# Plant Exosomal Vesicles: Perspective Information Nanocarriers in Biomedicine
## 7 Characterization of Exosomes  
The characterization of plant nanovesicles includes a wide spectrum of morphological, physical, and biochemical analytical approaches. One of the most common imaging techniques used is scanning and transmission electronic microscopy (SEM and TEM, respectively) [83]. SEM provides information about the three-dimensional structure of the exosome surface, however, as soon as drying is applied during sample preparation, their natural morphology could be changed, leading to the formation of cup-shaped structures [84]. In turn, TEM is a more accurate method, as nanovesicles are not deformed during processing. Moreover, staining with heavy metals, such as osmium tetroxide and uranyl acetate, creates lipid membrane contrasts allowing distinguishing exosomes from impurities [85]. Cryo-SEM, or cryomicroscopy, implies freezing and sample analysis at a very low temperature (below \(-100\)\({}^{\circ}\)C) [86]. It is used to assess the morphology of nanovesicles in a state close to the native one due to their perfect preservation, as well as the absence of pre-fixation operations or the addition of heavy metals [21,83]. Atomic force microscopy can also be used as an additional indirect method for assessing the morphological and physical properties (e.g., adhesion and stiffness) of nanovesicles [87]. The method does not require extensive sample preparation: exosomes could be rapidly adsorbed and dried on glass or mica surfaces. The hydrodynamic sizes of exosomes are evaluated by using dynamic light scattering (DLS) or nanoparticle tracking analysis (NTA). DLS measures the volumetric scattered light from nanovesicles when illuminated with a monochromatic light source. Since the particles are in Brownian motion, scattered light from all particles interferes and intensity fluctuates with time, and information about the particles is obtained from the autocorrelation of the oscillation intensity recorded during the experiment [88]. At the same time, NTA is based on the use of a concentrated beam of light to illuminate the particles in the sample. As the particles scatter light and undergo Brownian motion, the camera records the path of each individual particle to determine the average speed and size [85]. As the measurement of nanoparticles using the latter method is more accurate when analyzing polydispersed samples [89,90], NTA is now considered a gold standard for exosome characterization [91]. The identification of specific protein markers is one of the most important biochemical characteristics of exosomes. Usually, Western blotting with antibodies specific to such proteins as PEN3, TET8, and HSP70 indicates the plant-derived nanovesicle fraction [92]. However, in the case of exosomes from the non-model plants, the commercially available antibodies for _Arabidopsis thaliana_ may have a low affinity to the proteins from evolutionarily distant species. Another versatile way to identify proteins, including marker ones, is mass spectrometry [93]. In addition to the purification, the peptide fractionation prior to mass spectrometric analysis is considered an important prerequisite for the identification of vesicular proteins with high confidence. In terms of detection sensitivity, mass spectrometry is not as sensitive as antibody-based methods, but it allows analysis of a whole spectrum of exosomal proteins at once [94]. As mentioned above, the purity of the exosomes must be checked very strictly, since the identification of many hundreds of proteins in exosomes is probably due to contamination [42]. Mass spectrometry can also be used to analyze the low molecular weight components, such as secondary metabolites, fatty acids, and sugars [95,96]. New generation sequencing (NGS) methods are used to identify the nucleic acid fractions, primarily the small RNAs [97]. Taking into account that a huge fraction of small RNAs is coprecipitated with exosomes in the complexes with proteins, protease trypsin digestion should precede RNase treatment to ensure degradation of RNAs located outside vesicles [48].