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(申请人提供)：低温电子显微镜是一种强大的技术，可以生成大分子及其与细胞内精细结构的相互作用的3D图像。这些材料以它们的自然状态嵌入在一层薄薄的无定形冰层中，以便在透射电子显微镜中成像。通过拍摄不同角度的一系列图像，可以进行三维重建。然而，这些低密度材料在透射电子显微镜图像中产生的对比度很小，这种影响与有限的电子电流相结合，这些电子电流可以施加到这些脆弱的样品上，然后才可能发生损伤。提高图像清晰度的最有希望的方法之一是使用相衬成像，即使用专门设计的相位板(在本工作中为Zernike相位板)来在样品散射的电子和直接通过的电子之间形成对比度。不幸的是，尽管有先进的显微镜可以接受相位板，但研究人员对缺乏始终如一的制造高质量相位板感到沮丧。在这个拟议的项目中，在制造用于EM栅极应用的超薄材料方面的广泛的微制造专业知识正被应用于相位板的生产问题。通过备受尊敬的超薄膜制造商TEMWindows.com和低温和相衬电子成像领域的先驱沃兹沃斯中心的合作，将使用可控和可制造的方法生产一系列相位板设计，并将这些器件与当前的碳基相位板进行直接比较。我们的目标是生产稳定、一致和低成本的相位板，在透射电子显微镜的长时间内几乎没有背景充电。 与公众健康相关：本提案中描述的项目将提供增强对比度的相位板的商业来源，以消除瓶颈并推动低温电子显微镜的发展。这项技术被用来研究大分子的三维结构以及它们如何与细胞内的复杂结构相互作用。这种结构性信息对于理解影响公共健康的各种疾病状态所涉及的基本过程至关重要。