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强大的技术，用于生成大分子的3-D图像及其与细胞内的精细结构的相互作用。这些材料被嵌入其本地状态，在薄层的无定形冰中，用于在透射电子显微镜中成像。通过从各个角度拍摄一系列图像，可以进行3-D重建。但是，这些低密度材料在TEM图像中几乎没有对比度，并且这种效果被有限的电子电流所复杂化，该电流可能会在可能发生损坏之前将其应用于这些脆弱的样品。提高图像清晰度的最有前途的方法之一是使用相比成像，其中使用特殊设计的相板（这项工作中的Zernike相板）用于在样品散布的电子和直接通过的电子之间发展对比度。 不幸的是，尽管有旨在接受相板的复杂显微镜可用性，但研究人员对缺乏始终制造的高质量相板感到沮丧。在这个拟议的项目中，用于用于EM网格应用的超薄材料中广泛的微加工专业知识已应用于相板生产问题。 通过Temwindows.com，受人尊敬的超薄膜制造商的合作，以及Wadsworth Center（沃兹沃思中心（Wadsworth Center）开发了冷冻和相反电子成像的先驱，将使用良好的控制和制造方法生产一系列的相板设计，并将这些设备直接与当前的基于碳基于碳相的配置材料进行比较。目的是产生稳定，一致和低成本的板块，在TEM中长时间持续时间显示背景很少。 公共卫生相关性：本提案中描述的项目将提供对比度增强相对板的商业来源，以去除瓶颈并推动冷冻电子显微镜的发展。该技术用于研究大分子的3-D结构以及它们如何与细胞内的复杂结构相互作用。这些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。