Development of Laser-Based Phase Contrast for Biological Electron Microscopy

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，结构的三维重建所需的颗粒的最小数量，以及适合于重建的颗粒的最小尺寸，仍然远远高于基本限制。在过去的四年中，我们已经开发出一种基于激光的相位板（LPP），可以有助于达到标准的量子（散粒噪声）限制的成像在冷冻EM。我们已经在光学台上对其进行了测试，展示了相衬成像，并超过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获得已知结构的密度图;我们完全期望在第四年年底完成这一目标。 在这个更新提案中，我们的目标是实现更高水平的性能，这将为结构生物学中的许多类问题增加重要价值，并且将受到整个cryo-EM社区的欢迎，作为用户友好的商业产品的基础。为此，我们将通过创建新的数据驱动反馈工具来部分自动化数据收集，以保持LPP与电子衍射图案的对齐。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彗差对准。这次升级将利用我们最近证明的相对论反转效应来消除微弱的鬼像。此外，为了补偿我们的显微镜在相位板模式下的较大色差，我们将安装一个枪单色仪。 使用LPP预计能够在被认为理论上可能用于冷冻EM的较低尺寸极限下重建颗粒。我们希望这也能提高3D分类的能力，将更大的颗粒分配到明显不同的构象和组成状态。在整个项目中，我们将通过对各种生物标本进行重建来确定LPP在多大程度上提高了冷冻EM能力。我们将确定产生高分辨率密度图所需的非对称单元的数量，以及可以重建的最小颗粒的大小。随着LPP的发展，我们将使用越来越具有挑战性的测试样本，从脱铁铁蛋白和人类微管相关蛋白到极小的蛋白质，如肌红蛋白或溶菌酶。

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.