Table 4.

Current techniques for uEV characterisation.

Characterisation	Technique	Information giving	Pros and cons
Morphology	Transmission electron microscopy (TEM)	Images of a heterogeneous group of EVs of different sizes and shapes for sample purity; TEM also shows EV heterogeneity by different staining densities to highlight morphological characteristics and surface features.	Pros: easier and more accessible than cryo-EM; commonly used for EV morphology. Cons: expensive and time consuming; must have a very thin layer.
	Cryogenic electron microscopy (cryo-EM)	Cryo-EM shows the lipid-bilayer and all particles in a given volume can be imaged, not just those that adhere to a surface (the grid) (108, 109).	Pros: preserves EV size better than the dehydrating conditions used to fix samples for TEM and may be more quantitative. Cons: costly equipment that requires specialized staff to setup.
	Atomic force microscopy (AFM)	Visualisation of uEVs with sub-nanometer resolution in three dimensions in atmospheric or submerged conditions (110).	Pros: samples do not require any special treatments that would irreversibly change or damage the sample; most AFM modes work well in ambient air or a liquid environment. Cons: can only obtain surface information from samples; also limited by the single scan image size and the relatively slow scanning speed.
	Super resolution fluorescence microscopy	Direct visualization of fluorescently labelled molecules within vesicles with 20 nm resolution, revealing the biomarker distribution and expression levels on single vesicles (111–113).	Pros: provides better spatial resolution for observing exosomes and enables intracellular tracking of exosomes. Cons: special fluorophores required; phototoxicity associated with multiple imaging/quenching cycles; imaging close to coverslip.
Size distribution and counts	Nanoparticle tracking analysis (NTA)	Particle size distribution and particle concentration within a range.	Pros: accessible and commonly used for EV morphology. Cons: cannot exclude non-EV entities; particle count may be overestimated; may generate biased results due to calibrators in use; different software generates different absolute particle count.
	Tunable resistive pulse sensing (TRPS)	EV particle size distribution, particle number and surface charge (90)	Pros: rapid, convenient, accurate and reproducible. Cons: discrepancy in count numbers between TRPS and NTA.
EV content	Western blotting/ELISA	Specific uEV content	Pros: easy and accessible; widely used for analysis and validation of one or a few target proteins. Cons: requires validated antibodies.
	Flow cytometry	Single EV surface protein	Pros: bead-based commercial kit are available (114). Cons: requires experienced staff to setup instrument for sufficient resolution (115).
	Liquid chromatography-tandem mass spectrometry (LC-MS/MS)	Protein profile within uEVs (116)	Pros: precise, rapid and sensitive; requires small sample size to produce data that can reach high statistical power. Cons: expensive in terms of capital and running costs; needs a skilled technician.
	RNA-sequencing	Transcriptome of uEVs (117)	Pros: sequencing of small RNAs and targeted or capture sequencing of longer RNAs has proved to be successful. Cons: total RNA sequencing is limited by short fragment length, low number of quantified genes or a high level of ribosomal RNA contamination.
	Ultra-performance liquid chromatography coupled to mass spectrometry (UPLC–MS)	Lipids and metabolites of uEVs (77, 118).	Pros: fast analysis of small molecular weight samples. Cons: problems associated with dangerous organic solvents in use which are toxic and injurious to the environment.