Department of Cell and Chemical Biology, Leiden University Medical Centre, 2300 RC, Leiden, The Netherlands.
Department of Molecular Sociology, Max Planck Institute of Biophysics, Max-von-Laue-Straße 3, 60438, Frankfurt Am Main, Germany.
Sci Rep. 2023 May 22;13(1):8270. doi: 10.1038/s41598-023-35590-x.
Cryogenic transmission electron microscopy (cryo-TEM) and super-resolution fluorescence microscopy are two popular and ever improving methods for high-resolution imaging of biological samples. In recent years, the combination of these two techniques into one correlated workflow has gained attention as a promising route towards contextualizing and enriching cryo-TEM imagery. A problem that is often encountered in the combination of these methods is that of light-induced damage to the sample during fluorescence imaging that renders the sample structure unsuitable for TEM imaging. In this paper, we describe how absorption of light by TEM sample support grids leads to sample damage, and we systematically explore the importance of parameters of grid design. We explain how, by changing the grid geometry and materials, one can increase the maximum illumination power density in fluorescence microscopy by up to an order of magnitude. Finally, we demonstrate the significant improvements in super-resolution image quality that are enabled by the selection of support grids that are optimally suited for correlated cryo-microscopy.
低温传输电子显微镜（cryo-TEM）和超分辨率荧光显微镜是两种常用于高分辨率生物样本成像的热门且不断改进的方法。近年来，将这两种技术结合到一个相关联的工作流程中，作为一种有前途的方法，引起了人们对低温 TEM 图像进行情境化和丰富化的关注。在这些方法的组合中经常遇到的一个问题是，在荧光成像过程中，光对样品的诱导损伤会使样品结构不适合 TEM 成像。在本文中，我们描述了 TEM 样品支撑网格对光的吸收如何导致样品损伤，并且系统地探讨了网格设计参数的重要性。我们解释了如何通过改变网格的几何形状和材料，将荧光显微镜中的最大照明功率密度提高一个数量级。最后，我们通过选择最适合相关低温显微镜的支撑网格，展示了超分辨率图像质量的显著改善。
