To reveal the morphology of the single extracellular vesicles (EVs), transmission electron microscopy (TEM) ^[1][35], cryo-EM ^[2][36] as well as atomic force microscopy (AFM) ^[3][37] have been widely used to observe EVs in different conditions. In fact, it was in the 1960s ^[4][38] that EVs were first-time observed under an EM when they were described as “platelet dust”. In the 1980s, Pan et al. ^[5][6][39,40] described EVs as small dense bodies observed under TEM with a size of 50 nm and demonstrated that the transferrin receptor was externalized in these vesicles. EVs exhibit a saucer-like structure under TEM caused by the collapse of the samples during sample drying treatment, while cryo-EM can completely preserve their original morphology, as they were imaged in their native aqueous status without fixation. In addition, cryo-EM enables the precise observation of the EV morphology and heterogeneity ^[7][41]. Poliakov et al. ^[8][42] reported the detailed structures of small exosome-like vesicles isolated from human seminal fluid for the first time. They analyzed 301 cryo-EM images of vesicles purified by a sucrose gradient, and described their morphological characteristics in detail, including multiplicity, shape, external features and the overall density of the vesicles.

In order to accurately analyze EVs in three dimensions, AFM was utilized to disclose the structural and nanomechanical features of EVs. Yurtsever et al. ^[9][43] found distinct local domains on the surface of exosomes using 3D-AFM. The exosome samples were prepared by ultracentrifugation and a MagCapturerm exosome isolation kit. They revealed that exosomes have an elastic modulus ranging from 50 MPa to 350 MPa. Moreover, they also found that the exosome mechanical properties are related to the malignancy of tumor cells and confirmed that the increased elastic modulus of exosomes derived from metastatic tumor cells contained rich specific proteins related to the elastic fiber formation. These findings are important for future studies on exosome biofunctions and provide a different strategy for using exosomes as cancer diagnostic biomarkers. Ye et al. ^[10][44] employed AFM to investigate the physical properties of single EVs released by cancer cells. The EV samples were prepared by ultracentrifugation. The relationship between the tumor malignancy and the EV size was explored. The EVs of greater malignancy and smaller size exhibit an increased stiffness and osmotic pressure but a lower bending modulus, establishing a relationship between the tumor malignancy and EV nanomechanical signatures.

Since EVs are a group of nano- to micro-sized particles varying from 30 nm to 5000 nm, it is still difficult to accurately enumerate pure EVs. One of the most widely used techniques for the enumeration of mono-dispersed nanoparticles is dynamic light scattering (DLS) ^[11][12][45,46]. DLS detects the scattered light intensity from particles undergoing the Brownian motion in a solution that fluctuates over time. The particle size is measured indirectly, based on the movement of the particles, so the resolution of the DLS ^[13][47] is limited when applied to characterize the polydisperse sample with heterogeneous EVs in size. A nanoparticle tracking analysis (NTA) ^[14][15][48,49] also utilizes the properties of the Brownian motion, as well as light scattering, to estimate the particle size in a solution. The scattered light from the particles is captured by a digital camera, and then computer software is used to analyze the Brownian motion of each particle. This particle-by-particle analysis eliminates the inherent limitation in the DLS. Due to the unique advantage, the NTA has been favorably evaluated when used for the EV characterization.

Resistive pulse sensing (RPS) ^[16][50] is an efficient technique for the particle enumeration and size measurement in electrolyte solutions, based on the Coulter counter principle. A tiny insulating aperture is submerged in an electrolyte solution containing suspended particles. When a particle passes through the orifice, the changes, in terms of the ion current pulse, can be detected. The size of the particles that can be detected is determined by the diameter of the aperture. The use of RPS for single EV counting ^[17][51] attracts considerable interest. To improve the feasibility of the particle detection and the flexibility of the pore size, tunable resistive pulse sensing (TRPS) ^[18][52] was then proposed, and the pore size can be reversibly adjusted, which enables the flexible detection of single particles of different sizes. Yet, the EV characterization using TRPS is still suffering from the heterogeneity problem and the unknown buffer components in the biological samples ^[19][53]. De Vrij et al. ^[20][54] spiked the internal control beads into EV samples for a variation correction, enabling the quantification of EVs in biological samples by a scanning ion occlusion sensing (SIOS) technology without EV labeling. This method provides a valuable strategy to the TRPS approach for the EV quantification.

