Microfabricated porous TEM grids for improved phase contrast and CryoEM imaging

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相互作用的三维图像。这些材料被嵌在一层薄薄的非晶冰中，在透射电子显微镜下进行成像。通过从不同角度拍摄一系列图像，可以进行3-D重建。然而，这些低密度材料在TEM图像中产生的对比度很小，并且在可能发生损坏之前，可以应用于这些脆弱样品的有限电子电流使这种影响更加复杂。提高图像清晰度最有希望的方法之一是通过使用相位对比成像，其中特别设计的相位板（Zernike相位板在这项工作中）用于在样品散射的电子和直接通过的电子之间形成对比。不幸的是，尽管设计用于接受相板的精密显微镜可用，但研究人员因缺乏一贯制造的高质量相板而感到沮丧。在这个拟议的项目中，广泛的微加工专业知识在制造超薄材料的EM网格应用被应用到相板生产的问题。通过TEMWindows.com（一家受人尊敬的超薄膜制造商）和Wadsworth中心（冷冻和相衬电子成像的先驱）之间的合作，一系列相板设计将使用良好控制和可制造的方法生产，这些设备将直接与当前的碳基相板进行比较。目标是生产稳定、一致和低成本的相板，在TEM中长时间内显示很少的背景电荷。

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.