Table 2.

Single EV characterization methods

Method	Advantages	Disadvantages	Detection Limit	References
flow cytometry	high-throughput multiparametric analysis surface marker-specific detection using fluorescent antibodies	limited sensitivity to small EVs (<100 nm), specificity depends on antibody quality and labeling signal overlap and background can affect accuracy	~100-150 nm	94-96
NTA	size distribution of individual EVs high-throughput fluorescent mode allows limited marker-specific detection	low resolution for heterogeneous or small EVs requires large sample volumes	~30-100 nm	97, 98
AFM	high spatial resolution label-free morphological analysis high specificity for physical properties (stiffness, size, surface structure)	low throughput technical expertise required	<10 nm	99
Raman spectroscopy/SERS	label-free chemical composition high biochemical specificity	low throughput sensitive to noise and sample preparation artifacts		27, 100, 101
TEM	high morphological resolution high specificity for EV structure and morphology selective molecular identification via immunogold labeling enables	labor-intensive low throughput	<10 nm	102
Super-Resolution Microscopy	allows investigation of EVs functions in vivo/in situ with molecular resolution	requires labels		103, 104
SPRi	real-time monitoring of EVs-ligand binding kinetics, small sample size, label free	labor intensive, limited by use of capturing molecules		105, 106
Digital ELISA	detection of EV surface or cargo proteins	requires optimized antibody pairs	<100 EVs/mL	95, 107
Nano-FTIR	label-free quantitative and qualitative characterization of EV biochemical content, minimal preprocessing, small sample size	technically demanding low throughput	~50-100 nm	108
qSMLM	Quantitative characterization of the size, shape and protein content	requires optimized antibody pairs, low throughput, complex sample preparation, and limited multiplexing capabilities	~10 nm	109, 110