Table 2.

Advantages and Disadvantages of the Described EV Characterization Methods

Method	Principle	Advantage	Disadvantage	Potential for quantitationa	Costa	Refs
Fluorescence/confocal microscopy	Fluorescence/light radiation	Nondestructive analysis, EV uptake/degradation can be monitored, semiquantitative	Lengthy procedure, dye aggregates result in overestimation, reporters need to be specific	++	$$$	[108., 109., 110.,119., 120., 121., 122., 123., 124., 125., 126.]
TEM and cryo-TEM	Electron radiation	Direct imaging of EVs, nondestructive	High computational cost, challenging sample preparation, low throughput, not quantitative, reproducibility might be an issue	+	$$$$	[113., 114., 115.,135]
AFM	Hooke’s law	High resolution, provides details of EV morphology	Low throughput, specialized equipment	+	$$$	[108., 109., 110.,117,118]
DLS	Brownian motion	Simple and fast	Nonideal for heterogeneous and polydisperse samples, low resolution	++	$$	[113., 114., 115.,119., 120., 121., 122., 123., 124., 125., 126.]
NTA	Light scattering/Brownian motion; dark microscopy and fluorescence	Size and concentration measured simultaneously	Biased toward smaller particles	+++	$$$	[117,118,135]
FCM and nano-FCM	Fluorescence/light scattering, Coulter principle	No sample preparation necessary, fast, EV specific, reproducible, quantitative, low sample volume	Size standards do not correlate correctly, restrictions on lower size limits of detection	+++	$$$$	[128,133]
RPS	Coulter principle	High throughput, measures concentration, size, and charge simultaneously, low sample volume	Biased toward larger particles, nonstandardized settings, cannot determine particle type, requires frequent calibration; reproducibility and robustness can be an issue	+++	$$	[135,141]
RS	Light scattering	Reports chemical composition (for simple sample systems), no sample preparation, small sample volume	Challenging data interpretation, medium throughput, characterization of composition is challenging for complex samples	+	$$	[128,133,135]
FLOWER	Immunoaffinity interactions and resonance frequency shifts	Requires further evaluation	Requires further evaluation	++	$$	[135,141,143]
SP-IRI	Immunoaffinity interactions and interference of light	Requires further evaluation	Requires further evaluation	++	$$	[135,144,145]

^aPotential for quantitation and cost rankings shown in arbitrary units using a range of 0–4 units.