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

摘要： 单光子(1P)荧光微型化显微镜与基因编码钙传感器的耦合 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，回答神经科学中的基本问题。我们团队与加州大学洛杉矶分校的合作 Miniscope Project已经让600多个实验室建造和使用了2500多台开源微型显微镜 扩展的功能只需商业版本所提供功能的一小部分，因此 使接入民主化。然而，1P微型望远镜缺乏对精细结构中的活动进行成像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移除 覆盖在组织上，用于对更深层的神经元进行成像。双光子(2P)显微镜具有精致的横向和轴向 解决办法，绕过所有这些障碍。最近的技术进步使建造 小鼠的双光子微型显微镜成为可能。然而，视场(FOV)仍然有限，而且 这些显微镜需要定制的光学元件，需要花费数十万美元才能购买 在商业上。我们为小鼠设计和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，允许对800微米的视场进行2P成像，使视场从 最新推出的2P微型显微镜(Mini2P-V1)。在这项提案中，我们将优化这台显微镜并 在行为自由的小鼠身上进行轴突、树突和深层躯体成像。这台显微镜将在 三个实验室。戈尔沙尼实验室将用丘脑前扣带回轴突的钙成像来测试这一范围 在社会互动中的大脑皮层。席尔瓦实验室将通过树枝状钙和谷氨酸来测试示波器 记忆连接过程中脾后皮质的成像。什特拉曼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 适用于老鼠和非人类灵长类动物，具有扩展的能力，包括更高的数值孔径(NA)， 大视场和时间多路传输能力，允许以高帧速率(MiniMux2P)进行体积成像。 该显微镜将由布莱尔实验室测试，以解剖大鼠脑内浅层和深层CA1神经元在脑内的作用 导航。它还将在Churchland实验室进行测试，以在 决策任务。最后，我们将使用我们的开源维基来传播这项技术 向成千上万的用户传播迷你望远镜技术。我们将提供零件清单、光学设计和 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，演示成像基础知识、手术技术和分析工具。 我们希望这些尖端的、新颖的和开源的工具将允许调查人员将他们的研究扩展到更远的地方 这是目前可用的技术所能做到的。

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.