Structural Analysis of Exosomes Using Different Types of Electron Microscopy

Abstract

Negative staining has been traditionally used for exosome imaging; however, the technique is limited to surface topology only and can cause staining artifacts. Therefore, to analyze the internal structure of exosomes, we employed a method of block preparation, thin sectioning, and electron tomography. In addition, an automatic serial sectioning technique with 15-nm thickness through focused ion beam was employed to observe the three-dimensional structure of exosomes of various sizes. Cryo-transmission electron microscopy revealed the near-to-native structure of exosomes.

Characterization of Urine Stem Cell-Derived Extracellular Vesicles Reveals B Cell Stimulating Cargo

“…Nano–Sight analysis of the EV pellets revealed a homogenous population of EVs less than 150 nm in diameter. This was further confirmed by TEM, where we observed an EV size range of 60–130 nm, and the classical cup–shaped morphology described with a similar staining method [ 48 ].…”

Early life stress affects the miRNA cargo in epididymal extracellular vesicles in mouse

“…The presence and purity of epididymosomes was further validated by staining with the EV-specific marker CD9 and absence of the cellular marker GAPDH (Figure 1B). Size analysis by nanoparticle-tracking indicated that the collected particles are 50-300 nm in diameter (Figure 1D), and imaging by transmission electron microscopy showed the typical cup-shaped structures of epididymosomes (Figure 1C, Supplementary Figure 1A) [18]. RNA profiling by high-resolution automated electrophoresis showed enrichment for small RNAs of different lengths, similar to previous studies on cauda epididymosomal RNA content (Supplementary Figure 1B) [2,13].…”

Imaging extracellular vesicles: current and emerging methods

“…In cryo-electron microscopy (cryo-EM), samples will be fixed by cryo-immobilization where waters are vitrified instead of ice crystal formation in the sample by liquid ethane cooling. Cryo-immobilizing allows samples to be preserved in their native hydrated state, thus avoiding artifacts commonly caused by conventional fixation method such as cup shaped EVs [46, 47]. Combined with immunogold labeling, cryo-TEM can image EVs containing proteins and track EV uptake by recipient cells [48], as well as distinguishing EV subgroups by their size [49, 50].…”

“…After cryo-immobilization, samples can also undergo freezing substitution with fixing and embedding reagents for the specimens to be imaged under traditional TEM in room temperature. Since cryo-EM yields superior sample quality and morphology preservation over traditional EM methods [47], it is increasingly being applied to study EVs.…”