High-Throughput, Highly Multiplexed In Situ Proteomic Imaging of Human Tissues

人体组织的高通量、高度多重原位蛋白质组成像

• 批准号: 10026444
• 负责人: Peng Yin
• 金额: $ 57万
• 依托单位: HARVARD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-15 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10026444
• 关键词: Antibodies; Area; Atlases; Bar Codes; Binding Sites; Biological Assay; Biology; Chromosomes; Clinical Pathology; Collaborations; Color; Communities; DNA; Data; Detection; Development; Fluorescence; Freezing; Hour; Human; Human BioMolecular Atlas Program; Image; Imaging technology; In Situ; Label; Maps; Mediating; Methods; Microscope; Molecular; Multiplexed Ion Beam Imaging; Phase; Proteins; Proteomics; Protocols documentation; Publishing; RNA; Reaction; Sample Size; Sampling; Scanning; Signal Transduction; Slide; Stains; System; Techniques; Testing; Thick; Time; Tissue Sample; Tissue imaging; Tissues; Tonsil; Validation; Vision; Work; automated image analysis; base; cost; data resource; data submission; data visualization; design; disease diagnosis; drug discovery; fluorescence imaging; fluorophore; human imaging; human tissue; imager; imaging approach; imaging modality; imaging system; improved; in situ imaging; indexing; instrument; interest; multiplex detection; novel; single cell proteins; vibration

Summary High multiplexing capability is indispensable for high-content mapping. However, spectral overlap and lack of orthogonal labeling create severe limitations for conventional fluorescence imaging. For protein imaging, these limitations have been circumvented through multiplexed detection via unconventional probes and specialized instruments, iterative sequential antibody labeling and imaging, or sequential detection through DNA-barcoding. Although all of these techniques can theoretically achieve high multiplexing, they come at a cost of limited throughput. Utilizing in situ signal amplification would substantially reduce exposure - and thus imaging - time per frame, allowing for high throughput as well as improved sensitivity. Although amplification methods exist, they have not been robustly multiplexed beyond 5-8 spatially overlapping targets. A critical unmet technical need for fully realizing the HuBMAP vision is a highly multiplexed signal amplification technique that can simultaneously amplify tens of distinct targets by tens- to hundreds-fold, thereby enabling highly multiplexed, high throughput, in situ imaging of proteins in human tissues. We propose such an in situ signal amplification method, based on a novel molecular mechanism that we recently published. In the new method, staining with multiple probes (DNA-barcoded primary antibodies) will be performed simultaneously, and then all barcodes will be simultaneously extended into long concatemers in situ. Finally, mapping of concatemers will be sequentially performed through rapid exchange and imaging cycles, improving throughput by 10-fold while enabling detection of rare targets in tissues. Beyond proteins, the method will be applicable to RNA and DNA (chromosome) targets. We will integrate the method with commercially available, automated staining and imaging systems, and apply it to image diverse human tissues via broad collaboration within and beyond HuBMAP community.

总结 高复用能力对于高内容映射是必不可少的。然而，光谱重叠和缺乏 正交标记对常规荧光成像产生了严重的限制。对于蛋白质成像，这些 通过非常规探针和专用探针的多重检测， 这些方法可以通过以下方式进行：使用常规的免疫分析仪器、迭代的顺序抗体标记和成像或通过DNA条形码的顺序检测。 尽管所有这些技术理论上都可以实现高复用，但它们的代价是有限的 吞吐量利用原位信号放大将大大减少曝光-从而成像-时间， 帧，允许高吞吐量以及改进的灵敏度。尽管存在扩增方法，但它们具有 在5-8个空间上重叠的靶之外没有被稳健地多路复用。一个关键的未满足的技术需求， 实现HuBMAP愿景是一种高度复用的信号放大技术， 数十个不同的靶标，从而能够在原位实现高度多重化、高通量 人体组织中的蛋白质成像。我们提出了这样一种原位信号放大方法，基于一种新的 我们最近发表的分子机制。在新方法中，用多个探针（DNA条形码）染色， 第一抗体）同时进行，然后所有条形码将同时原位延伸成长多联体。最后，将通过快速交换和成像循环顺序进行多联体的映射，将通量提高10倍，同时能够检测组织中的稀有靶标。除了蛋白质，该方法还将适用于RNA和DNA（染色体）靶标。我们将把这种方法与 商业上可获得的自动染色和成像系统，并将其应用于成像不同的人体组织， 在HuBMAP社区内外开展广泛合作。