Dual beam fib sem

RSMs of these samples were taken around the asymmetric (−105) plane, using a Bruker AXS D8 Discover HRXRD. The TEM-ready samples were prepared using the in situ FIB lift out technique on an FEI Dual Beam FIB/SEM. Structural characterizations were realized by a FEI Tecnai Osiris TF-20 FEG/TEM equipped with EDS.

In order to observe the deformation process beneath the indenter tip, as well as the shear plasticity, a bright field cross sectional transmission electron microscopy (XTEM) images are carried out on selected indents using FEI Tecnai TF-20 FEG/TEM operated at 200 kV in bright-field (BF). The TEM-ready sample was prepared using the in-situ FIB lift out technique on a FEI Dual Beam FIB/SEM. The sample was capped with sputtered C and e-Pt/I-Pt prior to milling to protect the surface of the area of interest.

For ultrastructural analysis, cells were plated in a Chamber Slide™ system and fixed in 0.5% glutaraldehyde and 2% paraformaldehyde, 0.1 M cacodylate buffer, pH 7.4 for 1 h, then post fixed in 1% osmium tetroxide in the same buffer for 45 min, in the dark. Samples were washed for 30 min and contrasted en bloc with 1% uranyl acetate in the dark, then gradually dehydrated in ethanol. All the steps of the above procedure were performed at 4°C. Cells were infiltrated with a mixture of ethanol and Epoxy Embedding Medium (Sigma-Aldrich™, Cat# 45359-1EA-F), then embedded in the same resin, allowing specimens to polymerize at 60°C, for 3 days. Resin-embedded cells were mounted on stubs using a self-adhesive carbon disk and gold sputtered by an Emithech K550. Regions of interest were cross-sectioned by the focused gallium ion beam of the Dualbeam FIB/SEM (Helios Nanolab, FEI, Hillsboro, OR, USA). Images of cross sections were acquired at a working distance of 2 mm using backscattered electrons and a through-the-lens detector in immersion mode with an operating voltage of 2 kV and an applied current of 0.17 Na. Images were composed in an Adobe Photoshop CS6 format.