Practical Phase-Plate Imaging for Cryo-EM

实用的冷冻电镜相位板成像

• 批准号: 8535802
• 负责人: MICHAEL MARKO
• 金额: $ 29.07万
• 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/8535802
• 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三维结构以及生物大分子的近原子级三维结构。虽然光学显微镜的令人印象深刻的进步能够以纳米级的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，冷冻电磁断层扫描技术立即揭示了细胞所有结构成分之间的三维相互作用。虽然X射线晶体学可以以原子分辨率解析大分子结构，但“单粒子”低温-EM技术可以实现近原子分辨率而无需使大分子结晶;并且虽然NMR可以提供溶液中小的大分子的原子分辨率，但低温-EM可以提供高达兆道尔顿范围的大分子的近原子分辨率。 Cryo-EM依赖于玻璃体冷冻标本的相衬成像，玻璃体冷冻标本散射弱，对电子辐照非常敏感。因此，关键是用最小的电子剂量获得最大的对比度。然而，目前采用的相衬成像方法需要显微镜强烈散焦，这导致不同尺寸范围内的特征具有不同的对比度。结果，存在相当大的整体对比度损失，并且在进行3-D重建时需要复杂的图像处理。这些缺点对增加冷冻EM断层摄影中的细胞分辨率、增加大分子的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的大分子造成严重障碍。 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，如在最近的原理验证研究中所证明的。我们建议继续发展相位板成像，以使其成为一个实用的，常规的技术冷冻EM。我们将（1）改进薄膜相位板的设计和制造，使其能够广泛和经济地供应，并具有足够的寿命，（2）调整现有的自动冷冻EM数据收集软件与相位板一起使用，使相位板保持居中，并保持最佳照明条件，（3）建立协议和指南，以优化特定类别标本的相位板成像。这项开发工作将对cryo-EM在细胞和分子水平上提供有关生物结构的详细知识的能力产生重大影响，并将显着提高通量。