AFM-based High-Throughput Nanomechanical Screening of Single Extracellular Vesicles

Abstract

We herein describe an Atomic Force Microscopy (AFM)-based experimental procedure which allows the simultaneous mechanical and morphological characterization of several hundred individual nanosized vesicles within the hour timescale.When deposited on a flat rigid surface from aqueous solution, vesicles are deformed by adhesion forces into oblate spheroids whose geometry is a direct consequence of their mechanical stiffness. AFM image analysis can be used to quantitatively measure the contact angle of individual vesicles, which is a sizeindependent descriptor of their deformation and, consequently, of their stiffness. The same geometrical measurements can be used to infer vesicle diameter in its original, spherical shape.We demonstrate the applicability of the proposed approach to natural vesicles obtained from different sources, recovering their size and stiffness distributions by simple AFM imaging in liquid. We show how the combined EV stiffness/size readout is able to discriminate between subpopulations of vesicular and nonvesicular objects in the same sample, and between populations of vesicles with similar sizes but different mechanical characteristics. We also discuss a force spectroscopy calibration procedure to quantitatively link the stiffness of EVs to their average contact angle.Finally, we discuss expected extensions and applications of the methodology.

Gold nanoparticles interacting with synthetic lipid rafts: an AFM investigation

“…The SLB was formed on functionalised borosilicate glass coverslips, by injecting the liposomes (this time suspended in ultrapure water) in the buffer solution where they experienced an osmotic imbalance across the membrane, decreasing their pressurisation (please refer to 'Materials and methods' section for the details). As a result, following the adhesion to the substrate, liposomes will deform adopting more oblate shapes (Ridolfi et al, 2019), increasing the area occupied by each vesicle and favouring the previously described vesicle fusion mechanism. As shown in Figure 1(C), consistently with NR data the surface is almost completely covered by a lipid bilayer, which presents nanometric domains of different heights, with the brighter areas corresponding to thicker membrane regions and the darker ones to thinner SLB portions.…”

Augmented COlorimetric NANoplasmonic (CONAN) Method for Grading Purity and Determine Concentration of EV Microliter Volume Solutions

“…Relevant to this protocol paper, the EV average size was estimated through in-liquid AFM measurements (Figure 4). Full details on the EV separation protocols and characterization are given in Ridolfi et al (2019). Figure 2A shows the characterization of freshly synthesized, citrate-capped AuNPs, performed with the UV-Vis spectroscopy.…”

Gold Nanoparticles interacting with Synthetic Lipid Rafts: an AFM investigation

“…The SLB was formed on functionalized borosilicate glass coverslips, by injecting the liposomes (this time suspended in ultrapure water) in the buffer solution where they experienced an osmotic imbalance across the membrane, decreasing their pressurization (please refer to Materials and Methods for the details). As a result, following the adhesion to the substrate, liposomes will deform adopting more oblate shapes 41 , increasing the area occupied by each vesicle and favoring the previously described vesicle fusion mechanism. As shown in Figure 1c, consistently with NR data the surface is almost completely covered by a lipid bilayer, which presents nanometric domains of different heights, with the brighter areas corresponding to thicker membrane regions and the darker ones to thinner SLB portions.…”

“…From a simple AFM topography, small lipid vesicles can be confused with AuNPs or AuNPs clusters; this could introduce statistical noise to the localization and morphometrical analysis. We recently developed an AFM-based nanomechanical characterization able to discriminate lipid vesicles from objects with the same morphology but different mechanical behavior 41 . This method evaluates the deformation that lipid vesicles undergo once adsorbed on a surface, by calculating their contact angle (α).…”