Multiscale Imaging of Cryptococcus Cells

For standard scanning electron microscopy (SEM), sample preparation and visualization were performed as described in reference 41 (link). For SBF-SEM, a single block was produced for analysis by mixing together aliquots from strains known to produce appreciable numbers of the different cell phenotypes (H99E, H99W, H99O, H99S, and KN99a) following capsule induction. Cells were harvested and washed with 0.1 M phosphate buffer (PB), covered with 1 mL of fixative (2.5% glutaraldehyde, 2% paraformaldehyde, 0.1 M PB), and left overnight at 4°C. Secondary fixation, staining, ethanol dehydration, resin infiltration, and embedding were performed as described in reference 18 (link). The final sample block contained approximately 5,000 cells. The sample block was then loaded onto a Zeiss Sigma variable-pressure SEM equipped with Gatan 3View 2XP. Inverted backscattered electron images (8,192 by 8,192 pixels, 16-bit) were acquired with an xy pixel size of 100 nm and z pixel size (slice thickness) of 10 nm.