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: 9427705
• 负责人: Holger Mueller
• 金额: $ 44.65万
• 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 2021-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9427705
• 关键词: Attenuated; Back; Biological; Carbon; Cells; Classification; Communities; Custom; Data; Development; Devices; Disease; Electron Beam; Electron Microscope; Electron Microscopy; Electrons; Equipment; Exhibits; Freezing; Frequencies; Goals; Hydration status; Image; Lasers; Maps; Methods; Molecular Machines; Molecular Weight; Motion; Optics; Phase; Physics; Proteins; Research; Resolution; Specimen; Structure; System; Techniques; Technology; Thinness; Transmission Electron Microscopy; Work; base; contrast imaging; density; design; flexibility; high resolution imaging; imaging system; improved; lens; macromolecule; microscopic imaging; particle; protein complex; prototype; quantum; reconstruction; simulation; structural biology; three dimensional structure; tool; transmission process

PROJECT SUMMARY Recent technological advances in transmission electron microscopy of frozen-hydrated specimens (cryo-EM) have made it possible to retrieve the three-dimensional structure of biological macromolecules with near- atomic resolution. In conventional cryo-EM, the thin, transparent protein assemblies are made visible by defocusing the imaging system. The drawback of this method is that the low spatial frequency components of the image, which are essential for identification and classification of the particles, remain heavily attenuated. Poor contrast at low spatial frequencies makes it difficult to reconstruct protein complexes with a molecular weight below 100-200 kDa, or even larger assemblies that exhibit significant structural variability. On the other hand, cryo-EM reconstruction of particles as small as 40 kDa is theoretically possible with an in-focus phase contrast device, such as a Zernike phase plate. Recently, the capabilities of Zernike phase contrast in cryo-EM single particle analysis have been demonstrated with a “Volta” phase plate, based on a thin amorphous carbon foil. However, a potential for improvement still exists, in particular in achieving a constant, stable phase shift. In this project, we set out to build a Zernike phase plate for transmission electron microscopy (TEM) based on a concept borrowed from the field of atomic physics: coherently controlling the motion of quantum particles with lasers. In this approach, a laser beam focused in the back focal plane of a TEM objective lens creates an effective potential, which retards the phase of the transmitted wave relative to the scattered wave and thus acts as a Zernike phase plate. Since no material objects are inserted in the beam path, a laser-based electron phase plate is not susceptible to electron beam damage. Importantly, it allows for a stable, controllable phase The necessary high-intensity, continuous laser focus will be created by amplifying a laser beam in a near-concentric Fabry–Pérot optical resonator with a small mode waist, which we have developed in our recent work. While we have already demonstrated a sustained optical intensity sufficient to retard a 300 keV electron beam by 9°, a full 90° phase shift, optimal for a Zernike phase plate, is well within reach with state of the art cavity mirrors. Our numerical simulations show that with a cavity-based laser phase plate, phase contrast extends to sufficiently low spatial frequencies to allow for nearly full-contrast imaging of protein complexes smaller than 5-10 nm. The initial development and characterization of the laser phase plate's efficacy as a tool for protein reconstruction will take place in a custom-built TEM, designed specifically for phase plate development. We will then proceed to build a laser phase plate compatible with standard cryo-EM equipment, with the goal of making the laser-based Zernike phase contrast technology shift to be applied to the transmitted wave. available to the broad structural biology community.

项目摘要 冷冻-水化样品透射电子显微术（cryo-EM）的新技术进展 已经使得有可能检索生物大分子的三维结构， 原子分辨率在传统的冷冻电镜中，薄的、透明的蛋白质组装体通过以下方式变得可见： 使成像系统散焦。这种方法的缺点是， 对于颗粒的识别和分类至关重要的图像仍然严重衰减。 在低空间频率下的差对比度使得难以用分子重建蛋白质复合物。 重量低于100 - 200 kDa，或甚至更大的组装体，其表现出显著的结构可变性。另 另一方面，理论上，用聚焦相可以重建小至40 kDa的颗粒 对比度装置，例如Zernike相位板。最近，Zernike相位衬度在冷冻EM中的能力 单颗粒分析已经用基于薄无定形碳的"Volta"相位板进行了证明 箔然而，仍然存在改进的潜力，特别是在实现恒定、稳定的相移方面。 在该项目中，我们着手构建用于透射电子显微镜（TEM）的Zernike相位板，基于 这是一个从原子物理学领域借来的概念：用量子力学来控制量子粒子的运动。 激光在该方法中，聚焦在TEM物镜透镜的后焦平面中的激光束产生透射光。 有效势，相对于散射波延迟透射波的相位，从而起作用 Zernike相位板。由于在光束路径中没有插入物质物体，因此基于激光的电子 相位板不易受电子束损坏的影响。重要的是，它允许一个稳定的，可控的阶段 必要的高强度，连续的激光焦点将被创建 通过在具有小模腰的近同心法布里-珀罗光学谐振腔中放大激光束， 我们在最近的工作中开发的。虽然我们已经证明了持续的光强度 足以使300 keV电子束延迟9 °，对于Zernike相位板最佳的完全90 °相移是 在现有技术腔镜的范围内。我们的数值模拟表明， 激光相位板，相位对比度扩展到足够低的空间频率，以允许几乎全对比度 小于5 - 10 nm的蛋白质复合物的成像。激光器的最初发展和特性 相位板作为蛋白质重建工具的功效将在定制的TEM中发生， 特别是用于相位板显影。然后，我们将继续构建与以下兼容的激光相位板： 标准冷冻EM设备，目标是使基于激光的Zernike相衬技术 将被施加到发射波的偏移。 提供给广大的结构生物学社区。