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.

用于调查脑细胞类型和回路的工具必须具有可扩展性，无论是在分子目标的数量上 可以立即可视化，并且可以评估组织的大小，它们还必须具有高分辨率， 尽管轴突、树突和突触等细胞区室表现出纳米级的特征尺寸。 许多团队在完整脑回路中表达的 RNA 多重成像方面取得了快速进展，或“空间 转录组学”，完整脑回路蛋白质多重成像技术仍然落后，尽管 事实上，了解定义的突触、轴突、树突和神经元中蛋白质的精确身份和位置 亚细胞区室是理解神经功能的关键之一，从而推导细胞类型 此外，鉴于许多蛋白质位于特定的纳米级区室中。 神经元，并且许多神经元只有在密集的纳米级复合物的背景下才能实现其全部功能 对于许多蛋白质7，我们对蛋白质的纳米级绘图的需求比对 RNA 的需求更为强烈。 建议通过创建（目标 1）一个完整的工具箱来解决这些限制，该工具箱至少用于多重成像 通过优化 DNA 条形码抗体的使用进行快速连续成像，一次检测 30 个（最好是 50 个）蛋白质 许多不同的神经蛋白（又名Immuno-SABER，由PI Peng Yin小组开发），在上下文中 膨胀显微镜是一种利用样品物理膨胀进行纳米成像的全新方法 （由 PI Ed Boyden 小组开发）我们还将开发用于Immuno-SABER 的目的。 多重抗体成像，一种新型的扩展显微镜，可以使蛋白质彼此疏散， 为了更好地利用抗体（目标 2），我们将整合纳米级、高度多重的空间。 我们将整合上述蛋白质组学方法与空间转录组学（目标 3）。 扩大标本，博伊登实验室的一个现有项目（手稿正在准备中），与免疫 目标1的SABRE协议。这样我们就能够同时研究蛋白质组学和转录组学 我们的目标是提供具有纳米级精度的信息贯穿神经架构。 在同一大脑中调查至少 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 我们将在 BICCN U19 的背景下验证和展示我们技术的力量。 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，我们的目标不是为神经科学界提供服务。 只是一个易于使用但非常强大的工具箱，我们的工具箱将直面以下局限性。 以前的蛋白质成像策略在可成像蛋白质的数量、分辨率和成像质量方面缺乏规模 通过与我们的 BICCN 网络定期举行会议和讨论。 合作者，我们将确保我们的技术将无缝融入管道、工作流程和 协调 BICCN 使命的框架。