*2.4.1 Physical analysis*

The physical analyses of EVs involve determining a size range, shape and concentration. The size of EVs can be determined directly by high-resolution imaging, or indirectly, by using optical or electrical readouts. Direct high-resolution imaging includes microscopy methods, such as EM or AFM, to obtain an accurate estimate of individual EVs in nanoscale resolution [18]. EM is used to determine the size and morphology of individual EVs. This method employs an electron beam instead of light to obtain high-resolution images in nanoscale. The most commonly used EM techniques are scanning (SEM) and transmission (TEM). Scanning electron microscopy image will explore the topography of the EVs surface. Since electrons pass through the sample in TEM, a 2D image of EVs will be obtained, which will


*Abbreviations: AFM: atomic-force microscopy; DLS: dynamic light scattering; ELISA: enzyme-linked immunosorbent assay; EM: electron microscopy; IF: immunofluorescence microscopy; NTA: nanoparticle tracking analysis; RPS; restrictive pulse sensing; SEC: size exclusion chromatography: EM: transmission electron microscopy WB: western blotting.*

#### **Table 1.**

*Most commonly used methods for isolation and characterization of EVs.*

also provide the information on the inner structure [26]. These electron microscopy methods require fixation or drying of the sample which complicates the translation of observed structures to the native morphology of the EVs. To avoid sample dehydration variations of electron microscopy techniques, such as cryogenic TEM, have been evolved [27]. In the AFM, an extremely sharp tip scans the area and its deflection is translated into topology information. It provides additional information about mechanical properties, such as stiffness and elasticity of the vesicles. In most cases, AFM is performed on dry, immobilized surfaces, which in turn may damage the EVs [28]. This can be prevented by analyzing EVs in a solution [29]. Indirect methods estimate the size and concentration based on the interaction of EVs with light (DLS and flow cytometry), their diffusion trajectories (NTA or their effect on the electrical current (tRPS). DLS is based on the analysis of temporal intensity fluctuation of laser light scattered by a dispersion of freely diffusing EVs. Unlike EM and AFM it measures the collective mobility (diffusion coefficient) of scattering EVs that are present in the measured volume. Flow cytometry is often used to analyze the number of cells and their biochemical composition. EVs are much smaller than cells and are usually not detected due to the low sensitivity of the method. However, adapted protocols have been developed to enable the analysis of EVs [30]. In flow cytometry, the flow of cells is hydrodynamically focused in a flow chamber and enables the illumination of a single cell by several different lasers. The forward light scatter on the cell will allow information on the cells' sizes while the side scatter will gave information on the granularity and composition [31]. Because the EVs are very small and have a low refractive index, flow cytometers can more accurately determine the EVs larger than 500 nm. Smaller EVs are detected in the background signal and collectively due to the swarm effect, which happens when multiple EVs are simultaneously and not separately illuminated by the laser, creating a swarm [32]. The recent advances in the field of flow cytometry enable to detect also populations as small as 100 nm [33]. NTA measures how fast a particle diffuses in a static solution due to the principle of Brownian particle motion. By analyzing its motion trajectories, it determines the size distribution of vesicles. tRPS is a technique that measures changes in electrical current as each particle passes through an adjustable nanopore [18]. The heterogeneity of the samples is a major problem with all indirect methods. Compared to direct methods the number of EVs that can be analyzed is typically higher, which allows a better estimate of the concentration. This is also due to the fact that these vesicles are in their original state. However, these methods are not able to provide information on the presence of contaminants, such as lipoproteins.
