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

为自由行为的动物开发开源、高性能微型多光子显微镜系统

• 批准号: 10490819
• 负责人: Blake Alexander Madruga
• 金额: $ 3.86万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-30 至 2024-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10490819
• 关键词: Adopted; Adoption; Algorithms; Animal Model; Animals; Anterior; Axon; Behavior; Brain; Brain region; Calcium; Caliber; Cells; Collaborations; Communities; Computer software; Computer-Aided Design; Custom; Dendrites; Development; Devices; Dimensions; Electronics; Elements; Engineering; Face; Fluorescence; Frequencies; Geometry; Goals; Head; Image; In Vitro; Individual; Instruction; Investigation; Label; Lasers; Literature; Location; Measures; Mechanics; Methods; Microscope; Microscopy; Mus; Neurons; Neuropil; Neurosciences; Optics; Pattern; Performance; Photons; Polychlorinated Biphenyls; Population; Procedures; Process; Protocols documentation; Publishing; Research; Resolution; Rest; Role; Sampling; Scanning; Signal Transduction; Slice; Social Interaction; Structure; System; Techniques; Technology; Testing; Thalamic structure; Thick; Tissues; Training; Transgenic Animals; Validation; Weight; awake; calcium indicator; cingulate cortex; cost; cost effective; design; design-build-test; direct application; experimental study; fluorescence imaging; free behavior; imaging system; in vivo; information processing; innovation; learning algorithm; lens; miniaturize; multi-photon; multiphoton microscopy; nanoscale; neuronal cell body; novel; open source; performance tests; prevent; prototype; relating to nervous system; response; sensor; simulation; social; submicron; technology validation; tool; two-photon

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.

项目总结/摘要 光学系统的发展，能够记录钙动力学从大型网络的胞体， 轴突和树突在自由行为动物中的作用对于理解它们在行为中的功能作用至关重要。 虽然多光子微型显微镜已经被开发并在文献中提出，但是它们或者 不能有效地解决细胞投射或在其他方面受到限制，使其直接 应用到神经科学的问题很难。 为了克服这些挑战，我提出了一种新型的微型多光子显微镜的发展 该技术能够在自由行为的动物体内解析700 um大视野内的亚微米细胞特征。 这样做需要设计和制造定制物镜，一种创新的光学几何形状， 高度调谐的扫描仪控制信号和定制的中继透镜。一旦组装完成，拟议的系统将提供 新的能力，通过技术进步，导致一个理想的适合系统的神经科学。 在目标1中，显微镜的组件将单独开发和测试，然后组合在一起， 一个完整的系统。定制物镜、扫描仪硬件/控制算法和中继透镜将 实现，并在组装显微镜之前进行单独测试。目标2是以验证和使用为中心 在控制和实验条件下的技术。首先，将分衍射荧光珠 用于在对厚的荧光标记组织切片成像之前测量系统性能和PSF。 一旦验证，将在表达荧光标记的转基因动物中进行头部固定成像实验。 钙指标，并将动态测量和分析。最后，动物将在自由活动期间进行成像。 社交互动任务中的行为，以研究前扣带皮质中的丘脑投射动态。 目标3的重点是优化硬件，并创建新的子系统，以适应现有的2 P 实验室中的显微镜以高效和具有成本效益的方式进行微型2 P显微镜。Aim 3 详细介绍了所有关键信息的广泛传播，这些信息对创建和使用发达的微型 范围，遵循加州大学洛杉矶分校的微型项目的方法。机械设计文件，分析/控制软件， PCB制造文件，沿着关于实验期间系统对准和使用的教学视频a- 将开放获取，以便研究界可以访问和采用所设计的技术 进行神经科学的关键实验