Detection and Characterization of Extracellular Vesicles by Transmission and Cryo-Transmission Electron Microscopy

Abstract

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.

Preliminary investigation of extracellular vesicles in mammary cancer of dogs and cats: Identification and characterization

“…In the present study, in EV‐enriched pellets TEM highlighted the presence of membrane‐bound particles, which size and morphology are similar to that found in many studies on hEVs and equine EVs . In the UCM pellet no membrane‐bound particles were found, confirming the purity of the samples, that can be verified by the absence of growth medium/FBS‐derived EVs.…”

“…Additionally, the entire procedure (ultracentrifugation, dehydration and fixation) might alter the size and morphology, producing artefacts, such as the typical "cup shape" morphology of exosomes after fixation. 1,20 In the present study, in EV-enriched pellets TEM highlighted the presence of membrane-bound particles, which size and morphology are similar to that found in many studies on hEVs 39,45,46 and equine EVs. 31 In the UCM pellet no membrane-bound particles were found, confirming the purity of the samples, that can be verified by the absence of growth medium/FBS-derived EVs.…”

Extracellular Vesicles as Signaling Mediators and Disease Biomarkers across Biological Barriers

“…The most direct method to determine EV size and morphology is electron microscopy (EM), including both transmission EM (TEM) [133,134] and scanning EM (SEM) [39,135,136]. To avoid sample dehydration, cryogenic EM techniques have been developed, of which cryogenic TEM (cryo-TEM) is the most appropriate [137] (Figure 1). Atomic Force Microscopy (AFM), which is also used [138,139], provides information about specific properties, such as EV stiffness and elasticity [140].…”

Chicken seminal fluid lacks CD9‐ and CD44‐bearing extracellular vesicles

“…A 5 μl drop of the fixed fractions were deposited on 200‐mesh EM copper grids with formvar coating, the excess fluid removed by blotting and incubated for 7 min at room temperature. The grids were thereafter transferred to 2% uranyl acetate (w/v) drops for negative staining (Cizmar & Yuana, ). Electron micrographs were obtained using a transmission electron microscope (EM JEM 1230; JEOL Ltd.), operated at 100 kV.…”