Development of Open-Source, High Performance Miniature Multiphoton Microscopy Systems for Freely Behaving Animals

项目摘要/摘要 能够从SOMAS的大型网络记录钙动态的光学系统的发展， 自由行为动物的轴突和树突对于理解它们在行为中的功能作用至关重要。 虽然在文献中已经开发和呈现了多光子微型显微镜，但它们或者 无法有效地解析细胞投射，或者在其他方面受到极大的限制，使其直接 在神经科学问题上的应用很困难。 为了克服这些挑战，我提出了一种新型微型多光子显微镜的开发 它能够在自由行为的动物身上解析跨越700微米大视野的亚微米细胞特征。 这样做需要设计和制造定制物镜，一种创新的光学几何结构， 高度调谐的扫描仪控制信号，以及定制的继电器镜头。一旦组装完成，拟议的系统将提供 通过技术进步带来的新能力，形成了一个非常适合神经科学的系统。 在目标1中，显微镜的部件将被单独开发和测试，然后组装成 一个完整的系统。自定义物镜、扫描仪硬件/控制算法和中继镜将 在组装显微镜之前，要实现和单独测试。目标2以验证和使用为中心 在控制和实验条件下的技术。首先，将亚衍射荧光微珠 用于在厚的、荧光标记的组织切片成像之前测量系统性能和PSF。 一旦被证实，头部固定成像实验将在表达荧光的转基因动物中进行。 钙指示剂，并对其动力学进行测量和分析。最后，动物将在免费期间被成像 社会互动任务中的行为，以研究丘脑在前扣带回皮质的投射动力学。 目标3侧重于硬件的优化和创建新的子系统以适应现有的2P 实验室中的显微镜，以高效和经济的方式进行微型2P显微镜。AIM 3也 详细说明广泛传播对建立和使用已开发的微型信息系统至关重要的所有关键信息 范围，遵循加州大学洛杉矶分校迷你望远镜项目的方法。机械设计文件、分析/控制软件、 印刷电路板制造文件，以及有关系统在实验期间的对准和使用的教学视频- 将开放获取，以便研究界可以访问和采用设计的技术 进行对神经科学至关重要的关键实验。

PROJECT SUMMARY / ABSTRACT The development of optical systems that are able to record calcium dynamics from large networks of somas, axons, and dendrites in freely behaving animals is critical to understanding their functional roles in behavior. While multi-photon miniature microscopes have been developed and presented in the literature, they are either unable to effectively resolve cellular projections or are prohibitively limited in other ways, making their direct application to neuroscience questions difficult. In order to overcome these challenges, I propose the development of a novel miniature multiphoton microscope which is able to resolve submicron cellular features across large, 700um fields of view in freely behaving animals. Doing so necessitates the design and fabrication of custom objective lenses, an innovative optical geometry, highly-tuned scanner control signals, and custom relay lenses. Once assembled, the proposed system will offer new capabilities through technical advances, resulting in an ideally suited system for neuroscience. In Aim 1, the components of the microscope will be individually developed and tested before coming together as a complete system. The custom objective lenses, scanner hardware / control algorithms, and relay lenses will be realized, and individually tested before the microscope is assembled. Aim 2 is centered on validation and use of the technique both in controlled and experimental conditions. First, sub-diffraction fluorescent beads will be used to measure system performance and PSF, before thick, fluorescently labeled tissue-slices are imaged. Once validated, head-fixed imaging experiments will be conducted in transgenic animals expressing fluorescent calcium indicators, and the dynamics will be measured and analyzed. Lastly, animals will be imaged during free behavior in a social interaction task, to investigate thalamic projection dynamics in the anterior cingulate cortex. Aim 3 is focused on the optimization of hardware, and the creation of new subsystems to adapt existing 2P microscopes in labs to conduct miniature 2P microscopy in an efficient and cost-effective manner. Aim 3 also details extensive dissemination of all key information central to the creation and use of the developed micro- scope, following the UCLA miniscope project’s approach. Mechanical design files, analysis / control software, PCB manufacture files, along with instructional videos on alignment and use of the system during experimenta- tion, will be made open-access such that the research community can access and adopt the designed technology for conducting critical experiments central to neuroscience.