Du Ming, Jacobsen Chris
Department of Materials Science and Engineering, Northwestern University, 2145 Sheridan Road, Evanston IL 60208, USA.
Advanced Photon Source, Argonne National Laboratory, 9700 South Cass Avenue, Argonne IL 60439, USA; Department of Physics & Astronomy, Northwestern University, 2145 Sheridan Road, Evanston IL 60208, USA; Chemistry of Life Processes Institute, Northwestern University, 2170 Campus Drive, Evanston IL 60208, USA.
Ultramicroscopy. 2018 Jan;184(Pt A):293-309. doi: 10.1016/j.ultramic.2017.10.003. Epub 2017 Oct 7.
Electron and x-ray microscopes allow one to image the entire, unlabeled structure of hydrated materials at a resolution well beyond what visible light microscopes can achieve. However, both approaches involve ionizing radiation, so that radiation damage must be considered as one of the limits to imaging. Drawing upon earlier work, we describe here a unified approach to estimating the image contrast (and thus the required exposure and corresponding radiation dose) in both x-ray and electron microscopy. This approach accounts for factors such as plural and inelastic scattering, and (in electron microscopy) the use of energy filters to obtain so-called "zero loss" images. As expected, it shows that electron microscopy offers lower dose for specimens thinner than about 1 µm (such as for studies of macromolecules, viruses, bacteria and archaebacteria, and thin sectioned material), while x-ray microscopy offers superior characteristics for imaging thicker specimen such as whole eukaryotic cells, thick-sectioned tissues, and organs. The required radiation dose scales strongly as a function of the desired spatial resolution, allowing one to understand the limits of live and frozen hydrated specimen imaging. Finally, we consider the factors limiting x-ray microscopy of thicker materials, suggesting that specimens as thick as a whole mouse brain can be imaged with x-ray microscopes without significant image degradation should appropriate image reconstruction methods be identified.
电子显微镜和X射线显微镜能让人们以远超可见光显微镜的分辨率对水合材料的完整、未标记结构进行成像。然而，这两种方法都涉及电离辐射，因此辐射损伤必须被视为成像的限制因素之一。借鉴早期的工作，我们在此描述一种统一的方法，用于估计X射线显微镜和电子显微镜中的图像对比度（进而估计所需的曝光量和相应的辐射剂量）。这种方法考虑了诸如多重散射和非弹性散射等因素，以及（在电子显微镜中）使用能量过滤器来获取所谓的“零损失”图像。不出所料，结果表明，对于厚度小于约1微米的标本（如用于研究大分子、病毒、细菌和古细菌以及薄切片材料），电子显微镜提供的剂量更低，而对于成像较厚的标本，如整个真核细胞、厚切片组织和器官，X射线显微镜具有更优越的特性。所需的辐射剂量与所需的空间分辨率密切相关，这使人们能够了解活体和冷冻水合标本成像的限制。最后，我们考虑了限制对较厚材料进行X射线显微镜成像的因素，表明如果能找到合适的图像重建方法，像整个小鼠大脑这么厚的标本也能用X射线显微镜成像，且不会有明显的图像退化。
