Table 5.

Summary of methodological AFM papers investigating EV biomechanics.

Paper	Sample/Purification	AFM Methods	Mechanical Findings	Impact and Application
Sharma 2010 [190]	EXOs extracted from Saliva by Ultracentrifugation (UC).	EXOs were absorbed overnight on mica. Measurements were performed in PBS using a soft (k = 0.02 N/m) MSCT cantilever (Veeco) at 0.25 Hz. AFM was used in the amplitude and phase modulation mode. Applied forces ranged from 1.4 nN to 2.4 nN.	EXOs deform under applied forces. Deformation is accompanied by a tri-lobed-shaped depression region in the particle center. The larger the applied force in the 1.4–2.4 nN, the deeper the depression (Figure 6b). Strong specific adhesions forces were measured using CD63-conjugated tips.	One of the first papers focused on EXO biomechanics highlighting their deformation under applied force and pointing out the need to use low forces for imaging purposes. The paper also shows that AFM can be used for specific exosome detection, through tip conjugation, thus being potentially useful in diagnostics.
Li 2021 [194]	EXOs extracted by UC from the bone marrow of lymphoma patients.	EXOs were absorbed on Poly-L-Lysine-coated slides and characterized with peak force tapping (PFT) AFM in PBS with a silicon nitride tip (k = 0.7 N/m, f = 150 kHz, and tip radius 20 nm).	Poly-L-Lysine facilitates AFM measurements of EXOs improving the height/diameter ratio (~0.3) compared to air-drying. PFT is suitable for the quantitative imaging of EXO topography, stiffness, adhesion and dissipative properties, also highlighting the contrast with the substrate. Dissipation is not homogeneous and symmetric on EXO surfaces.	These results demonstrate that AFM in the PFT mode is suitable for a quick and effective quantitative analysis of EXOs, and thus is of potential use for the search of EXO-based biomarkers.
Parisse 2013 [195]	SKBR3-derived EXOs.	The IT-AFM experiment was performed in liquid using a cantilever with k = 0.1 N/m and f = 32 kHz.	The typical shape of EXO FD curves is reviewed, highlighting their resemblance with curves acquired on artificial lipid vesicles.	In-depth theoretical models developed for artificial vesicles can be used to analyze extracellular vesicles.
Vorselen 2020 [174]	EXOs extracted from serum of healthy donors and patients diagnosed with hereditary spherocytosis.	EVs were absorbed on a poly-L-lysine coverslip. Imaging was performed using FD-based AFM with forces < 0.1 nN, reducing EV compression. Mechanics were measured through FD curves at 0.5 nN at a slow speed of 0.2–1 Hz (elastic response). Higher forces were used to penetrate the lipid bilayer and record membrane tethering.	The paper validates a protocol based on the Canham–Helfrich model to decouple EV bending modulus and La Place pressure. The method uses information from indentation and membrane tethering, which provides surface tension, σ. Steps are detailed for measuring σ, a non-trivial task because of tip contamination. The correct FD curve hallmarks are discussed: (i) a smooth indentation between the contact point and the EV height; (ii) an abrupt increase in reaction force when the two bilayers are pressed together; (iii) two discontinuities indicating 1st and 2nd bilayer rupture; and (iv) the interaction with the substrate.	A key paper in EXO biomechanics, as it provides a unified protocol for performing nanoindentation on vesicles and the subsequent data analysis. The paper guides scientists step by step through sample preparation, FD curve acquisition, model choice and application and statistical analysis of the data.
Ridolfi 2019 [193]	EXOs extracted by UC-SEC from milk and nematodes	EXOs were absorbed on Poly-L-Lysine. Measurements were performed in peak force mode in DI water using Bruker SNL-A probes (tip radius 2–12 nm, k = 0.35 N/m). Applied force was 0.15–0.25 nN, cantilever speed < 5 µm/s	The authors developed an AFM protocol to characterize artificial and natural vesicles mechanics in liquid by AFM imaging: (i) the method allowed them to retrieve EV unperturbed geometry and contact angle α in terms of particle height and surface projected radius; (ii) α depends on stiffness and it is roughly independent of EV size; (iii) a deviation from point ii indicates the presence of contaminants; (iv) a calibration curve is provided that allows calculating stiffness (a mechanical parameter) from contact angle (a morphological parameter).	A potential game-changer in the field, this paper presents the first high-throughput method for EXO mechanics based on imaging. The study derives stiffness from contact angles, and it is based on the understanding that EVs are deformed by adhesion forces into an equilibrium shape that is a direct consequence of EV stiffness. The method also allows the removal of contaminants, a key issue in EXO research.