Table 3.

Main optical and nonoptical methods for EV analysis

Methods	Principle	Limitations	Reference
Fluorescence-activated cell sorter	Laser-interrogated particle fluorescence method with a nondestructive and quantitative manner	Beads with known size necessary; low refractive signal of EVs; high background; nonoptimum for particles below 300 nm	(64)
Dynamic light scattering	Reflects scattering light intensity distribution under Brownian motion of suspended particles, providing size from those particles	Less accurate with heterogeneous size population; biases toward larger particles	(21, 44)
Nanoparticle tracking analysis	Dark-field microscope that combines laser light scattering with charge-coupled device for detection of particles smaller than 1,000 nm; calculates diameter of each vesicle with Stokes-Einstein equation	Need a “right” dilution to not underestimate smaller vesicles; low sensitivity to fluorescence signals	(15, 20)
Transmission electron microscopy	High-resolution microscope with resolution down to 1 nm; image is created by electron interference when the electron beam crosses the sample	Not applied for high-throughput profiling of EV; high quality of EV preparation is necessary; preparation could change EV morphology	(50, 67, 92)
Atomic force microscopy (AFM)	Super high resolution that can provide size, distribution, morphology and map mechanical properties with nanometric precision; a technique that detects and records interactions between the probing tip and the sample surface	Slow speed and limited imaging area; influenced by AFM probes	(94)

EV, extracellular vesicle.