Despite the fact that the NTA and RPS have been requested for the EV characterization, they are still unable to accurately determine the EV size, due to the existence of complicated background nanoparticles in the EV samples, including lipoproteins and protein aggregates. Flow cytometry might be an ideal method for the EV analysis at the single particle level, to obtain more specific information. However, conventional flow cytometry was designed for the microparticle characterization, such as cells, which may be not suitable for the nano-sized EVs. Van der Vlist et al. ^[21][55] reported a protocol to set up a high-resolution flow cytometry for the individual nano-sized vesicle analysis. In this method, the cell-derived vesicles are labeled with bright fluorescent, then an optimized configuration of the flow cytometer was employed for the flow cytometric analysis. The high-resolution flow cytometer enables the precise identification and analysis for the nano-sized vesicles, as small as 100 nm in diameter. Yan’s group ^[22][23][56,57] built a high-sensitivity flow cytometer (HSFCM) that achieved a limit of detection for single gold nanoparticles of 7 nm. They then employed the HSFCM for the single EV detection that can detect EVs between 40 nm and 200 nm in diameter with an analysis rate of 10,000 particles/min. HSFCM offers a sensitive way to measure the size of single EVs and analyze the surface proteins, which might considerably enhance the understanding of the cell-cell communication mediated by EVs and facilitate the development of new diagnostic techniques.

Lately, droplet technology ^[24][58] has been used for the counting of single EVs, showing a dramatic improvement in the detection sensitivity. Liu et al. ^[25][59] reported an immunosorbent test for the digital validation of the target EVs. Briefly, EVs were first captured on the magnetic beads and labeled with a reporter which generates a fluorescent signal. These beads were then encapsulated separately into droplets with just one bead enclosed in each droplet. The droplet-based single-exosome-counting enzyme-linked immunoassay (droplet digital ExoELISA) achieved a limit of detection, down to 10 exosomes/μL in an absolute counting way for the single exosomes. Compared with the signal amplification method of the ELISA, the signal amplification method of DNA in an in vitro amplification, may be more stable. They successfully quantified EVs in serum samples using a droplet digital ExoELISA. Yang et al. ^[26][60] developed a droplet-based extracellular vesicle analysis (DEVA) assay, which enables the quantification of EV subpopulations with a high throughput of 20 million droplets per minutes, which is 100 times greater than microfluidic systems. Notably, a LOD of 9 EVs/μL was achieved when processing EVs in the PBS samples. The application of the droplet digital technology provides a completely new way that allows for the ultrasensitive EV detection.

The techniques for the molecular analysis of bulk EVs have been widely reported, such as the western blot (WB) and the enzyme linked immunosorbent assay (ELISA), but it is limited in the analysis of the surface proteins or nucleic acids for the single EVs, due to the low detection sensitivity and the heterogeneity of the single EVs. Here, the following contentse summarize the advances of the current technologies developed for the molecular analysis at the single EV level.

Although there is an intrinsic limitation on the detectable size for flow cytometry, when directly detecting exosomes, it has been extensively used in studying the surface marker on exosomes through various instrumentation development, as well as exosome labeling strategies. Shen et al. ^[27][32] designed a probe with switchable conformations that recognizes CD63, to visualize individual EVs. The anti-CD63 aptamer was introduced in the probe for the target recognition, and a trigger domain was designed for initiating the DNA growth via the hybridization chain reaction (HCR). Thereby, the DNA nanostructures resulting from the target-initiated engineering (TIE) could enlarge the labeled EVs, making them detectable by a confocal fluorescence microscope. A simultaneous analysis of the dual surface marker expression on a single EV was also demonstrated in this study. The multiparameter analysis for the single EVs could also been achieved by using imaging flow cytometry (IFCM) ^[28] [61]. IFCM facilitates the analysis of the surface protein profiles of CD9, CD63 and CD81 on glioblastoma EVs. They found that the number and proportion of CD63+ and CD81+ EVs in cancer patients, were significantly increased.

