A robust platform for multiplexed, subcellular proteomic imaging in human tissue

用于人体组织多重亚细胞蛋白质组成像的强大平台

• 批准号: 9894465
• 负责人: Robert michael Angelo
• 金额: $ 59万
• 依托单位: STANFORD UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-11 至 2022-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9894465
• 关键词: Allergic; Antibodies; Archives; Area; Atlases; Back; Basic Science; Biopsy; Cell Nucleus; Cells; Clinical; Cloud Computing; Communities; Data; Data Set; Decidua; Development; Disease; Equipment; Event; Extramural Activities; Feedback; First Pregnancy Trimester; Formalin; Foundations; Freeze Drying; Funding; Goals; Granuloma; Health; Hippocampus (Brain); Histologic; Hour; Human; Image; Image Analysis; Imaging Techniques; Imaging technology; Immune; Immunosuppression; Individual; Institutes; Ions; Letters; Link; Machine Learning; Medical center; Messenger RNA; Metals; Morphology; Multiplexed Ion Beam Imaging; Noninfiltrating Intraductal Carcinoma; Optics; Organ; Paraffin Embedding; Pathology; Peer Review; Phenotype; Population; Proteins; Proteomics; Protocols documentation; Pulmonary Tuberculosis; Readiness; Reagent; Reproducibility; Resolution; Resources; Sampling; Scanning; Signal Transduction; Site; Spectrometry, Mass, Secondary Ion; Stains; Standardization; Structure; Technology; Three-Dimensional Image; Time; Tissue Embedding; Tissues; Translational Research; United States National Institutes of Health; Work; cancer immunotherapy; cohort; computational platform; computerized tools; design; graphical user interface; high dimensionality; high resolution imaging; human imaging; human tissue; imaging Segmentation; imaging modality; imaging platform; insight; instrumentation; ion source; learning strategy; multiplexed imaging; new technology; next generation; programs; protein expression; quantitative imaging; reagent standardization; technology validation; tool; tumor microenvironment; user-friendly; virtual

Project Summary Multiplexed Ion Beam Imaging by Time of Flight (MIBI-TOF) uses secondary ion mass spectrometry and metal conjugated primary antibodies to simultaneously visualize dozens of proteins at subcellular resolution in a single tissue section. This technology is back compatible with archival formalin fixed, paraffin embedded tissue (FFPE) and has been used in peer-reviewed work to simultaneously visualize and quantify 36 proteins in retrospective human tissue cohorts. In line with the stated goals of the HuBMAP consortium to develop both “High-sensitivity, high-resolution imaging techniques that can rapidly provide spectral data over large areas of tissue” and “Quantitative imaging analysis tools, including automated 3D image segmentation, feature extraction, and image annotation,” the work outlined here will create a standardized, high throughput, and user-friendly workflow for using MIBI-TOF in basic and translational research to gain insight into how single cell phenotype and tissue structure are functionally-linked in health and disease. To achieve this, we will validate 100 FFPE antibodies and optimize ready-to-use multiplexed staining panels in lyophilized format that will permit storage for at least two years. Protocols and reagents for multiplexed signal amplification of protein and mRNA targets will be further refined, while next generation instrumentation will increase sample throughput to permit full tissue section imaging of up to 40 proteins in 1 hour. Standardized reagents and more robust instrumentation will be accompanied by an automated computational pipeline that utilizes a standard set of segmentation markers and machine learning to accurately identify nuclei and cell borders in any non-neural human tissue. This data will be used to cluster single cell events into functionally distinct populations according to morphology, protein expression, and histological distribution. The reagents and computational pipeline proposed here synergize with existing HuBMAP-funded platforms and could be readily generalized to virtually any high dimensional imaging modality. Thus, this work will not only provide a practical, back compatible imaging platform for high throughput multiplexed imaging, but will also accelerate development of other complimentary imaging technologies as well.

项目摘要 通过飞行时间（MIBI-TOF）使用二次离子质谱和金属的多路复用离子光束成像（MIBI-TOF） 共轭的原代抗体，以简单地在单一分辨率下可视化数十个蛋白质 组织部分。该技术与福尔马林固定的石蜡嵌入式组织（FFPE）兼容 并已在同行评审工作中用于同时可视化和量化36个蛋白质 人组织队列。与Hubmap联盟的既定目标相一致，以发展“高敏，， 高分辨率成像技术可以快速提供在大面积组织上的光谱数据”和 “定量成像分析工具，包括自动3D图像分割，特征提取和图像 注释：“此处概述的工作将创建标准化，高通量和用户友好的工作流程 在基础和翻译研究中使用MIBI-TOF来洞悉单细胞表型和组织如何 结构在健康和疾病中与功能连接。为了实现这一目标，我们将验证100个FFPE抗体 并以冻干格式优化现成的多重染色面板，至少可以存储 两年。蛋白质和mRNA靶标的多路复用信号扩增的方案和试剂将进一步 精制，而下一代仪器将增加样品吞吐量以允许完整的组织部分 1小时内最多40种蛋白质成像。标准化试剂和更健壮的仪器将是 伴随着自动化计算管道，该管道利用一组标准的分割标记和 机器学习以准确识别任何非神经人类组织中的核和细胞边界。这些数据将是 根据形态，蛋白质，用于将单细胞事件聚集到功能上不同的群体中 表达和组织学分布。这里提出的试剂和计算管道与 现有的Hubmap资助平台，可以很容易地将其概括为几乎任何高维成像 方式。那就这项工作不仅将为高通量提供一个实用的背部兼容成像平台 多路复用成像，但也将加速其他免费成像技术的发展。