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

项目摘要 这一重点技术开发项目将推进到第一次实践一个变革性的新成像 电子显微镜（ColorEM）技术，实现高分辨率和3 细胞和组织中不同分子复合物的三维（3D）定位和分化。与此 技术，我们将大幅改善目前实行的，多标签策略，包括 使用胶体金、量子点等检测的抗体标记方法。我们将进一步提供更多 可访问的，更高的通量和剂量有效的替代光谱方法，如能量色散 X射线（EDX）和电子能量损失谱（EELS）/能量过滤透射电子 显微镜（EFTEM）。在这项工作的核心，我们将利用性能特点的下一个- 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 扫描透射电子显微镜（STEM）是一种广泛部署的 现代EM列的能力。这种新型像素化STEM检测器的优化应用 技术，结合扩展的元素探针和先进的软件分析， 将允许前所未有的新能力，用于执行原子序数（Z）对比度成像- a 我们称之为ColorSTEM。为了将这种新的综合方法付诸实践，我们将建立在 最近的可行性研究并克服剩余的技术障碍，以1）制作多标记EM标本 与优化的元素探针组合; 2）故障排除的第一次使用的潜在变革性的新的 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， 用元素探针选择性标记的分子复合物的明确区分。

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.