Microstructural Analysis by Scanning Electron Microscopy

Following microcomputed tomography to confirm proper orientation of region of interest, specimens were mounted on aluminum pins with conductive silver epoxy (Ted Pella, Redding, CA). The specimens were trimmed to remove excess resin above ROI and to remove silver epoxy from sides of specimen. The specimens were sputter coated with gold-palladium and then imaged using a Gemini scanning electron microscope (Zeiss) equipped with a 3View2XP and OnPoint backscatter detector (Gatan). Images were acquired at 2.5 kV accelerating voltage with a 30 μm condenser aperture and 1 μsec dwell time; Z step size was 50 nm; raster size was 12k × 9k and Z dimension was 1200 sections. Volumes were either collected in variable pressure mode with a chamber pressure of 30 Pa and a pixel size of 3.8 nm (Figure 2—video 1 and Figure 3D) or using local gas injection (Deerinck et al., 2018 (link)) set to 85% and a pixel size of 6.5 nm (Figures 2A, B and 3C). Volumes were aligned using cross correlation, segmented, and visualized using IMOD (Kremer et al., 1996 (link)).