Practical Phase-Plate Imaging for Cryo-EM

实用的冷冻电镜相位板成像

• 批准号: 8335498
• 负责人: MICHAEL MARKO
• 金额: $ 30.96万
• 依托单位: WADSWORTH CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-20 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8335498
• 关键词: 3-Dimensional; Acceleration; Automation; Biological; Biomedical Research; Carbon; Cells; Cellular Structures; Computer software; Consensus; Cryoelectron Microscopy; Crystallography; Data Collection; Detection; Development; Disadvantaged; Dose; Educational workshop; Electrons; Electrostatics; Elements; Farming environment; Film; Fluorescence; Freezing; Guidelines; Housing; Image; In Situ; Individual; Ions; Knowledge; Label; Laboratories; Lighting; Location; Macromolecular Complexes; Methodology; Methods; Microscope; Microtubules; Modeling; Molecular; Molecular Structure; Negative Staining; Noise; Organelles; Particle Size; Peptide Elongation Factor G; Performance; Phase; Phytochrome; Preparation; Protocols documentation; Reporting; Resolution; Risk; Signal Transduction; Solutions; Specimen; Structure; System; Techniques; Testing; Thick; Time; Work; aqueous; base; cost; dalton; density; design; dimer; electron tomography; image processing; improved; instrument; interest; irradiation; light microscopy; macromolecule; nanometer; nanoscale; particle; reconstruction; software systems; theories; tomography; voltage

DESCRIPTION (provided by applicant): Cryo-electron microscopy (cryo-EM) is used to study the native, nanometer-scale 3-D structure of cells and cell organelles as well as the near-atomic-scale 3-D structure of biological macromolecules. While impressive advances in light microscopy enable detection and location of macromolecules in cells with nanometer-scale precision, fluorescence-based techniques detects only structures that are labeled. On the other hand, the technique of cryo-EM tomography reveals at once the 3-D interaction between all structural components of the cell. While x-ray crystallography can solve macromolecular structure at atomic resolution, the technique of "single-particle" cryo-EM can achieve near-atomic resolution without the need to crystallize the macromolecule; and while NMR can provide atomic resolution of small macromolecules in solution, cryo-EM can provide near- atomic resolution of macromolecules up to the mega-Dalton range. Cryo-EM depends on phase-contrast imaging of vitreously frozen specimens, which are weakly-scattering and very sensitive to electron irradiation. Thus it is critical to obtain the maximum contrast with the minimum electron dose. However, the currently employed method of phase-contrast imaging requires that the microscope be strongly defocused, which causes features in different size ranges to have different contrast. As a result, there is considerable overall loss of contrast, and complicated image-processing is needed when making a 3-D reconstruction. These shortcomings pose serious obstacles to increasing cellular resolution in cryo-EM tomography, to increasing image-processing throughput in single-particle reconstruction of macromolecules, and to extending single-particle reconstruction to macromolecules smaller than about 150 kDa,. The disadvantages of the defocus method of cyo-EM phase-contrast imaging can be overcome by in-focus imaging using a phase plate, as demonstrated in recent proof-of-principle studies. We propose to continue development of phase-plate imaging in order to make it a practical, routine technique for cryo-EM. We will (1) improve thin-film phase plate design and manufacture so that they can be widely and economically supplied and have adequate lifetimes, (2) adapt existing automated cryo-EM data-collection software for use with phase plates so that the phase plate stays centered and the optimum illumination conditions are maintained, and (3) establish protocols and guides to optimize phase-plate imaging for specific classes of specimens. This developmental work will have a major impact on the ability of cryo-EM to provide detailed knowledge about biological structures, both at cellular and molecular levels, and it will significantly increase throughput.

描述（由申请人提供）：冷冻电子显微镜 (cryo-EM) 用于研究细胞和细胞器的天然纳米级 3-D 结构以及生物体的近原子级 3-D 结构大分子。虽然光学显微镜技术取得了令人印象深刻的进步，能够以纳米级精度检测和定位细胞中的大分子，但基于荧光的技术只能检测标记的结构。另一方面，冷冻电镜断层扫描技术可以立即揭示细胞所有结构成分之间的 3D 相互作用。 X射线晶体学可以以原子分辨率解​​析大分子结构，而“单粒子”冷冻电镜技术可以实现近原子分辨率，而无需使大分子结晶； NMR 可以提供溶液中小大分子的原子分辨率，而冷冻电镜可以提供高达兆道尔顿范围的大分子的近原子分辨率。 冷冻电镜依赖于玻璃体冷冻样本的相差​​成像，玻璃体冷冻样本的散射较弱，对电子辐射非常敏感。因此，以最小的电子剂量获得最大的对比度至关重要。然而，目前采用的相差成像方法需要显微镜强烈散焦，这导致不同尺寸范围的特征具有不同的对比度。结果，整体对比度损失相当大，并且在进行 3D 重建时需要复杂的图像处理。这些缺点对提高冷冻电镜断层扫描中的细胞分辨率、提高大分子单粒子重建中的图像处理吞吐量以及将单粒子重建扩展到小于约150 kDa的大分子构成了严重障碍。 正如最近的原理验证研究所证明的那样，cyo-EM 相衬成像的散焦方法的缺点可以通过使用相位板的聚焦成像来克服。我们建议继续开发相位板成像，使其成为冷冻电镜的实用常规技术。我们将 (1) 改进薄膜相位板的设计和制造，以便能够广泛、经济地供应它们并具有足够的使用寿命，(2) 采用现有的自动化冷冻电镜数据收集软件与相位板一起使用，以便相位板板保持居中并保持最佳照明条件，并且 (3) 建立协议和指南以优化特定类别样本的相位板成像。这项开发工作将对冷冻电镜在细胞和分子水平上提供有关生物结构详细知识的能力产生重大影响，并将显着提高通量。