Crossbeam 540 focused ion beam scanning electron microscope

The samples were processed as previously described [56 (link)]. They were trimmed with a 90° diamond trimming knife (Diatome, Biel, Switzerland) and attached to the SEM stub (Science Services, Pin 12.7 mm × 3.1 mm) by a silver-filled epoxy (Epoxy Conductive Adhesive, EPO-TEK EE 129–4; Electron Microscopy Sciences) and polymerized at 60°C overnight. Following polymerization the samples were coated with a 10-nm gold layer using the sputter coater EM ACE600 (Leica) at 35 mA current and placed into the Crossbeam 540 focused ion beam scanning electron microscope (Carl Zeiss Microscopy). To protect the surface and ensure even milling, a 400-nm platinum layer was deposited on top of the region of interest. Atlas 3D software (Atlas 5.1, Fibics, Canada) was used to collect the 3D data. Samples were exposed with a 15-nA current, and a 7-nA current was used to polish the surface. The images were acquired at 1.5 kV with the ESB detector (450 V ESB grid, 600 pA, pixel size x/y 5 nm) in a continuous mill and acquire mode using 1.5 nA for the milling aperture (z-step 25 nm).