3D histology and provide a crucial tool for researchers for a vast array of applications in neuroscience and other fields.