Development of Laser-Based Phase Contrast for Biological Electron Microscopy

低温电子显微镜（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。然而，三维重建结构所需的最小粒子数和可重建的最小粒子尺寸仍然远远超过基本极限。在过去的四年里，我们开发了一种基于激光的相位板（LPP），可以帮助达到低温电子显微镜成像的标准量子（短噪声）极限。我们已经在光学台上对其进行了测试，演示了相衬成像，并且超出了我们设定的所有性能参数。现在，在第四年的开始，我们已经迈出了第一步，用LPP获得了一个已知结构的密度图；我们完全期望在第四年结束前完成这一目标。

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.