Microfabricated porous TEM grids for improved phase contrast and CryoEM imaging

微加工多孔 TEM 网格可改善相衬和 CryoEM 成像

• 批准号: 8252786
• 负责人: THOMAS R GABORSKI
• 金额: $ 15.58万
• 依托单位: SIMPORE, INC.
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2012-12-19
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8252786
• 关键词: 3-Dimensional; Advanced Development; Appearance; Biological; Carbon; Cells; Cellular Structures; Characteristics; Charge; Collaborations; Complex; Cryoelectron Microscopy; Development; Devices; Disease; Dose; Electron Microscope; Electrons; Equipment; Evaluation; Feedback; Film; Freezing; Goals; Ice; Image; Ions; Mechanics; Membrane; Metals; Methods; Microfabrication; Microscope; Movement; Pattern; Phase; Positioning Attribute; Process; Production; Property; Public Health; Recording of previous events; Research Infrastructure; Research Personnel; Sampling; Scanning; Series; Source; Specimen; Structure; Techniques; Technology; Test Result; Thinness; Three-Dimensional Image; Translating; Transmission Electron Microscopy; Vacuum; Water; Work; aqueous; base; cost; density; design; electron tomography; improved; macromolecule; nanoscale; next generation; operation; particle; prevent; prototype; reconstruction; scaffold; silicon nitride; structural biology; success; tomography; tool; transmission process

DESCRIPTION (provided by applicant): Cryo electron microscopy is a powerful technique for generating 3-D images of macromolecules and their interactions with fine structure within cells. These materials are imbedded in their native state within a thin layer of amorphous ice for imaging in a transmission electron microscope. By taking a series of images at various angles, 3-D reconstructions can be made. However, these low-density materials produce little contrast in TEM images, and this effect is compounded by the limited electron current that can be applied to these fragile samples before damage is likely to occur. One of the most promising methods to increase image clarity is through the use of phase contrast imaging where specially designed phase plates (Zernike phase plates in this work) are used to develop contrast between electrons that are scattered by the sample and those that pass directly through. Unfortunately, despite the availability of sophisticated microscopes that are designed to accept phase plates, researchers are frustrated by the lack of consistently manufactured, high-quality phase plates. In this proposed project, extensive microfabrication expertise in the manufacture of ultrathin materials for EM grid applications is being applied to the problem of phase plate production. Through a collaboration between TEMWindows.com, a respected ultrathin membrane fabricator, and the Wadsworth Center, a pioneer in the development of cryo and phase contrast electron imaging, a series of phase plate designs will be produced using well-controlled and manufacturable methods, and these devices will be directly compared to current carbon-based phase plates. The goal is to produce stable, consistent, and low cost phase plates that show little background charging over long duration in a TEM. PUBLIC HEALTH RELEVANCE: The project described in this proposal will provide a commercial source of contrast enhancing phase plates to remove a bottleneck and advance the development of cryo electron microscopy. This technique is used to study the 3-D structure of large molecules and how they interact with the complex structures within cells. This structural information is critical to understanding fundamental processes involved in various disease states that impact public health.

描述（由申请人提供）：冷冻电子显微镜是一种用于生成大分子及其与细胞内精细结构相互作用的 3D 图像的强大技术。这些材料以其自然状态嵌入一层薄薄的无定形冰中，以便在透射电子显微镜中成像。通过从不同角度拍摄一系列图像，可以进行 3D 重建。然而，这些低密度材料在 TEM 图像中产生的对比度很小，而且在可能发生损坏之前施加到这些易碎样品上的电子电流有限，从而加剧了这种效应。提高图像清晰度最有前途的方法之一是使用相衬成像，其中使用专门设计的相板（本工作中的泽尼克相板）来增强样品散射的电子与直接穿过的电子之间的对比度。 不幸的是，尽管有设计用于接受相位板的复杂显微镜，但研究人员因缺乏一致制造的高质量相位板而感到沮丧。在这个拟议的项目中，用于电磁网格应用的超薄材料制造的广泛微加工专业知识被应用于解决相位板生产问题。 通过受人尊敬的超薄膜制造商 TEMWindows.com 与低温和相衬电子成像开发先驱沃兹沃斯中心之间的合作，将使用良好控制和可制造的方法生产一系列相位板设计，这些设备将直接与当前的碳基相位板进行比较。目标是生产稳定、一致且低成本的相位板，在 TEM 中长时间显示出很少的背景充电。 公共健康相关性：本提案中描述的项目将提供对比度增强相位板的商业来源，以消除瓶颈并促进冷冻电子显微镜的发展。该技术用于研究大分子的 3D 结构以及它们如何与细胞内的复杂结构相互作用。这种结构信息对于理解影响公共卫生的各种疾病状态的基本过程至关重要。