Advancing Multi-Color EM via Direct Detector-enabled 4D-STEM

通过支持直接检测器的 4D-STEM 推进多色 EM

• 批准号: 10248396
• 负责人: Mark H Ellisman
• 金额: $ 35.39万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN DIEGO
• 依托单位国家: 美国
• 财政年份: 2020
• 资助国家: 美国
• 起止时间: 2020-09-01 至 2024-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10248396
• 关键词: 3-Dimensional; Advanced Development; Algorithms; Antibodies; Big Data; Biological; Cell Nucleus; Cells; Characteristics; Chemicals; Color; Complex; Computer Analysis; Computer software; Computing Methodologies; Cryoelectron Microscopy; Data Set; Detection; Development; Devices; Dimensions; Dose; Dose-Rate; Electron Beam; Electron Microscopy; Electron energy loss spectroscopy; Electrons; Elements; Energy-Filtering Transmission Electron Microscopy; Feasibility Studies; Gold Colloid; Heart; Image; Imaging technology; Label; Laboratories; Measures; Metals; Methodology; Methods; Modernization; Molecular; Molecular Structure; Monte Carlo Method; Nature; Noise; Osmium; Outcome; Performance; Positioning Attribute; Preparation; Process; Quantum Dots; Resolution; Roentgen Rays; Sampling; Scanning; Signal Transduction; Specimen; Speed; Stains; Technology; Testing; Thick; Tissues; Transmission Electron Microscopy; Validation; Visualization; Work; base; computerized data processing; contrast imaging; detector; flexibility; improved; industry partner; interest; light microscopy; materials science; metal chelator; microscopic imaging; new technology; next generation; novel; sensor technology; simulation; software development; structural biology; technology development

PROJECT SUMMARY This focused technology development project will advance into first practice a transformative new imaging technology for multicolor electron microscopy (ColorEM), enabling new capabilities for high resolution and 3 dimensional (3D) localization and differentiation of different molecular complexes in cells and tissues. With this technology, we will substantively improve upon presently practiced, multiple labeling strategies involving antibody labelling approaches detected using colloidal gold, quantum dots, etc.. We will further provide a more accessible, higher throughput, and dose efficient alternative to spectroscopic methods, like energy dispersive x-ray (EDX) and electron energy loss spectroscopy (EELS) / energy filtered transmission electron microscopy (EFTEM). At the heart of this effort, we will exploit the performance characteristics of a next- generation, ultra-high speed direct detection device (DDD), now being optimized and extended (by us with an industry partner) for use with scanning transmission electron microscopy (STEM), a broadly deployed capability on modern EM columns. The optimized application of this new pixelated STEM detector technology, in combination with an extended palette of elemental probes and advanced software analysis, will allow for unprecedented new capabilities for performing atomic number (Z) contrast imaging – a process we call ColorSTEM. To bring this new integrated methodology into first practice, we will build on recent feasibility studies and surmount remaining technical hurdles to 1) make multi-labelled EM specimens with optimized elemental probe combinations; 2) troubleshoot the first use of a potentially transformative new sensor technology and work out the methodology for employing it for Z contrast imaging; and 3) devise the associated analytical/computational methodology for processing the very large resultant data sets to enable unambiguous differentiation of molecular complexes selectively marked with elemental probes.

项目概要 这一重点技术开发项目将推进变革性新成像的首次实践 多色电子显微镜 (ColorEM) 技术，实现高分辨率和 3 的新功能 细胞和组织中不同分子复合物的维度（3D）定位和分化。 技术，我们将对目前实践的多种标签策略进行实质性改进，涉及 使用胶体金、量子点等检测抗体标记方法。我们将进一步提供更多 光谱方法（如能量色散法）的可访问、更高通量和剂量效率的替代方案 X 射线 (EDX) 和电子能量损失光谱 (EELS)/能量过滤透射电子 显微镜（EFTEM）是这项工作的核心，我们将利用下一代的性能特征。 一代，超高速直接检测设备（DDD），现在正在优化和扩展（由我们与 行业合作伙伴）与扫描透射电子显微镜 (STEM) 一起使用，这是一种广泛部署的技术 这种新型像素化 STEM 探测器的优化应用。 技术，结合元素探针的扩展调色板和先进的软件分析， 将实现前所未有的原子序数 (Z) 对比成像新功能 - 为了将这种新的集成方法付诸实践，我们将在此基础上进一步发展。 最近的可行性研究并克服剩余的技术障碍：1) 制作多标记的 EM 样本 通过优化的元素探针组合；2）解决潜在变革性新产品的首次使用问题 传感器技术并制定出将其用于 Z 对比度成像的方法；以及 3) 设计 用于处理非常大的结果数据集的相关分析/计算方法，以实现 用元素探针选择性标记的分子复合物的明确区分。