Lucas Miriam S, Günthert Maja, Bittermann Anne Greet, de Marco Alex, Wepf Roger
ETH Zurich, Zurich, Switzerland.
Monash University, Clayton, VIC, Australia.
Methods Cell Biol. 2017;140:123-148. doi: 10.1016/bs.mcb.2017.03.001. Epub 2017 Apr 6.
Live-cell imaging is one of the most widely applied methods in live science. Here we describe two setups for live-cell imaging, which can easily be combined with volume SEM for correlative studies. The first procedure applies cell culture dishes with a gridded glass support, which can be used for any light microscopy modality. The second approach is a flow-chamber setup based on Ibidi μ-slides. Both live-cell imaging strategies can be followed up with serial blockface- or focused ion beam-scanning electron microscopy. Two types of resin embedding after heavy metal staining and dehydration are presented making best use of the particular advantages of each imaging modality: classical en-bloc embedding and thin-layer plastification. The latter can be used only for focused ion beam-scanning electron microscopy, but is advantageous for studying cell-interactions with specific substrates, or when the substrate cannot be removed. En-bloc embedding has diverse applications and can be applied for both described volume scanning electron microscopy techniques. Finally, strategies for relocating the cell of interest are discussed for both embedding approaches and in respect to the applied light and scanning electron microscopy methods.
活细胞成像技术是生命科学领域应用最为广泛的方法之一。在此，我们介绍两种活细胞成像设置，它们能够轻松地与体扫描电子显微镜相结合，用于相关研究。第一种方法是使用带有网格玻璃支撑的细胞培养皿，可用于任何光学显微镜模式。第二种方法是基于Ibidi μ载玻片的流动腔设置。这两种活细胞成像策略均可后续采用连续块面或聚焦离子束扫描电子显微镜进行研究。本文介绍了两种在重金属染色和脱水后进行树脂包埋的方法，充分利用了每种成像模式的独特优势：经典的整体包埋和薄层塑化。后者仅适用于聚焦离子束扫描电子显微镜，但在研究细胞与特定底物的相互作用或底物无法去除的情况下具有优势。整体包埋具有多种应用，可用于上述两种体扫描电子显微镜技术。最后，针对两种包埋方法以及所应用的光学显微镜和扫描电子显微镜方法，讨论了重新定位感兴趣细胞的策略。
