Digital micrograph software

The nanospheres were extracted from the white maxillipeds and antennae of a cleaner shrimp (Supplementary Fig. 15). To do this, the maxillipeds and antennae were sliced into ~1–2-mm pieces, placed in a glass vial with a few drops of hexane, and sonicated for 30 s in a sonication bath, then 3 µl of the resulting suspension was dropped on a carbon-coated Cu-meshed TEM grid and allowed to dry. The resulting samples were observed with a ThermoFisher Scientific (FEI) Tecnai T12 G2 TWIN TEM operating at 120 kV. Images and electron-diffraction patterns were recorded using a Gatan 794 MultiScan CCD camera. Electron-diffraction analysis was done using Gatan DigitalMicrograph software with the DIFPack module. Particle diameters were measured using Gatan DigitalMicrograph software, and 146 particles were measured to find the average size and standard deviation (Supplementary Table 1 and Supplementary Figs. 16 and 17).

To reduce observer bias, all the samples were double blinded and randomized (https://www.randomizer.org/) for microscopy and evaluation. The evaluation of the samples was done by four different experts. The sections were examined with an FEI Tecnai G2 20 (Thermo Fischer Scientific, USA) at 120 kV. Two consecutive sections with a thickness of 70 nm were examined for each sample. Serial EM software was used to create a map of both sections. Three points within the maps were randomly selected. To obtain a better overview of the general amount of lysis in the samples, a montage with nine images (3 × 3) at magnification X1500 was created using Serial EM Software and a 2 K × 2 K CCD camera (Ultrascan 1000, Gatan, Pleasanton, USA) at each of these three points. To further analyze the ultrastructure and to determine the g-ratios and the quality of the preservation of the myelin sheaths, three micrographs with a magnification of X6500 were captured with the same camera using DigitalMicrograph™ software (Gatan, USA), amounting to a total of 18 micrographs at X6500 magnification for each of the 16 specimens. The brightness and the contrast of the images were adjusted with Adobe Photoshop (Adobe Systems, MountainView, USA).

MEFi were seeded in 10-cm dishes (7.5 × 10⁶ cells) 48 h before treatment. Cells were then rinsed twice with PBS and fixed for 1 h in cacodylate sodium buffer 0.2 M, pH 7.2, containing 2.5% glutaraldehyde, 8% paraformaldehyde and 0.01% CaCl₂. Samples were then treated as described previously (Baron Gaillard et al, 2011 (link)). Observations were performed on an EM 912 electron microscope (Zeiss) at 100-kV acceleration equipped with a BioScan camera (Model 792; Gatan, Warrendale, PA, USA). Images were acquired with the Digital Micrograph software (Gatan). Quantification of the number of mitochondria, mitophagic vacuoles and lipid droplets normalized by cytoplasmic surface area was done by counting these structures on 40 images for which surface occupied by cell cytoplasm has been determined. Quantification of mean size of mitochondria was done by using area of an ellipse formula (area = longer diameter × smaller diameter × π), longer diameter of mitochondria being determined on images and smaller diameter considered as half of longer diameter.