Development of Laser-Based Phase Contrast for Biological Electron Microscopy

生物电子显微镜激光相衬技术的发展

• 批准号: 10491050
• 负责人: Holger Mueller
• 金额: $ 37.66万
• 依托单位: UNIVERSITY OF CALIF-LAWRENC BERKELEY LAB
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-09-15 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10491050
• 关键词: 12 year old; 3-Dimensional; Apoferritin; Automation; Biological; Biological Testing; Cellular biology; Classification; Coma; Communities; Cryoelectron Microscopy; Data; Data Collection; Data Set; Development; Electron Beam; Electron Microscope; Electron Microscopy; Electrons; Feedback; Frequencies; Goals; Guns; Human; Image; Lasers; Manuals; Maps; Mechanics; Microscope; Microtubule-Associated Proteins; Modification; Molecular Biology; Molecular Conformation; Muramidase; Noise; Optics; Particle Size; Pattern; Performance; Phase; Proteins; Resolution; Source; Specimen; Speed; Structure; Testing; Titan; arm; base; contrast imaging; cost; data quality; density; design; electron diffraction; improved; particle; quantum; reconstruction; structural biology; tool; transmission process; user-friendly; ward

Cryo-electron microscopy (cryo-EM) has already had a revolutionary impact on cell and molecular biology and become a major source of structural information. Still, the minimum number of parti- cles needed for a three-dimensional reconstruction of a structure, and the minimum size of the particles amenable to reconstruction, remains far above fundamental limits. Over the past four years, we have developed a laser-based phase-plate (LPP) that can contribute to reaching the standard quantum (shot-noise) limit of imaging in cryo-EM. We have tested it on the optical bench, demonstrated phase-contrast imaging, and exceeded all performance parameters that we set out to achieve. Now, at the beginning of the fourth year, we have already made first steps to- wards obtaining a density map of a known structure with the LPP; we fully expect to complete this goal by the end of the fourth year. In this renewal proposal, we aim to achieve an even higher level of performance, one that will add significant value for many classes of problems in structural biology, and that will be well-received by the entire cryo-EM community as a basis for a user-friendly, commercially available product. To do this, we will partially automate data collection by creating new, data-driven feedback tools to maintain alignment of the LPP to the electron diffraction pattern. Upgrading the mechanical and optical design of the LPP will allow us to maintain stable coma-free alignment of the microscope. This upgrade will leverage the relativistic reversal effect, which we recently demonstrated, to elim- inate weak ghost images. In addition, to compensate for the larger chromatic aberration of our microscope in phase-plate mode, we will install a gun monochromator. Using the LPP is expected to enable reconstructions for particles at the lower size limit of what is believed to be theoretically possible for cryo-EM. We expect this to also improve the power of 3D- classification to assign much larger particles into distinctly different conformational and composi- tional states. Throughout the project, we will establish the extent to which the LPP improves cryo- EM capabilities by performing reconstructions of a wide variety of biological specimens. We will determine the number of asymmetric units needed to produce high-resolution density maps, at equivalent values of the resolution, as well as the size of the smallest particles that can be recon- structed. As we advance the LPP, we will use more and more challenging test specimens, from apoferritin and a human, microtubule-associated protein to extremely small proteins, such as my- oglobin or lysozyme.

冷冻电子显微镜（Cryo-EM）已经对细胞和分子生物学产生了革命性的影响，并成为结构信息的主要来源。尽管如此，三维重建结构所需的最小数量，以及可延伸至重建的颗粒的最小尺寸仍然远高于基本限制。在过去的四年中，我们开发了一种基于激光的相板（LPP），该相位板（LPP）可以达到低温EM中成像的标准量子限制的限制。我们已经在光学台上测试了它，展示了相对比较成像，并超过了我们设定的所有性能参数。现在，在第四年初，我们已经迈出了第一步，该步骤是获得了使用LPP的已知结构的密度图。我们完全期望在第四年末完成这一目标。 在此续签建议中，我们旨在达到更高的绩效，这将为结构生物学的许多类别带来重要的价值，并且将由整个Cryo-EM社区充分收获，以此作为用户友好，商业上可用的产品的基础。为此，我们将通过创建新的，数据驱动的反馈工具来部分自动化数据收集，以保持LPP与电子衍射模式的一致性。升级LPP的机械和光学设计将使我们能够保持显微镜的稳定无昏迷对齐。该升级将利用我们最近证明的相对论逆转效应来消除弱的幽灵图像。此外，为了补偿在相板模式下显微镜更大的色差，我们将安装枪支单色器。 预计使用LPP可以在较低尺寸的极限上重建颗粒，这在理论上可能是冷冻EM的可能性。我们希望这还可以提高3D分类的能力，以将更大的颗粒分配为明显不同的构象和组成状态。在整个项目中，我们将通过进行各种生物标本的重建来确定LPP改善冷冻功能的程度。我们将确定在分辨率的等效值以及可以重新构造的最小颗粒的大小上，生成高分辨率密度图所需的不对称单元数量。当我们推进LPP时，我们将使用越来越具有挑战性的测试标本，从丙酰铁蛋白和人类，微管相关的蛋白质到极小的蛋白质，例如my- oglobin或lysozyme。