Exosomes, a novel tumor biomarker after circulating tumor cells and circulating tumor DNA ^[1][2][3][1,2,3], can be used in tumor diagnosis [4]. Biosignaling molecules of exosomes are exchanged by endocytosis, and thereby tumor cell activities such as growth, metastasis, drug resistance, and immune evasion are regulated ^[5][6][5,6]. Therefore, exosomes have important value in the early diagnosis of tumors, the monitoring of treatment progress, and prognosis as shown in Figure 1 ^[4][7][8][9][4,7,8,9].

Exosomes are membranous extracellular vesicles (EV) secreted by cells with a particle size of 30 to 150 nm [10]. The study found that the function of exosomes depends on the cell type from which they are derived, while it maintains the same genetic material as the donor cells [11]. Different analyses become one of the tumor-specific research targets during exosome activity. For example, exosomes derived from tumor cells carry many types of proteins, such as surface proteins, inclusions, enzymes, etc. Among them, the surface proteins such as CD9, CD63, and CD81 and the inclusion factors such as HSP70 and Alix are representative proteins for the isolation and identification of exosomes ^[12][13][14][12,13,14]. The differences in proteins can reflect information exchange between tumor cells and basal cells and between tumor cells and tumor cells, which regulates immune response, migration, differentiation, and other basic cellular functions ^{[15][16][17][18]}[15,16,17,18]. For example, studies have found that exosomes derived from different cells are different in size, morphology, and composition [19]. The exosomes derived from tumor cells are large and contain more lipids and outer membrane proteins, which promote tumor cell growth, invasion, and metastasis [20]. The surface proteins of cancer exosomes are often different in different stages, which indicates that these proteins are closely related to the process of cancer. Similarly, the surface proteins of cancer exosomes from different sources are also different, which can be used for the early diagnosis of cancer ^[21][22][21,22]. Research has proven that ADAM10, metalloprotease, CD9, Annexin-1, and HSP70 are enriched in exosomes isolated from the pleural effusion or serum of breast cancer patients [23]. However, the exosomes derived from immune cells are smaller and include a variety of immune molecules, such as cytokines, antigens, antibodies, etc., which could regulate immune responses and antitumor effects [24]. More importantly, quantitative analysis is necessary to identify exosomes accurately. In addition, some studies believe that the morphology, quantity, and concentration of exosomes are different in the process of secretion [25], so real-time tracking and quantification can objectively analyze the delicate mechanism to achieve optimal anti-tumor effect. In conclusion, the morphological characterization and function of exosomes are the basis for exploring the fine physiological information and biochemical mechanisms in cellular biology ^[26][27][26,27]. Exosomes are emerging biomarkers of tumor liquid biopsy, and therefore it is particularly important to explore their biological information such as function tracing, protein quantification at unit point, and microstructural changes ^[28][29][28,29]. At present, much attention has been paid to their basic characteristics such as concentration, diameter, morphology, and particle tracking in high-throughput samples ^[30][31][32][30,31,32]. In this review, we propose that the characterization techniques for obtaining this information are exosome detection techniques. Conventional characterization methods can achieve the basic characterization of exosomes, yet they are subject to certain limitations. DLS can only obtain the size distribution of exosomes but cannot detect concentration [33]. Although flow cytometry (FCM) realizes multi-parameter detection, the analysis results are based on high-throughput samples and cannot obtain the morphological characteristics of a single EV [34]. EM can visually observe the morphological characteristics of a single EV, but it is not suitable for living cells, and the direction is limited in biological research [35]. Therefore, there is an urgent need for an exosome detection technique that can preserve the fluorescence specificity and achieve high-throughput sample single-molecule detection. In recent years, researchers have paid more attention to fluorescence microscopy. The fluorescence microscope has the advantage of live cell imaging and specific labeling; however, its resolution ranges from 200 nm to 500 nm, which cannot be applied to detecting exosomes. Therefore, how to improve the imaging resolution of optical microscopy and apply it to the imaging observation of subcellular structures such as exosomes has become a research hotspot in recent years. With the development of fluorescence microscopy, super-resolution imaging technology breaks through the diffraction limitation, which is conducive to the field of exosomes ^[26][36][26,36]. Compared with others, it has the advantages of high resolution, specific detection, and live-cell imaging to realize the qualitative and quantitative analysis of proteins of living or fixed cells and track the mechanism of exosomes [36]. In order to better guide the research on exosomes, we systematically discussed ^[36]and compared characterization techniques for exosomes. This review system elaborates on the principles and applications of conventional characterization technologies for exosomes such as dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), flow cytometry (FCM), electron microscopy (EM), and scanning electron microscopy (SEM), as well as super-resolution technologies represented by stochastic optical reconstruction microscopy (STORM), photoactivated localization microscopy (PALM), stimulated emission depletion (STED), and structured illumination microscopy (SIM). More importantly, this paper discusses the medical problems and characterization techniques of exosomes from morphological characterization to the functional expression of exosomes, from complex samples to single exosomes, and from multiple protein quantification to a single protein of a single exosome. The mechanism and information communication of exosomes were systematically settled from the perspective of technology development, which provides more systematic and comprehensive guidance in the field of basic research.

2. Conventional Characterization Technologies

Due to the unique biological function of exosomes, an increasing amount of basic research is being concentrated on it ^{[37][38][39][40][41]}[37,38,39,40,41]. Characterization technologies play important roles in the study of exosomes [30]. Generally speaking, various approaches for analysis are categorized into two primary types: biochemical analysis and physical analysis. Biochemical analysis mainly determines the source and composition of exosomes, including Western blot and enzyme-linked immunosorbent assay (ELISA), in which the specific binding of antibody antigens decides the effect qualitatively or quantitatively [42]. However, the disadvantage is that the morphological characteristics and concentration of exosomes cannot be obtained.  We introduce electron technologies such as tunable resistive pulse sensing (TRPS) and electron microscope (EM) in the first section. Then, we compare optical analysis technologies including dynamic light scattering (DLS), nanoparticle tracking analysis (NTA), and flow cytometry (FCM). Last but not least, we discuss the main parameters of different technologies, providing technical guidance for the fundamental research on exosome characterization.

2.1. Tunable Resistive Pulse Sensing

Tunable Resistive Pulse Sensing (TRPS) is based on Coulter’s principle. The suspension was mixed in the electrolyte, which could go through the nanopore chip with a specific aperture. The resistance between the two electrodes inside and outside changes instantaneously at the moment of passing through the nanopore, the result of which is a pulse signal as shown in Figure 2a. The intensity and frequency of the signal are related to the size and number of exosomes. The expression of exosomes was counted by the pulse signal. In 2014, Maas proposed a method to characterize the concentration and size of EVs by the TRPS based on the qNano system [43]. In 2017, researchers pointed out that TRPS has promise in the quantitative and dimensional analysis of single-particle EV [44]. In 2018, Durak-Kozica analyzed EVs from endothelial cells for a short time and found that the diameter of EVs was 121.84 ± 0.08 and 115.82 ± 0.96 nm from microvascular and big vessels, respectively [45]. This technology enables the efficient quantification of size and number, which cannot be specifically analyzed for exosomes due to the principle of potential pulses.