Institute of Bioengineering of Catalonia (IBEC), Barcelona Institute of Science and Technology, Barcelona, Spain.
Leiden Institute of Chemistry and The Institute for Chemical Immunology, Leiden University, Leiden, The Netherlands.
Methods Cell Biol. 2021;162:303-331. doi: 10.1016/bs.mcb.2020.09.001. Epub 2020 Oct 16.
Correlative light and electron microscopy (CLEM) entails a group of multimodal imaging techniques that are combined to pinpoint to the location of fluorescently labeled molecules in the context of their ultrastructural cellular environment. Here we describe a detailed workflow for STORM-CLEM, in which STochastic Optical Reconstruction Microscopy (STORM), an optical super-resolution technique, is correlated with transmission electron microscopy (TEM). This protocol has the advantage that both imaging modalities have resolution at the nanoscale, bringing higher synergies on the information obtained. The sample is prepared according to the Tokuyasu method followed by click-chemistry labeling and STORM imaging. Then, after heavy metal staining, electron microscopy imaging is performed followed by correlation of the two images. The case study presented here is on intracellular pathogens, but the protocol is versatile and could potentially be applied to many types of samples.
相关光镜和电子显微镜（CLEM）涉及一组多模态成像技术，这些技术被组合在一起，以确定荧光标记分子在其超微结构细胞环境中的位置。在这里，我们描述了 STORM-CLEM 的详细工作流程，其中 Stochastic Optical Reconstruction Microscopy（STORM）是一种光学超分辨率技术，与透射电子显微镜（TEM）相关联。该方案的优点是两种成像方式都具有纳米级分辨率，从而在获得的信息上具有更高的协同作用。样品按照 Tokuyasu 方法制备，然后进行点击化学标记和 STORM 成像。然后，进行重金属染色后，进行电子显微镜成像，然后对两张图像进行相关处理。这里呈现的案例研究是关于细胞内病原体的，但该方案具有通用性，可能适用于许多类型的样本。
