Jpk software

Atomic force microscopy (AFM) images of the Ect-enriched and Ect-depleted plasma fractions were obtained using NanoWizard (JPK Instruments AG, Berlin, Germany). All experiments were performed in distilled water using a commercial liquid cell (JPK Instruments, Berlin). V-shape gold-coated cantilevers (MLCT cantilever; Bruker Nano Inc., Billerica, MA, USA) with nominal spring constants of 0.01 N/m were used for scanning in fluid. Surface images (256 × 256 pixels) were obtained from a scan size of 10 × 10 μm and a scan rate of 0.4 Hz. Approximately 10-15 images of the surface were obtained for each sample. The size, cross-section, height, and diameter of all the EVs, including Ects, were calculated using JPK software (JPK Instruments).

Force mapping provides a network of force-distance curves, measuring the dependence of the tip-sample interaction force on the tip height above the surface. The absolute value of Young's modulus can be determined by fitting the FD curve to the modified Hertzian-Sneddon equation: $F(\delta )=\frac{2Etan\alpha }{\pi (1-{\upsilon }^2)}\delta$ where F is the measured force, E is the Young's modulus, ν is the Poisson's ratio (0.5 for incompressible materials), δ is the tip-sample separation (obtained by correction of the cantilever height to its bending) and α is the half-angle to face of pyramidal tip (reflects the tip geometry). The data processing module of the JPK software was used to process the maps of FD curves. The resulting Young's modulus maps were exported in order to be post-processed using the Gwyddion software. Data masking and final export of stiffness maps were performed also using the Gwyddion software.