Vesicles are giant unilamellar vesicles made of DOPC, supplemented with 0.1% DOPE-Atto647N (ref AD-647N, Atto-tec, Germany) and 0.03% DSPE-PEG(2000) Biotin (ref 880129, Avanti Polar Lipids, USA) electroformed during 1 h at 1V RMS [44 (link)] in a sucrose buffer at 250 milliosmoles. Vesicules were adhered on avidin coated glass coverslips, deflated with an hyperosomotic shock due to buffer evaporation and imaged with a Yokogawa spinning-disc CSU-X1 mounted on a Nikon Ti-Eclipse microscope stand using a 100x objective with NA 1.3 (z spacing 340 nm, xy pixel size 122 nm).
MRI dataset was acquired from a normal healthy person, using a FLAIR sequence.
FIB-SEM 80% confluent HeLa cells were rinsed once with PBS, fixed for 3h on ice using 2.5% glutaraldehyde/2% paraformaldehyde in buffer A (0.15M cacodylate, 2mM CaCl2). Then cells were extensively washed on ice in buffer A, pelleted and incubated 1h on ice in 2% osmium tetroxide and 1.5% potassium Ferro cyanide in buffer A and finally rinsed 5 times in distilled water at room temperature. Cells were then incubated 20min at room temperature in 0.1M thiocarbohydrazide, which had been passed through a 0.22 μm filter, and extensively washed with water. Samples were incubated overnight at 4° C protected from light in 1% uranyl-acetate, washed in water, further incubated in 20mM lead aspartame for 30min at 60°C and finally washed in water. Samples were dehydrated in a graded series ethanol, embedded in hard Epon and incubated for 60h at 45°C then for 60 h at 60°C. A small bloc was cut and mounted on a pin, coated with gold and inserted into the chamber the HELIOS 660 Nanolab DualBeam SEM/FIB microscope (FEI Company, Eindhoven, Netherlands). ROI were prepared using focused ion beam (FIB) and ROI set to be approximatively 20 microns wide. For imaging, electrons were detected using Elstar In-Column secondary electrons Detector (ICD). During acquisition process, the thickness of the FIB slice between each image acquisition was 5 nm.
The drosophila egg chamber is dissected from a drosophila ovary. Cell nuclei were stained with DAPI and cell membranes labeled with the fusion proteins Nrg::GFP and Bsg::GFP [45 (link)]. The egg chamber was embedded in Vectashield and spacers were used to prevent tissue deformation. Images were acquired using an inverted Olympus point scanning confocal microscope IX81 with a 60x objective NA 1.42(z spacing 750 nm, xy pixel size 265 nm).
