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.

项目摘要 /摘要 能够从大型索马斯网络记录钙动力学的光学系统的开发， 轴突和自由行为动物中的树突对于理解其在行为中的功能作用至关重要。 虽然在文献中已经开发并介绍了多光子微型显微镜，但它们是 无法有效解决蜂窝项目或以其他方式受到禁止的限制，使其直接 对神经科学问题的应用很难。 为了克服这些挑战，我提出了新型微型多光显微镜的发展 能够在自由行为的动物中解决跨700UM观点的次子细胞特征。 这样做的必要是设计和制造自定义目标镜片，一种创新的光学几何形状， 高度调整的扫描仪控制信号和自定义继电器镜头。一旦组装，建议的系统将提供 通过技术进步的新功能，导致了适合神经科学系统的理想系统。 在AIM 1中，显微镜的组件将进行单独开发和测试，然后再一起进行 完整的系统。定制目标镜头，扫描仪硬件 /控制算法和中继镜头将 在组装显微镜之前，可以实现并单独测试。 AIM 2以验证和使用为中心 在受控条件和实验条件下的技术。首先，亚分量荧光珠将是 用于测量系统性能和PSF之前，在成像较厚，荧光标记的组织切片之前。 经过验证后，将在表达荧光的转基因动物中进行头部固定成像实验 钙指标，将测量和分析动力学。最后，在自由期间会成像 社交互动任务中的行为，研究前扣带回皮层中的丘脑投射动力学。 AIM 3专注于硬件的优化，以及创建新子系统以调整现有2P 实验室中的显微镜以有效且具有成本效益的方式进行微型2P显微镜。 AIM 3也是 详细详细介绍了所有关键信息的广泛传播，这是创建和使用开发的微型 - 范围，遵循UCLA Miniscope项目的方法。机械设计文件，分析 /控制软件， PCB制造文件，以及有关在实验期间对齐和使用系统的教学视频 将进行开放访问，以便研究界可以访问和采用设计的技术 进行神经科学中心的关键实验。