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根本不同类型的微制造相衬设备的免充电版本的有效性，这两种微制造相衬设备设计用于透射电子显微镜。聚焦相位对比度在生物医学研究中的应用的优点是：由于聚焦相位对比度在低空间频率下提供了改进的对比度传递函数(CTF)，因此可以更容易地识别和选择小至200 kDa的蛋白质复合体的图像进行计算机处理。高分辨率的结构信息将不再由于使用大的散焦值来实现所需的相位对比度而被破坏或丢失。改进的图像质量可以导致检测结构非均质样品中的颗粒之间的结构(组成)和构象差异的能力的提高。一种将被评估的设备是一种微型制造的孔径，它可以阻挡以小角度散射的半平面电子波，同时允许其余的散射波以通常的方式对成像做出贡献。第二种类型的器件是一种新颖的双电极“静电漂移管”设计，它对未散射的电子相对于散射的电子施加90度的相移。我们制造的免充电器件将采用高温热处理和材料和器件的精细化，表面的化学和物理清洁，以及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。这些设备的有效性将通过生物测试样本的冷冻-EM图像来确定，例如链霉亲和素和烟草花叶病毒的单层晶体。当前研究的应用将包括微管的结构可塑性和各种调节蛋白与微管的相互作用的研究。

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.