Multiplexed Nanoscale Protein Mapping Through Expansion Microscopy and Immuno-SABER

通过膨胀显微镜和免疫 SABRE 进行多重纳米级蛋白质图谱

• 批准号: 10088537
• 负责人: Edward S. Boyden
• 金额: $ 269.07万
• 依托单位: MASSACHUSETTS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-16 至 2024-09-15
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10088537
• 关键词: Acute; Address; Antibodies; Antigens; Architecture; Axon; Bar Codes; Brain; Censuses; Collaborations; Communities; Complex; DNA; Data; Dendrites; Disease; Exhibits; Forelimb; Head; Human; Image; Letters; Location; Manuscripts; Maps; Methods; Microscopy; Mission; Molecular Target; Motor Cortex; Mus; Neurons; Neurophysiology - biologic function; Neurosciences; Oligonucleotides; Preparation; Primates; Proteins; Proteomics; Protocols documentation; RNA; Research; Resolution; Retrieval; Sampling; Specimen; Speed; Surveys; Synapses; Technology; Tissues; Transcript; Yin; base; biological systems; brain cell; cell type; imaging modality; in situ sequencing; interest; meetings; multiplexed imaging; nanoimaging; nanoscale; new therapeutic target; novel; relating to nervous system; serial imaging; success; tool; transcriptomics

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.

测量脑细胞类型和电路的工具必须是可扩展的，这既在分子靶标数量中 可以立即可视化，并且可以评估的组织大小。它们也必须是高分辨率， 由于轴突，树突和突触暴露的纳米级尺寸。尽管 许多团队在完整脑电路中表达的RNA的多重成像中的许多团队的快速进步，或 转录组学”，用于蛋白质多形成像的技术完整脑电路滞后，dospite 事实是，知道蛋白质在定义的突触，轴突，树突状的确切身份和位置 亚细胞隔室是理解神经功能的关键之一，从而得出了A的细胞类型 系统的人口普查。此外，鉴于许多蛋白质都位于特定的纳米级室 神经元，许多人只有在怀疑的纳米级复合体的背景下才能达到其全部功能 许多蛋白质7，纳米级映射的需求比RNA更为敏锐。我们在这里 提案通过创建（AIM 1）至少用于多路复用成像的完整工具箱来解决这些限制 30，理想情况下是50蛋白，通过优化使用DNA-barcoded抗体快速串行成像的蛋白质 在上下文中 扩展显微镜，一种利用样品物理扩展的纳米影像的根本新方法 （由Pi Ed Boyden组开发）。我们还将开发出于免疫麻将的目的 多路复用抗体成像，这是一种新形式的膨胀显微镜，彼此删除蛋白质， 为了通过抗体进行更好的访问（AIM 2）。最后，我们将整合纳米级，高度多路复用，空间 上面用空间转录组学描述的蛋白质组学方法（AIM 3）。我们将集成原位测序 Boyden Lab的现有项目扩展标本（准备中的手稿），免疫 - AIM 1的SABER协议。通过这种方式，我们将能够轻松调查蛋白质组学和转录组学 信息，整个神经体系结构，具有纳米级精度。我们将旨在实现 在同一大脑中，调查至少100份成绩单和30种蛋白质，理想情况下是150个成绩单和50个蛋白质 标本。我们将在BICCN U19的背景下验证和证明我们技术的力量 合作者感兴趣的大脑电路。重要的是，我们从一开始就专注于 比例和准确性，是BICCN成功的关键。我们旨在交付神经科学社区 只是一个易于使用但功能非常强大的工具箱。我们的工具箱将面对，前往 以前的蛋白质成像策略在可成像，分辨率和蛋白质数量方面缺乏规模 与转录组学的组合性。通过与我们的BICCN网络的定期会议和讨论 合作者，我们将确保我们的技术将无缝纳入管道，工作流和 BICCN任务的协调框架。