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：
10026444
负责人：
Peng Yin
金额：
$ 57万
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 2022-06-30
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

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传统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门的多重检测，已经克服了限制仪器、迭代连续抗体标记和成像，或通过 DNA 条形码进行连续检测。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每次的曝光时间，从而减少成像时间帧，允许高吞吐量以及提高的灵敏度。尽管存在扩增方法，但它们没有被稳健地复用超过 5-8 个空间重叠的目标。未满足的关键技术需求实现 HuBMAP 愿景是一种高度复用的信号放大技术，可以同时放大数十到数百倍的不同目标，从而实现高度多重、高通量、原位人体组织中蛋白质的成像。我们提出了这样一种基于新颖的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（DNA 条形码一抗）将同时进行，然后所有条形码将同时原位延伸成长串联体。最后，将通过快速交换和成像循环依次进行多联体的绘制，将通量提高 10 倍，同时能够检测组织中的稀有目标。除了蛋白质之外，该方法还将适用于 RNA 和 DNA（染色体）目标。我们将把该方法与市售的自动染色和成像系统，并将其应用于多种人体组织的成像 HuBMAP 社区内外的广泛合作。