Microfabricated porous TEM grids for improved phase contrast and CryoEM imaging

描述（由申请人提供）：冷冻电子显微镜是一种强大的技术，用于生成大分子及其与细胞内精细结构相互作用的3-D图像。这些材料以其天然状态嵌入在一层薄薄的无定形冰中，用于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，可以进行三维重建。然而，这些低密度材料在TEM图像中产生的对比度很小，并且这种效果由于在可能发生损坏之前可以应用于这些易碎样品的有限电子电流而变得复杂。提高图像清晰度的最有前途的方法之一是通过使用相衬成像，其中使用专门设计的相位板（在这项工作中为Zernike相位板）来开发被样品散射的电子与直接通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精密显微镜，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，广泛的微制造技术在制造电磁网格应用的MEMS材料被应用到相位板生产的问题。 通过TEMWindows.com（一家受人尊敬的薄膜制造商）和沃兹沃斯中心（低温和相衬电子成像的先驱）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板设计，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，其在TEM中在长时间内显示出很少的背景充电。 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位板的商业来源，以消除瓶颈并促进低温电子显微镜的发展。该技术用于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。这种结构信息对于理解影响公共卫生的各种疾病状态所涉及的基本过程至关重要。

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.