Open-source miniaturized two-photon microscopes for large field-of-view and volumetric imaging

用于大视场和体积成像的开源小型双光子显微镜

• 批准号: 10675751
• 负责人: Daniel Aharoni
• 金额: $ 98.22万
• 依托单位: UNIVERSITY OF CALIFORNIA LOS ANGELES
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-08-15 至 2026-07-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10675751
• 关键词: Animals; Anterior; Axon; Behavior; Brain; Bypass; Calcium; Cells; Collaborations; Communities; Coupled; Custom; Cytoplasmic Granules; Decision Making; Dendrites; Educational process of instructing; Educational workshop; Elements; Ensure; Excision; Fiber; Glutamates; Hippocampus; Image; Lateral; Letters; Link; Memory; Methods; Microscope; Microscopy; Mus; Neurons; Neurosciences; Operative Surgical Procedures; Optics; Parietal Lobe; Pattern; Persons; Photons; Population; Production; Publishing; Rattus; Recording of previous events; Research; Research Personnel; Resolution; Role; Scanning; Social Interaction; Specific qualifier value; Structure; System; Techniques; Technology; Testing; Thalamic structure; Tissue imaging; Tissues; Work; cingulate cortex; cost; data acquisition; design; design,build,test; equipment acquisition; imaging capabilities; insight; lens; memory encoding; meter; miniaturize; neural network; nonhuman primate; novel; open source; open source tool; photonics; sensor; spatial memory; theories; tool; two photon microscopy; two-photon; wiki

Abstract: Single-photon (1P) epifluorescence miniaturized microscopy coupled with genetically encoded calcium sensors has allowed investigators to record the activity of large populations of identified neurons over days to weeks in freely behaving animals, answering fundamental questions in neuroscience. Our group's efforts with the UCLA Miniscope Project have allowed over 600 labs to build and use over 2500 open-source miniaturized microscopes with expanded capabilities at a small fraction of the cost of those offered by commercial versions, thus democratizing access. Yet, 1P miniscopes lack the lateral and axial resolution to image activity in fine structures such as dendrites and axons. In addition, 1P imaging is limited to superficial structures or requires removal of overlying tissue for imaging of deeper neurons. Two-photon (2P) microscopy has exquisite lateral and axial resolution and bypasses all of these obstacles. Recent advances in technology have made the construction of two-photon miniaturized microscopes for mice possible. However, the field of view (FOV) is still limited, and these microscopes require custom-built optics and cost several hundred thousand dollars to acquire commercially. We have designed and built a two-photon miniaturized microscope for mice, including a custom- made objective lens, that allows 2P imaging of an 800 micrometer FOV nearly quadrupling the FOV from the latest published 2P miniaturized microscope (Mini2P-V1). In this proposal, we will optimize this microscope and test it in freely behaving mice for axonal, dendritic and deep somatic imaging. This microscope will be tested in three labs. The Golshani Lab will test the scope with calcium imaging of thalamic axons in anterior cingulate cortex during social interaction. The Silva Lab will test the scope by performing dendritic calcium and glutamate imaging in retrosplenial cortex during memory linking. The Shtrahman Lab will test deep imaging capability by imaging dentate granule neurons through an intact CA1. We will also build a larger miniaturized microscope suitable for rats and non-human primates with expanded capabilities, including a higher numerical aperture (NA), large FOV and temporal multiplexing capability to allow volumetric imaging at high frame rates (MiniMux2P). This microscope will be tested by the Blair Lab to dissect the role of superficial and deep CA1 neurons of rats in navigation. It will also be tested in the Churchland Lab to image rat posterior parietal cortical neurons during decision-making tasks. Finally, we will disseminate the technology using our open-source wiki that has already disseminated miniscope technology to thousands of users. We will provide parts-lists, optical designs and methods for obtaining custom lens elements. As we have done before, we will educate users through online videos and hands-on workshops where imaging basics, surgical techniques and analysis tools are demonstrated. We hope these cutting edge, novel and open-source tools will allow investigators to extend their research beyond that of what is possible with currently available technology.

抽象的： 单光子 (1P) 落射荧光微型显微镜与基因编码钙传感器相结合 使研究人员能够记录大量已识别神经元在几天到几周内的活动 自由行为的动物，回答神经科学的基本问题。我们小组与加州大学洛杉矶分校的合作 Miniscope 项目已允许 600 多个实验室构建和使用 2500 多个开源微型显微镜 具有扩展的功能，而成本只是商业版本提供的功能的一小部分，因此 使访问民主化。然而，1P 微型显微镜缺乏精细结构中图像活动的横向和轴向分辨率 例如树突和轴突。此外，1P 成像仅限于表面结构或需要去除 覆盖组织以对更深的神经元进行成像。双光子（2P）显微镜具有精致的横向和轴向 解决方案并绕过所有这些障碍。最近技术的进步使得建筑 用于小鼠的双光子微型显微镜成为可能。然而，视野（FOV）仍然有限，并且 这些显微镜需要定制光学器件，花费数十万美元购买 商业上。我们为小鼠设计并制造了一个双光子微型显微镜，包括一个定制的 制造的物镜，允许 800 微米 FOV 的 2P 成像，几乎是 FOV 的四倍 最新发布的2P微型显微镜（Mini2P-V1）。在这个提案中，我们将优化这个显微镜 在自由行为的小鼠中测试它的轴突、树突和深层体细胞成像。该显微镜将在 三个实验室。 Golshani 实验室将通过前扣带回丘脑轴突的钙成像测试范围 社交互动过程中的皮质。 Silva 实验室将通过进行树突状钙和谷氨酸测试来测试范围 记忆链接过程中压后皮质的成像。 Shtrahman 实验室将通过以下方式测试深度成像能力 通过完整的 CA1 对齿状颗粒神经元进行成像。我们还将建造一个更大的微型显微镜 适用于具有扩展能力的大鼠和非人类灵长类动物，包括更高的数值孔径（NA）， 大视场和时间复用功能可实现高帧速率的体积成像 (MiniMux2P)。 布莱尔实验室将对该显微镜进行测试，以剖析大鼠浅层和深层 CA1 神经元在 导航。它还将在丘奇兰实验室进行测试，以对大鼠后顶叶皮层神经元进行成像 决策任务。最后，我们将使用我们已经发布的开源维基来传播该技术 向数千名用户传播微型显微镜技术。我们将提供零件清单、光学设计和 获得定制镜片元件的方法。正如我们之前所做的那样，我们将通过在线教育用户 演示影像基础知识、手术技术和分析工具的视频和实践研讨会。 我们希望这些尖端、新颖和开源的工具将使研究人员能够将他们的研究扩展到其他领域 当前可用技术的可能性。