Digital micrograph software package

Alginate-iron samples used for STEM/EDX were made using the equilibrium dialysis technique as described earlier. Preparation of these samples was performed as follows; aqueous sodium alginate (10 mL, 0.1% w/v, pH 5.8) in DI H₂O was sealed within a dialysis membrane and immersed in a pre-mixed solution of FeCl₃∙6H₂O (10 mM, 750 ml, pH 1.7) for 120 min. The dialysis bag was removed, and subsequently immersed in pure DI H₂O (750 mL) and incubated for another 120 min. Due to the viscous nature of the sample, copper TEM grids coated with lacey carbon were loaded with 50 μl of sample and excess sample was drawn from underneath, effectively pulling the sample through the grid. This produced a thin sample coverage over the grid with many sampling areas.
Electron microscopy images were taken using a 200kV FEG Jeol 2100F scanning transmission electron microscope fitted with a CEOS aberration corrector. Images were simultaneously acquired in high angular annular dark field (HAADF) and bright field (BF) mode using the Gatan DigitalMicrograph software package.

The EELS analyses were performed with the Gatan GIF Quantum system on STEM mode. The EELS elemental mapping was simultaneously recorded with HAADF-STEM. The pixel size is 0.32 Å × 0.32 Å and a short dwell time of 0.01 s/pixel was used in order to avoid the spatial drift and irradiation damage. S L_2,3 edges presented in Fig. 1e are the integrated signals from all the pixels in 3 rows for each defined layer (Supplementary Fig. 14). The width of 3 rows (0.96 Å) is smaller than the atomic radius (S, 1.09 Å; Mo, 1.36 Å; Au, 1.44 Å). There is a gap spacing between two adjacent layers with more than 4 rows (1.28 Å) to avoid possible interference from scanning drifts. In this way, we got the original EELS spectra of these six layers (Supplementary Fig. 15). To better show the characteristic EELS edges and peaks, we smoothed these spectra with low-pass numerical filtering. The background of sulfur core-loss edges was subtracted using a power law fitting. All the smoothing and subtracting processes are performed using the Digital Micrograph software package (Gatan Inc.).

Transmission electron microscopy (TEM) was carried out on a FEI Tecnai TF20 X-twin microscope (FEI, Hillsboro, Oregon, USA) operated at 200 kV (FEG, 1.9 Å point resolution) with an EDAX Energy Dispersive X-ray (EDX) detector attached. Images were recorded on a Gatan CCD camera with resolution of 2048 × 2048 pixels using the Digital Micrograph software package (Gatan, Pleasanton, CA, USA). Electron diffraction patterns were evaluated using the CrysTBox software (Institute of Physics, Prague, Czech Republic) for an automated analysis of electron diffraction patterns. The lamella for TEM observation was prepared by focused-ion-beam milling in a FEI Quanta 3D.

SEC fractions showing the highest MFI for exosomal markers by bead-based assay in flow cytometry were pooled, the fraction obtained by PEG precipitation and the vesicles isolated using PROSPR were selected (n = 1 for each method) for cryo-TEM microscopy. Vitrified specimens were prepared by placing 3 μl of a sample on a Quantifoil^® 1.2/1.3 TEM grid, blotted to a thin film and plunged into liquid ethane-N₂(l) in the Leica EM CPC cryoworkstation. The grids were transferred to a 626 Gatan cryoholder and maintained at −179 °C. The grids were analyzed with a Jeol JEM 2011 transmission electron microscope operating at an accelerating voltage of 200 kV. Images were recorded on a Gatan Ultrascan 2000 cooled charge-coupled device (CCD) camera with the Digital Micrograph software package (Gatan).

EVs collected by ultracentrifugation were analysed at similar dilutions on electron cryomicroscopy (CryoEM) in the microscopy facility at the Universitat Autònoma de Barcelona. Vitrified specimens were prepared by placing 3 μL of a sample on a holey carbon TEM grid, blotted to a thin film and plunged into liquid ethane-N2(l) in the Leica EM GP cryoworkstation. The grids were transferred to a 626 Gatan cryoholder and maintained at −179 °C. The grids were analysed with a Jeol JEM 2011 transmission electron microscope operating at an accelerating voltage of 200 kV. Images were recorded on a Gatan Ultrascan 2000 cooled charge-coupled device (CCD) camera with the Digital Micrograph software package (Gatan).

Cells were fixed at 4 °C in a mixture of 2% PFA and 2.5% glutaraldehyde in 0.1 M phosphate buffer (PB), then scraped, pelleted, washed in PB and postfixed in 1% osmium tetroxide and 0.8% potassium ferrocyanide. Samples were dehydrated with acetone and embedded in Spurr resin. Ultrathin sections were obtained using an Ultracut E (Reichert), picked up on copper grids and observed with the JEM 1011 (JEOL) electron microscope, operating at 80 kV. Micrographs were taken with a camera (Orius 1200A; Gatan) using the DigitalMicrograph software package (Gatan). Electron micrographs were processed using Adobe Photoshop CS6 (v.13.0.1) (Adobe Systems).

VLP morphology was assessed by cryo-EM. Extracted VLPs were deposited on a carbon-coated copper grid and prepared using an EM GP workstation (Leica). Vitrified VLPs were prepared on a Lacey Carbon TEM grid (copper, 400 mesh) and immediately plunge into liquid ethane. The grids were viewed on a JEOL 2011 transmission electron microscope operating at an accelerating voltage of 200 kV. Electron micrographs (Gatan US4000 CCD camera) were recorded with the Digital Micrograph software package (Gatan).