Dimensional characterization of extracellular vesicles using atomic force microscopy

Abstract

Extracellular vesicles (EV) are small biological entities released from cells into body fluids. EV are recognized as mediators in intercellular communication and influence important physiological processes. It has been shown that the concentration and composition of EV in body fluids may differ from healthy subjects to patients suffering from particular disease. So, EV have gained a strong scientific and clinical interest as potential biomarkers for diagnosis and prognosis of disease. Due to their small size, accurate detection and characterization of EV remain challenging. The aim of the presented work is to propose a characterization method of erythrocyte-derived EV using atomic force microscopy (AFM). The vesicles are immobilized on anti-CD235a-modified mica and analyzed by AFM under buffer liquid and dry conditions. EV detected under both conditions show very similar sizes namely ~30 nm high and ~90 nm wide. The size of these vesicles remains stable over drying time as long as 7 d at room temperature. Since the detected vesicles are not spherical, EV are characterized by their height and diameter, and not only by the height as is usually done for spherical nanoparticles. In order to obtain an accurate measurement of EV diameters, the geometry of the AFM tip was evaluated to account for the lateral broadening artifact inherent to AFM measurements. To do so, spherical polystyrene (PS) nanobeads and EV were concomitantly deposited on the same mica substrate and simultaneously measured by AFM under dry conditions. By applying this procedure, direct calibration of the AFM tip could be performed together with EV characterization under identical experimental conditions minimizing external sources of uncertainty on the shape and size of the tip, thus allowing standardization of EV measurement.

The Mechanism of Vesicle Solubilization by the Detergent Sodium Dodecyl Sulfate

“…We note that the measured LUV heights ranged from approximately 18 -30 nm, similar to those previously reported for extracellular vesicles of comparable size. The observed reduction in LUV height relative to the diameter measured by DLS was assigned to elastic deformation of the structures induced by mechanical indentation exerted by the AFM tip as previously reported [28][29][30] .…”

Extracellular Vesicle Quantification and Characterization: Common Methods and Emerging Approaches

“…Most typically, AFM is used on dry immobilized EV samples, which allows estimating their size and structure. Sample damage during drying can be prevented by analysing EVs in solution, by first immobilizing them on a surface via electrostatic interactions or via binding to complementary antibodies [86,87,88]. Casado et al used AFM to study the dynamics of EV secretion via shedding in living cells and found good correlation between the size of observed protrusions of the cell membrane and the size of EVs produced by these cells [74].…”

Mechanical characterization of vesicles and cells: A review

“…For sample immobilization, the surface must be very flat (roughness <0.5 nm). While immobilization of the sample on bare surfaces like muscovite mica is common, there are recent attempts to improve the stability of immobilization and initiate selective binding by functionalizing the surface with a charged polymer deposition or an antibody coating . Though surface modification techniques would allow for better immobilization, it demands further expertise and preparations.…”