Lawrence Berkeley National Laboratory, University of California, Berkeley, CA 94720, USA.
European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany.
Ultramicroscopy. 2021 Mar;222:113213. doi: 10.1016/j.ultramic.2021.113213. Epub 2021 Jan 21.
The brightness of modern Schottky field-emission guns can produce electron beams that have very high spatial coherence, especially for the weak-illumination conditions that are used for single-particle electron cryo-microscopy in structural biology. Even so, many users have observed defocus-dependent Thon-ring fading that has led them to restrict their data collection strategy to imaging with relatively small defocus values. In this paper, we reproduce the observation of defocus-dependent Thon-ring fading and produce a quantitative analysis and clear explanation of its causes. We demonstrate that a major cause is the delocalization of high-resolution Fourier components outside the field of view of the camera. We also show that, to correctly characterize the phenomenon, it is important to make a correction for linear magnification anisotropy. Even when the anisotropy is quite small, it is present at all defocus values before circular averaging of the Thon rings, as is also true before merging data from particles in many orientations. Under the conditions used in this paper, which are typical of those used in single-particle electron cryomicroscopy, fading of the Thon rings due to source coherence is negligible. The principal conclusion is that much higher values of defocus can be used to record images than is currently thought to be possible, keeping in mind that the above-mentioned delocalization of Fourier components will ultimately become a limitation. This increased understanding should give electron microscopists the confidence to use higher amounts of defocus to allow, for example, better visibility of their particles and Ewald sphere correction.
现代肖特基场发射枪的亮度可以产生具有非常高空间相干性的电子束，特别是对于结构生物学中单粒子电子冷冻显微镜所使用的弱照明条件。即便如此，许多用户已经观察到与离焦相关的 Thon 环衰减，这导致他们将数据采集策略限制在具有相对较小离焦值的成像上。在本文中，我们再现了与离焦相关的 Thon 环衰减的观察结果，并对其原因进行了定量分析和清晰解释。我们证明，一个主要原因是高分辨率傅里叶分量的离域超出了相机的视场。我们还表明，为了正确描述该现象，对线性放大各向异性进行校正非常重要。即使各向异性非常小，在对 Thon 环进行环形平均之前以及在合并来自许多方向的粒子的数据之前，它始终存在于所有离焦值处。在本文中使用的条件下，这些条件是单粒子电子冷冻显微镜中常用的条件，源相干引起的 Thon 环衰减可以忽略不计。主要结论是，可以使用比目前认为可能的更高的离焦值来记录图像，同时要记住，傅里叶分量的上述离域最终将成为一个限制。这种更深入的理解应该使电子显微镜专家有信心使用更高的离焦值，例如，允许更好地观察他们的粒子和埃瓦尔德球校正。
