Detection and Characterization of Extracellular Vesicles by Transmission and Cryo-Transmission Electron Microscopy
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.
Key wordsImmunogold labeling Negative staining Lactadherin CD9 CD63 Exosomes Image processing
Both authors thank the technical supports given by A.E. Grootemaat from Department of Cell Biology and Histology, Academic Medical Centre, University of Amsterdam, Dr. R.I. Koning from the Molecular Cell Biology Department at the Leiden University Medical Center, and Dr. G. Posthuma from Cell Microscopy Center, Department of Cell Biology of University Medical Center Utrecht, The Netherlands.
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