Mourik M J, Faas F G A, Zimmermann H, Eikenboom J, Koster A J
Department of Molecular Cell Biology, Leiden University Medical Center, Leiden, the Netherlands.
Carl Zeiss Microscopy GmbH, Munich, Germany.
J Microsc. 2015 Aug;259(2):97-104. doi: 10.1111/jmi.12222. Epub 2015 Jan 23.
Electron microscopy is used in biological research to study the ultrastructure at high resolution to obtain information on specific cellular processes. Serial block face-scanning electron microscopy is a relatively novel electron microscopy imaging technique that allows three-dimensional characterization of the ultrastructure in both tissues and cells by measuring volumes of thousands of cubic micrometres yet at nanometre-scale resolution. In the scanning electron microscope, repeatedly an image is acquired followed by the removal of a thin layer resin embedded biological material by either a microtome or a focused ion beam. In this way, each recorded image contains novel structural information which can be used for three-dimensional analysis. Here, we explore focused ion beam facilitated serial block face-scanning electron microscopy to study the endothelial cell-specific storage organelles, the Weibel-Palade bodies, during their biogenesis at the Golgi apparatus. Weibel-Palade bodies predominantly contain the coagulation protein Von Willebrand factor which is secreted by the cell upon vascular damage. Using focused ion beam facilitated serial block face-scanning electron microscopy we show that the technique has the sensitivity to clearly reveal subcellular details like mitochondrial cristae and small vesicles with a diameter of about 50 nm. Also, we reveal numerous associations between Weibel-Palade bodies and Golgi stacks which became conceivable in large-scale three-dimensional data. We demonstrate that serial block face-scanning electron microscopy is a promising tool that offers an alternative for electron tomography to study subcellular organelle interactions in the context of a complete cell.
电子显微镜在生物学研究中用于以高分辨率研究超微结构，以获取有关特定细胞过程的信息。连续块面扫描电子显微镜是一种相对新颖的电子显微镜成像技术，它能够通过测量数千立方微米的体积，但以纳米级分辨率对组织和细胞中的超微结构进行三维表征。在扫描电子显微镜中，反复采集图像，然后用切片机或聚焦离子束去除嵌入生物材料的薄层树脂。通过这种方式，每个记录的图像都包含可用于三维分析的新结构信息。在这里，我们探索聚焦离子束辅助的连续块面扫描电子显微镜，以研究内皮细胞特异性储存细胞器——魏-帕小体在高尔基体生物发生过程中的情况。魏-帕小体主要含有凝血蛋白血管性血友病因子，该因子在血管损伤时由细胞分泌。使用聚焦离子束辅助的连续块面扫描电子显微镜，我们表明该技术具有清晰揭示亚细胞细节的敏感性，如线粒体嵴和直径约50nm的小泡。此外，我们揭示了魏-帕小体与高尔基体堆栈之间的大量关联，这在大规模三维数据中是可以想象的。我们证明，连续块面扫描电子显微镜是一种很有前途的工具，为电子断层扫描提供了一种替代方法，用于在完整细胞的背景下研究亚细胞器相互作用。
