In-Focus Phase Contrast for Cryo-EM

描述（由申请人提供）：拟议研究的目标是确定使用两种完全不同类型的微制造相对比器件的免充电版本的有效性，这些器件设计用于透射电子显微镜。在生物医学研究中使用焦内相衬的优势是：小到200 kDa的蛋白质复合物的图像应该更容易识别和选择用于计算机处理，因为焦内相衬在低空间频率下提供了改进的对比度传递函数（CTF）。高分辨率的结构信息将不再被破坏或丢失，因为使用大的散焦值来实现所需的相对比度。改进的图像质量可以提高在结构不均匀样品中检测颗粒之间结构（组成）和构象差异的能力。将被评估的一种设备是微制造孔径，它可以阻挡以小角度散射的电子波的半平面，同时允许其余的散射波以通常的方式贡献成像。第二种类型的装置是一种新颖的双电极“静电漂移管”设计，它对相对于散射电子的未散射电子施加90度相移。我们制造的免充电器件将采用高温退火和材料和器件的细化，表面的化学和物理清洁，并使用敏感的工具来检测表面污染。制造方法将包括传统的光刻湿法制造以及激光微加工和聚焦离子束铣削。这些装置的有效性将从生物测试标本的低温电镜图像来确定，如链霉亲和素和烟草花叶病毒的单层晶体。应用于当前的研究将包括研究微管的结构可塑性和各种调节蛋白与微管的相互作用。

DESCRIPTION (provided by applicant): The goal of the proposed research is to determine the effectiveness of using charging-free versions of two fundamentally different types of microfabricated phase-contrast devices, designed for use in the transmission electron microscope. The advantages of using in-focus phase contrast for applications in biomedical research are expected to be: Images of protein complexes as small as 200 kDa should be more easily identified and selected for computer processing, due to the improved contrast transfer function (CTF) at low spatial frequencies that is provided by in-focus phase contrast. Structural information at high resolution will no longer be corrupted or lost due to use of large values of defocus to achieve the needed amount of phase contrast. The improved image quality may result in improvement in the ability to detect structural (compositional) and conformational differences amongst particles in a structurally non-homogeneous sample. One type of device that will be evaluated is a microfabricated aperture that blocks a half-plane of the electron wave that is scattered at small angle, while at the same time allowing the rest of the scattered wave to contribute in the usual way to image formation. The second type of device is a novel, 2-electrode "electrostatic drift tube" design that applies a 90 degree phase shift to the unscattered electrons relative to the scattered electrons. Our fabrication of charging-free devices will employ high-temperature annealing and refinement of materials and devices, chemical and physical cleaning of surfaces, and the use of sensitive tools for detecting surface contamination. Fabrication methods will include traditional lithographic wet-fabrication as well as laser micromachining and focused-ion beam milling. The effectiveness of these devices will be determined from cryo-EM images of biological test specimens such as monolayer crystals of streptavidin and tobacco mosaic virus. Applications to current research will include studies on structural plasticity of microtubules and the interactions of various regulatory proteins with microtubules.