**2.1 Electron microscopies (EM)**

Electron microscopies (EM) can be used to study the morphology and size of EVs. Scanning electron microscopy utilizes beam of electrons to bombard the surface of samples and detect the backscattered electrons and secondary electrons to construct the image of the detected surface. EVs under SEM imaging are usually round shaped, but could be collapsed and irregular in shape, possibly due to the deformation of EV structure during sample preparation. Transmission electron microscopy (TEM) and cryo-TEM has become one of the gold standards for EV characterization according to MISEV2018 guideline suggested by International Society of Extracellular Vesicles (ISEV). Under TEM observation, EV are round spheres or cup-shaped with lipid membrane visible as bilayers. Immunogold labelling could be combined with EM to get the composition distribution on EVs (**Figure 1**) [1–3].
