Nanoscope image software

First 50 µL of EV resuspension from healthy and cancer samples was added to a freshly cleaved muscovite mica surface (NanoAndMore GmbH, Wetzlar, Germany) for 10 min, washed with 2.5 mL of deionized water, and dried with nitrogen. Then, EVs were observed under an atomic force microscope (Veeco, Oyster Bay, NY, USA) in tapping mode with aluminum-coated silicon probe tips (HQ:NSC35/Al BS, Mikromasch, Lady’s Island, SC, USA). The height and amplitude of the samples were recorded at 512 pixels × 512 pixels at a scanning rate of 1 Hz and processed with NanoScope Image Software (v5.10, Veeco, Metrology, NY, USA).

EV samples (1 µg protein) were diluted in a final volume of 200 µL PBS and 50 µL were adsorbed to freshly cleaved Ruby Muscovite mica (NanoAndMore GmbH, Germany) discs for 10 min. The discs were rinsed with deionized water and dried gently under nitrogen stream. Atomic force microscope (Veeco, Oyster Bay, NY, USA) in tapping mode and aluminum coated silicon probe tips (Mikromasch, Lady’s Island, SC, USA) were used for imaging. Topographic height, amplitude and phase images were recorded at 512 pixels × 512 pixels at a scanning rate of 1 Hz. Images were processed using NanoScope Image software (Veeco Metrology, NY, USA).