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

基本信息

批准号：
10215448
负责人：
Peng Yin
金额：
$ 58万
依托单位：
HARVARD UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-15 至 2022-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10215448
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 detection sensitivity 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

项目摘要

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.

概括对于高容器映射，高度多路复用能力是必不可少的。但是，光谱重叠和缺乏正交标记对常规荧光成像产生了严重的局限性。对于蛋白质成像，这些通过非常规探针和专业的多路复用检测，已经限制了局限性仪器，迭代顺序抗体标记和成像，或通过DNA-barcoding进行的顺序检测。尽管所有这些技术理论上都可以实现高倍增，但它们的成本有限吞吐量。利用原位信号放大将大大减少暴露于暴露，从而成像 - 框架，允许高吞吐量以及提高灵敏度。尽管存在扩增方法，但超过5-8个空间重叠的目标，并没有强力多路复用。完全未满足的技术需求意识到Hubmap视觉是一种高度多路复用的信号扩增技术，可以同时放大数十个不同的目标是数十亿到百倍，从而实现了高度多重的，高吞吐量的原位人类组织中蛋白质的成像。我们根据一种新颖的方式提出了这种原位信号放大方法我们最近发表的分子机制。在新方法中，用多个探针染色（DNA-Barcoded 初级抗体）将同时执行，然后所有条形码将同时延伸到原位的长串。最后，将通过快速交换和成像周期进行顺序进行辅导者的映射，从而将吞吐量提高了10倍，同时可以检测到组织中稀有靶标。除蛋白质外，该方法还适用于RNA和DNA（染色体）靶标。我们将将方法与市售，自动染色和成像系统，并将其应用于图像通过 Hubmap社区内外的广泛合作。