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

摘要： 单光子（1 P）落射荧光微型化显微镜与遗传编码钙传感器耦合 这使得研究人员能够在几天到几周的时间里记录大量已识别神经元的活动， 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项目允许600多个实验室建造和使用2500多个开源微型显微镜 与商业版本相比，其成本仅为一小部分， 民主化访问。然而，1 P微型镜缺乏横向和轴向分辨率，无法在精细结构中成像活动 例如树突和轴突。此外，1 P成像仅限于浅表结构或需要去除 用于更深层神经元的成像。双光子（2 P）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。最近的技术进步使建造 用于小鼠的双光子微型显微镜成为可能。然而，视场（FOV）仍然有限，并且 这些显微镜需要定制的光学系统， 在商业上。我们已经设计并建造了一个双光子微型显微镜的小鼠，包括定制- 制造的物镜透镜，允许800微米FOV的2 P成像，几乎是FOV的四倍。 最新发布的2 P微型显微镜（Mini2 P-V1）。在这个建议中，我们将优化这个显微镜， 在行为自由的小鼠中测试它的轴突、树突和深部体细胞成像。这台显微镜将在 三个实验室Golshani实验室将通过前扣带回中丘脑轴突的钙成像来测试该范围 在社交互动中。席尔瓦实验室将通过对树突状钙和谷氨酸盐进行测试来测试示波器 在记忆链接时的压后皮质成像。Shtrahman实验室将测试深度成像能力， 通过完整的CA 1成像齿状颗粒神经元。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灵长类动物，包括更高的数值孔径（NA）， 大FOV和时间多路复用能力，允许以高帧率进行体积成像（MiniMux 2 P）。 这台显微镜将由布莱尔实验室进行测试，以解剖大鼠的浅层和深层CA 1神经元， 导航它也将在Churchland实验室进行测试，以成像大鼠后顶叶皮层神经元， 决策任务。最后，我们将使用我们的开源wiki来传播这项技术， 向成千上万的用户传播微型望远镜技术。我们将提供零件清单，光学设计， 用于获得定制透镜元件的方法。正如我们以前所做的那样，我们将通过在线教育用户， 视频和实践研讨会，展示成像基础知识、手术技术和分析工具。 我们希望这些尖端、新颖和开源的工具能让研究人员将他们的研究扩展到 即现有技术的可能性。

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.