Cizmar Petr, Yuana Yuana
Imaging Division, Image Sciences Institute, University Medical Centre Utrecht, Heidelberglaan 100, 3584 CX, Utrecht, The Netherlands.
Methods Mol Biol. 2017;1660:221-232. doi: 10.1007/978-1-4939-7253-1_18.
Transmission electron microscopy (TEM) and transmission scanning electron Microscopy (TSEM), which denotes application of a scanning electron microscope (SEM) in the transmission mode, have been used to detect and characterize particles down to an imaging resolution of ~1 nm. In the field of EVs, TEM also has been valued for its capability to detect and characterize single EV. Furthermore, employing immunogold labeling in TEM could give information regarding biochemical properties of EV surface proteins. Significant shortcomings in TEM such as dehydration, chemical fixation, and/or staining of the biological specimens are eluded by the use of cryo-TEM. In cryo-TEM imaging, samples are directly applied onto an EM grid, vitrified and visualized, thus allowing for characterization of EVs near its native state. In this chapter, we describe a step-by-step guide for preparing EVs on the grid before TEM and cryo-TEM imaging. Finally, we provide a guide to an automated image-processing analysis to provide the size distribution of EVs.
透射电子显微镜（TEM）和透射扫描电子显微镜（TSEM，即在透射模式下应用扫描电子显微镜（SEM））已被用于检测和表征低至约1纳米成像分辨率的颗粒。在细胞外囊泡领域，TEM因其检测和表征单个细胞外囊泡的能力也受到重视。此外，在TEM中采用免疫金标记可以提供有关细胞外囊泡表面蛋白生化特性的信息。使用冷冻TEM可避免TEM中存在的诸如生物标本脱水、化学固定和/或染色等重大缺点。在冷冻TEM成像中，样品直接应用于电子显微镜网格上，进行玻璃化处理并可视化，从而能够在接近其天然状态下对细胞外囊泡进行表征。在本章中，我们描述了在进行TEM和冷冻TEM成像之前在网格上制备细胞外囊泡的分步指南。最后，我们提供了一个自动图像处理分析指南，以提供细胞外囊泡的大小分布。
