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: 10516900
• 负责人: Daniel Aharoni
• 金额: $ 110.93万
• 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/10516900
• 关键词: Animals; Anterior; Axon; Behavior; Brain; Bypass; Calcium; Cells; Collaborations; Communities; Coupled; Crystallization; Custom; Cytoplasmic Granules; Decision Making; Dendrites; Educational workshop; Elements; Ensure; Excision; Fiber; Glutamates; Hippocampus (Brain); Image; Lateral; Letters; Link; Memory; Methods; Microscope; Microscopy; Mus; Neurons; Neurosciences; Operative Surgical Procedures; Optics; Parietal; Parietal Lobe; Pattern; Persons; Photons; Population; Production; Publishing; Rattus; Recording of previous events; Research; Research Personnel; Resolution; Role; Scanning; Social Interaction; 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; miniaturize; neural network; neuronal cell body; nonhuman primate; novel; open source; open source tool; photonics; sensor; spatial memory; theories; tool; two photon microscopy; two-photon; wiki; Animals; Anterior; Axon; Behavior; Brain; Bypass; Calcium; Cells; Collaborations; Communities; Coupled; Crystallization; Custom; Cytoplasmic Granules; Decision Making; Dendrites; Educational workshop; Elements; Ensure; Excision; Fiber; Glutamates; Hippocampus (Brain); Image; Lateral; Letters; Link; Memory; Methods; Microscope; Microscopy; Mus; Neurons; Neurosciences; Operative Surgical Procedures; Optics; Parietal; Parietal Lobe; Pattern; Persons; Photons; Population; Production; Publishing; Rattus; Recording of previous events; Research; Research Personnel; Resolution; Role; Scanning; Social Interaction; 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; miniaturize; neural network; neuronal cell body; 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）落荧光微型显微镜与遗传编码的钙传感器相结合 允许研究人员在数天到几周内记录大量已鉴定神经元的活动 自由表现动物，回答神经科学中的基本问题。我们小组与加州大学洛杉矶分校的努力 Miniscope项目允许超过600个实验室构建和使用超过2500多个开源小型显微镜 随着商业版本提供的成本的一小部分，功能扩大，因此 民主化访问。然而，1便士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。另外，1p成像仅限于表面结构，或需要去除 上覆的组织，用于成像更深的神经元。两光子（2p）显微镜具有精美的侧向和轴向 解决并绕过所有这些障碍。技术的最新进展已建设 可能的小鼠的两光子微型显微镜。但是，视野（FOV）仍然有限，并且 这些显微镜需要定制的光学元件，并且需要花费数十万美元才能获取 商业上。我们已经为小鼠设计并建造了一个两光子的微型显微镜，包括定制 制作的物镜镜头，允许2p成像800千分尺的FOV几乎使FOV从 最新发表的2P小型显微镜（MINI2P-V1）。在此提案中，我们将优化此显微镜和 在自由表现的小鼠中对其进行测试，以进行轴突，树突状和深躯体成像。该显微镜将在 三个实验室。 Golshani实验室将用丘脑轴突的钙成像在前扣带回中测试范围 社交互动期间的皮质。 Silva实验室将通过执行树突状钙和谷氨酸测试范围 内存链接期间，在肾后皮质中进行成像。 Shtrahman实验室将通过 通过完整的Ca1成像牙齿颗粒神经元。我们还将构建更大的微型显微镜 适用于具有扩展能力的大鼠和非人类灵长类动物，包括较高的数值孔径（NA）， 较大的FOV和时间多路复用能力，以高框架速率（minimux2p）允许体积成像。 Blair Lab将测试该显微镜，以剖析大鼠浅表和深的CA1神经元在 导航。它也将在Churchland实验室进行测试 决策任务。最后，我们将使用已经已经有已经具有的开源Wiki传播技术 向成千上万的用户传播Miniscope技术。我们将提供零件列表，光学设计和 获取自定义镜头元素的方法。正如我们以前所做的那样，我们将通过在线教育用户 展示了成像基础，外科技术和分析工具的视频和动手研讨会。 我们希望这些前沿，新颖和开源工具能使研究人员将研究扩展到 当前可用的技术可能的可能性。