In-focus Phase Contrast for Cryo-EM

用于冷冻电镜的聚焦相差

• 批准号: 7680579
• 负责人: ROBERT M GLAESER
• 金额: $ 27.92万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2007
• 资助国家: 美国
• 起止时间: 2007-09-30 至 2011-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7680579
• 关键词: 3-Dimensional; Adopted; Automation; Biological; Biological Assay; Biomedical Research; Boxing; Charge; Collection; Complex; Computers; Condition; Data; Devices; Dimensions; Dose; Due Process; Effectiveness; Electrodes; Electron Microscopy; Electrons; Electrostatics; Ensure; Fourier Transform; Frequencies; Goals; Image; Methods; Microscope; Molecular Weight; Multiprotein Complexes; Optics; Pattern; Phase; Proteins; Research; Research Design; Research Project Grants; Resolution; Sampling; Technology; Testing; Tube; Work; base; design; desire; improved; macromolecule; novel; particle; performance tests; prevent; reconstruction; size

DESCRIPTION (provided by applicant): The goal of the proposed research is to determine whether technology can be developed to use an electrostatic phase-contrast aperture ("quarter-wave plate") for in-focus phase contrast in electron microscopy of biological macromolecules. The advantages of using in-focus phase contrast for applications in biomedical research are expected to be: Images of protein complexes as small as 250 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 a resolution of 0.8 nm will no longer be corrupted or lost due to use of large value 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 non-homogeneous sample. The final goal for the proposed research is to determine whether image-data collected with the help of a phase-contrast aperture can be used to obtain interpretable, three-dimensional reconstructions of multiprotein complexes as small as 250 kDa at a resolution of 0.8 nm. This work is based upon a novel, 2-electrode "drift tube" design that has some advantages in terms of fabrication at sub-micrometer dimensions. Based on our preliminary studies, we now propose to combine this device with an electron optical column that is modified in order to provide the desired match between the size of the electron diffraction pattern and the size of the microfabricated aperture. We will use the ratio of Fourier-transform amplitudes at a resolution "g" and at zero frequency [i.e. F(g)/F(0)] to quantitatively evaluate the effect that charging of the aperture has on contrast transfer at high resolution, and we will then use this quantitative assay to evaluate the effectiveness of alternative methods to avoid unwanted charging of the device. When we have demonstrated that the device can be used to produce a nearly flat CTF between ~1/(30 nm) and 1/(0.8 nm), we will automate all steps needed to use it for low-dose EM. The function of automation is to ensure that in-focus phase contrast is no more difficult to use than is defocus based phase contrast. In addition, we will test the performance of in-focus phase contrast on multiprotein complexes of various sizes in order to determine whether it is possible to obtain 3-D reconstructions at a resolution of 0.8 nm for particles as "small" as 250 kDa.

描述（由申请人提供）：拟议研究的目标是确定是否可以开发出使用静电相衬孔径（“四分之一波片”）在生物大分子电子显微镜中进行聚焦相衬的技术。在生物医学研究应用中使用焦点相差对比的优点预计是： 小至 250 kDa 的蛋白质复合物图像应该更容易识别和选择 计算机处理，由于低空间频率下改进的对比度传递函数 (CTF) 这是由焦点内相差提供的。 分辨率为 0.8 nm 的结构信息将不再因使用大尺寸 达到所需相差量的散焦值。 图像质量的提高可能会导致检测结构成分的能力提高） 以及非均质样品中颗粒之间的构象差异。 拟议研究的最终目标是确定图像数据是否在 相差孔径可用于以 0.8 nm 的分辨率获得小至 250 kDa 的多蛋白复合物的可解释的三维重建。这项工作基于一种新颖的 2 电极“漂移管”设计，该设计在亚微米尺寸的制造方面具有一些优势。根据我们的初步研究，我们现在建议将该设备与经过修改的电子光学镜筒结合起来，以便在电子衍射图案的尺寸和微加工孔径的尺寸之间提供所需的匹配。我们将使用分辨率“g”和零频率下的傅里叶变换幅度之比[即F(g)/F(0)] 定量评估孔径充电对高分辨率对比度传输的影响，然后我们将使用此定量测定来评估替代方法的有效性，以避免不必要的充电 设备的。当我们证明该设备可用于产生约 1/(30 nm) 和 1/(0.8 nm) 之间几乎平坦的 CTF 时，我们将自动执行将其用于低剂量 EM 所需的所有步骤。这 自动化的功能是确保对焦相衬并不比基于散焦的相衬更难使用。此外，我们将测试各种尺寸的多蛋白复合物上的焦内相差性能，以确定是否有可能以 0.8 nm 的分辨率对“小”至 250 kDa 的颗粒获得 3-D 重建。