Surface-enhanced Raman scattering (SERS) ^[29][62] is a potent surface-sensitive approach that can analyze the molecular spectral signals, even at the level of a single molecule, by a multi-orders-of-magnitude amplification. Tirinato et al. ^[30][63] used SERS to analyze the healthy cell-derived EVs and tumor cell-derived EVs, with a super-hydrophobic nanostructured substrate, that can control diluted solutions precisely. Tumor cell-derived exosomes, exhibiting a richer RNA content, can be differentiated from healthy cell-derived exosomes specifically. Raman tweezers microspectroscopy (RTM), which combines optical trapping with Raman probing, provides genuine Raman fingerprints of the sample’s constituent biomolecules. Kruglik et al. ^[31][64] demonstrated the utility of RTM in defining different subpopulations of exosomes. Notably, RTM provides a universal molecular signature for different EV subpopulations at the single-EV level. A Raman-enabled nanoparticle trapping analysis (R-NTA), proposed by Dai et al. ^[32][65], presented another method to access the chemical composition at the level of a single particle in a label-free way. They demonstrated the power of the R-NTA platform to characterize the morphology, as well as the chemical heterogeneity of the nanoparticles. Carney et al. ^[33][66] described the first application of the multispectral optical tweezers (MS-OTs) for the individual vesicle molecular profiling. This platform has the unique capability to multiplexing quantify the compositional difference across the EV groups.

Single particle interferometric imaging sensing (SP-IRIS) ^{[34][35][36][37][38]}[67,68,69,70,71] is of performance with the single-molecule detection capability. It enables the nanoparticle size measurement of 70 nm from complex biological samples, at low concentrations ^[39][40][72,73]. Recently, the potential of SP-IRIS on the single EV characterization, has been explored ^[41][42][74,75]. EVs are first isolated using immune recognition, and then the surface proteins and the RNA/DNA contents of the target EV are quantified. The technique has been shown to fractionate subpopulations of exosomes with specific markers, allowing for a better understanding on their heterogeneity ^[43][76]. An et al. ^[44][77] performed a preliminary screening of the common EV biomarkers CD9, CD63 and CD81 tetramers, using SP-IRIS. It was found that the CD81 expression levels were significantly higher in all EV samples, compared to CD9 and CD63.

Taking advantage of the high-resolution of AFM, individual EVs can be probed for the identification of their nanoscale composition ^[45][46][47][78,79,80]. The quantitative differences analyzed at the single-vesicle level between the normal and patient’s exosomes have been reported using high-resolution AFM ^[48][81]. Dazzi et al. ^[49][50][51][82,83,84] developed a technique coupling AFM and IR spectroscopy (AFM-IR), which enabled thenanoscale chemical component analysis ^[52][53][85,86]. AFM-IR offers the tremendous potential to detect the biomolecules inside individual EVs without labeling. The height images from AFM and AFM-IR spectra allow for the direct comparison of two different EV populations, which can be accomplished on a single EV ^[54][55][56][87,88,89]. In addition, the IR maps for the different EVs enable AFM-IR to generate specific fingerprints for individual EVs. AFM-IR is currently the most sensitive technique to investigate the heterogeneity across individual EVs and also EV subpopulations.

The Weissleder group ^[57][58][59][90,91,92] exploited the application of the antibody-based immuno-droplet digital polymerase chain reaction (iddPCR) for the protein analysis on single EVs. Followed by the PCR amplification, individual droplets with target EVs in them were imaged by fluorescence microscopy for the EV quantification. To specify the limitation from the fluorescence readout, they proposed an antibody-DNA barcode-based immunosequencing method (seiSEQ) ^[58][91]. The EVs attached with Ab-DNA are subsequently encapsulated into 70 µm droplets along with barcoded beads, where the PCR amplifies the DNA barcode signals, enabling the multiplexing of the protein profile in individual EVs with a high specificity. Banijamali et al. ^[60][93] further developed a droplet barcode sequencing technology to analyze multiple proteins without barcoded gel beads. The droplet digital technology has also been employed to amplify and characterize single EV nucleic acids. The traditional method for the EV nucleic acid analysis requires the EV lysis, followed by the quantitative real-time PCR (qPCR) or ddPCR in a population-based analysis. Pasini et al. ^[61][94] have reported that the droplet-based method enabled the detection of nucleic acid biomarkers in a single EV, which may explore more information on the specific EV subset that contains specific biomarkers.

Additionally, there are several further innovative strategies that have been proposed for the EV molecular analysis. Huang et al. ^[62][95] demonstrated a single EV counting system using lanthanide-doped upconversion nanoparticles (UCNPs). They showed the significant potential of the UCNPs to “digitally” quantify the surface proteins on the individual EVs, which provides an approach to monitor the EV heterogeneity changes during the tumor’s progression ^[63][96]. Dittrich’s team ^[64][97] fabricated a microfluidic platform that can capture, quantify and classify the EVs released by a single cell. Each detected EV can be assigned to one of 15 unique populations through multicolor immunostaining.