Multiplexed Nanoscale Protein Mapping Through Expansion Microscopy and Immuno-SABER

测量脑细胞类型和电路的工具必须是可扩展的，无论是在分子靶点的数量上 一次就可以看到，并且可以评估组织的大小。它们还必须是高分辨率的， 因为细胞隔间，如轴突、树突和突触显示出纳米级的特征尺寸。尽管 许多团队在对完整的大脑回路中表达的RNA进行多路成像方面取得了快速进展 转录学“，用于对完整的大脑电路进行蛋白质多路成像的技术落后，尽管 事实上，了解特定突触、轴突、树突和 亚细胞隔间是了解神经功能的关键之一，从而推导出细胞类型 系统的人口普查。此外，鉴于许多蛋白质位于特定的纳米级隔间中 神经元，许多只有在密集排列的纳米级复合体的背景下才能获得完整的功能 在许多蛋白质中，蛋白质比RNA更强烈地感受到对纳米尺度图谱的需求。我们在这里 建议通过创建(目标1)完整的工具箱来解决这些限制，该工具箱用于至少 通过优化使用DNA条形码抗体进行快速序列成像，一次30个蛋白质，理想情况下是50个蛋白质 许多不同的神经蛋白(又名免疫军刀，由皮鹏音小组开发)，在上下文中 一种激进的纳米成像新方法，它利用样品的物理膨胀 (由Pi Ed Boyden小组开发)。我们还将开发用于免疫军刀的 多路抗体成像，一种新形式的扩张显微镜，可以将蛋白质相互分离， 为了更好地获得抗体(目标2)。最后，我们将把纳米级、高度多元化的空间 上述蛋白质组学方法与空间转录组学(目标3)。我们将整合原位测序 扩大标本，博伊登实验室的现有项目(手稿正在准备中)，与免疫- SABER协议。这样，我们将能够同时研究蛋白质组学和转录组学 信息，贯穿整个神经架构，具有纳米级的精度。我们将致力于提供以下能力 在同一个大脑中调查至少100个转录本和30个蛋白质，最好是150个转录本和50个蛋白质 样本。我们将在我们的BICCN U19的背景下验证和展示我们的技术的力量 合作者感兴趣的大脑回路。重要的是，我们从一开始就关注以下问题 规模和准确性是BICCN成功的关键。我们的目标是向神经科学界传递 只是一个工具箱，易于使用，但非常强大。我们的工具箱将直面以下限制 以前的蛋白质成像策略在可成像的蛋白质数量、分辨率和 与转录组配合性。通过与我们的BICCN网络的定期会议和讨论 协作者，我们将确保我们的技术将无缝地整合到管道、工作流程和 BICCN特派团的协调框架。

Tools for surveying brain cell types and circuits must be scalable, both in the number of molecular targets visualizable at once, and in the size of the tissues that can be assessed. They also must be high resolution, since cellular compartments such as axons, dendrites, and synapses exhibit nanoscale feature sizes. Despite rapid progress by many teams in multiplexed imaging of expressed RNAs in intact brain circuits, or “spatial transcriptomics”, technologies for multiplexed imaging of proteins intact brain circuits lag behind, despite the fact that knowing the precise identity and location of proteins in defined synaptic, axonal, dendritic, and subcellular compartments is one of the keys to understanding neural function, and thus deriving cell types for a systematic census. Furthermore, given that many proteins are located in specific nanoscale compartments of neurons, and many attain their full functionality only in the context of densely packed nanoscale complexes of many proteins7, the need for nanoscale mapping is even more acutely felt for proteins than for RNAs. We here propose to address these limitations by creating (Aim 1) a full toolbox for the multiplexed imaging of at least 30, and ideally 50, proteins at once, by optimizing the use of DNA-barcoded antibodies for rapid serial imaging of many different neural proteins (aka Immuno-SABER, developed by the group of PI Peng Yin), in the context of expansion microscopy, a radical new method for nanoimaging that utilizes physical expansion of the sample (developed by the group of PI Ed Boyden). We will also develop, for the purposes of Immuno-SABER multiplexed antibody imaging, a new form of expansion microscopy that decrowds proteins from one another, for better access by antibodies (Aim 2). Finally, we will integrate the nanoscale, highly multiplexed, spatial proteomics methods described above with spatial transcriptomics (Aim 3). We will integrate in situ sequencing of expanded specimens, an existing project of the Boyden lab (manuscript in preparation), with the Immuno- SABER protocol of Aim 1. In this way we will be able to simultaneously survey proteomic and transcriptomic information, throughout neural architectures, with nanoscale precision. We will aim to deliver the ability to survey at least 100 transcripts and 30 proteins, and ideally 150 transcripts and 50 proteins, in the same brain specimen. We will validate and demonstrate the power of our technology in the context of our BICCN U19 collaborators' brain circuits of interest. Importantly, we are focusing from the beginning on the questions of scale and accuracy, key to the success of the BICCN. We aim to deliver to the neuroscience community not just a toolbox that is easy to use, but very powerful. Our toolbox will confront, head on, the limitations of previous protein imaging strategies that lack scale in terms of numbers of proteins imageable, resolution, and combinability with transcriptomics. Through regular meetings and discussions with our BICCN network collaborators, we will insure that our technologies will seamlessly incorporate into the pipelines, workflows, and coordinate frameworks of the BICCN mission.