High Speed Multiphoton Multifocal Whole Brain Imaging

项目摘要/摘要 在本应用中，我们描述了超高速串行双光子(STP)的发展。 断层扫描显微镜可以在不到一天的时间内以微米的分辨率对整个器官进行成像。 它将为荧光亚细胞提供最高的成像速度和灵敏度 整个器官成像。 我们将通过实现一种新型的多焦点多光子显微镜(MMM)来实现这一点 我们当前STP系统的版本，它构建在TIseVision的创始团队的广泛 有使用MMM系统的经验。它将解决现有MMM的两个主要问题： 1.由中间光学引起的像差导致的视场受限 2.去扫描路径中发射光子的损失。 我们将介绍一种新的激发方案的细节，该方案避免了像差和限制的视场 视图，并描述了使用高效光纤的非去扫描检测方案 将检测与原始隔行扫描策略结合在一起，最大限度地减少焦点串扰。 这一建议克服了MMM系统长期存在的几个技术问题 组织成像，具有一种新的MMM设计，非常适合体外整体器官成像。这个 能够以1微米的XY采样和2微米的Z对整个器官进行3D荧光成像 在不到12小时内取样，将对3D组织学和 为研究人员在神经科学和其他领域的大量应用提供了重要工具 菲尔兹。

PROJECT SUMMARY/ABSTRACT 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 12 hours and 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.