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.
Here, researchers from the University Medical Centre Utrecht describe a step-by-step guide for preparing EVs on the grid before TEM and cryo-TEM imaging. They also provide a guide to an automated image-processing analysis to provide the size distribution of EVs.
EVs were isolated from cell culture supernatant and stained with antibody CD63 (a), antibody CD9 (b), and lactadherin, a marker of a negatively charged phosphatidylserine (c). Antibodies CD63 and CD9 are coupled with 6 nm gold particle. Lactadherin is coupled with 10 nm gold particle. Double labeling of antibody CD63 and lactadherin (d) or antibody CD9 and lactadherin (e) are shown. These images are taken by using magnification 105,000× (a-d, scale bars 100 nm) and 60,000× (e, scale bar 200 nm)