Multiphoton In Vivo Microscopy (Core 2)

发现创伤后癫痫发生的基本分子和细胞机制， 创伤性脑损伤的生物病理学现实模型将需要具有纵向的、高分辨率的 进入回脑的大型动物大脑。荧光显微镜增强了这种能力， 通过使用基因靶向和离子选择性的 荧光团。在该项目的工具开发阶段I，我们开发了一种仪器， 支持技术为我们提供了执行此类测量的独特能力。这些 这些技术包括一种定制的双光子显微镜，其机架设计可以容纳 体重> 80 kg的动物脑的亚细胞分辨率体内成像。体内显微镜检查 在这种大小的动物（据我们所知，最大的是2- 4倍）中成像需要大量的 开发和优化运动伪影缓解工具，包括心跳触发、高 快速图像堆栈采集;灵活、可穿透、透明硬膜下窗口;以及事后成像 配准算法最后，显微镜可以进行数字荧光寿命成像， 使得能够使用新颖的单波长染料定量细胞外氯化物。显微镜 核心将实施第一阶段开发的成像工具，并优化钙成像， 长期重复访问。由于这些工具需要大量的成本和技术专长， 显微镜核心代表了一个具有成本效益的整合和一致的实施， 建议方案。

Discovering the basic molecular and cellular mechanisms of post-traumatic epileptogenesis in a biophysically realistic model of traumatic brain injury will require having longitudinal, high-resolution access to the gyrencephalic, large animal brain. Fluorescence microscopy enhances such capabilities, enabling multimodal data collection through the use of genetically targeted and ion-selective fluorophores. In the tool development Phase I of this project, we developed an instrument and supporting technologies that afford us the unique ability to perform such measurements. These technologies include a custom two-photon microscope with a gantry design that can accommodate imaging in vivo, with subcellular resolution, brains of animals weighing >80kg. Microscopic in vivo imaging in animals of this size (to our knowledge, the largest by a factor of 2-4x) required substantial development and optimization of motion artifact mitigation tools, including heartbeat-triggered, high speed image stack acquisition; flexible, penetrable, transparent sub-dural windows; and post-hoc image registration algorithms. Finally, the microscope can perform digital fluorescence lifetime imaging, enabling quantification of extracellular chloride using novel single-wavelength dyes. The Microscopy Core will implement the imaging tools developed in Phase I, as well as optimize for calcium imaging and long-term repeated access. As these tools require substantial cost and technical expertise, the Microscopy Core represents a cost-effective consolidation and consistent implementation for the proposed program.