Development of Laser-Based Phase Contrast for Biological Electron Microscopy

项目总结 冷冻水化样品(冷冻-EM)的电子显微镜技术进展 使我们能够用近乎... 原子分辨率。在传统的冷冻-EM中，薄而透明的蛋白质组件通过以下方式可见 使成像系统散焦。这种方法的缺点是其低空间频率分量 对于颗粒的识别和分类至关重要的图像仍然严重衰减。 低空间频率的对比度差使得用分子重建蛋白质复合体变得困难 重量在100-200 kDa以下，或更大的组件，表现出显著的结构变化。另一方面 另一方面，理论上可以使用聚焦相对小到40 kDa的粒子进行低温电磁重建 对比度装置，如泽尼克相位板。近年来，Zernike位相衬度在低温EM中的应用 单粒子分析已经用基于薄无定形碳的“Volta”相板进行了演示。 花剑。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。 在这个项目中，我们着手建立一个用于透射式电子显微镜的Zernike位相板，基于 从原子物理领域借用的一个概念：用相干控制量子粒子的运动 激光。在这种方法中，聚焦在瞬变电磁物镜的后焦平面上的激光束产生了 有效势，它延迟透射波相对于散射波的相位，从而起作用 作为Zernike相位板。由于光束路径中没有插入任何物质，因此基于激光的电子 相位板对电子束损伤不敏感。重要的是，它允许一个稳定、可控的阶段 将产生必要的高强度、连续的激光聚焦 通过在具有小模腰的近同心的法布里-佩罗光学谐振腔中放大激光，该腔 我们在最近的工作中有所发展。虽然我们已经证明了持续的光学强度 对于Zernike相位板，足以将300keV电子束延迟9°，即完全90°相移，是最理想的 与最先进的腔镜触手可及。我们的数值模拟表明，在基于腔的情况下 激光相位板，相位对比度扩展到足够低的空间频率，以允许几乎完全对比度 小于5-10 nm的蛋白质复合体的成像。激光器的初步发展和特性 相板作为蛋白质重建工具的功效将在定制的透射电子显微镜中进行，设计 专门用于相位板的显影。然后，我们将着手建立一个与 标准的低温电磁设备，目标是制造基于激光的泽尼克相衬技术 要应用于传输的波的移位。 可供广大结构生物学社区使用。

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.