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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要/摘要光学系统的发展能够记录大型体细胞网络中的钙动态，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。虽然多光子微型显微镜已经被开发出来并在文献中介绍，但它们要么无法有效解决细胞投射或在其他方面受到严重限制，使其直接应用于神经科学问题很困难。为了克服这些挑战，我建议开发一种新型微型多光子显微镜它能够解析自由行为动物在 700 微米大视野中的亚微米细胞特征。这样做需要设计和制造定制物镜、创新的光学几何形状、高度调谐的扫描仪控制信号和定制中继透镜一旦组装，拟议的系统将提供。通过技术进步获得新功能，从而形成一个非常适合神经科学的系统。在目标 1 中，显微镜的组件将在组装在一起之前单独开发和测试定制物镜、扫描仪硬件/控制算法和中继镜头将成为一个完整的系统。在组装显微镜之前实现并单独测试目标 2 的重点是验证和使用。首先，亚衍射荧光珠将在受控和实验条件下进行。用于在厚的荧光标记组织切片成像之前测量系统性能和 PSF。一旦得到验证，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，并对动态进行测量和分析，最后对动物进行自由成像。社交互动任务中的行为，以研究前扣带皮层的丘脑投射动力学。目标 3 侧重于硬件的优化，以及创建新的子系统以适应现有的 2P Aim 3 还可以在实验室中以高效且经济的方式进行微型 2P 显微镜检查。详细介绍了对开发的微型计算机的创建和使用至关重要的所有关键信息的广泛传播范围，遵循加州大学洛杉矶分校微型望远镜项目的方法，机械设计文件，分析/控制软件， PCB 制造文件，以及有关实验过程中系统对准和使用的教学视频化，将开放获取，以便研究界可以访问和采用设计的技术进行神经科学核心的关键实验。