Quanta 250 field emission gun scanning electron microscope

Bacterial cells were analyzed by scanning electron microscopy as previously described with some modifications.^{32 (link)} Briefly, bacteria were cultured in THB supplemented with 1% glucose in wells containing 12 mm glass coverslips coated with poly-L-lysine (Corning, Bedford MA) at 37°C for 24 hours. At 24 hours, supernatants were removed and samples were fixed with 2.0% paraformaldehyde, 2.5% gluteraldehyde in 0.05 M sodium cacodylate buffer for 24 hours. Secondary fixation with 0.1% osmium tetroxide was performed for 5 minutes prior to sequential dehydration with increasing concentrations of ethanol. After ethanol dehydration, samples were dried at the critical point using a critical point dryer machine (Tousimis), mounted onto aluminum sample stubs, and sputter-coated with 80/20 gold-palladium. Afterward, samples were painted with a thin strip of colloidal silver (Electron Microscopy Sciences) at the edge to facilitate charge dissipation. Samples were imaged with an FEI Quanta 250 field-emission gun scanning electron microscope. Images shown are representative of three separate experiments.

Left ventricular tissue was collected from human subjects under a protocol approved by the Vanderbilt Institutional Review Board. Non-failing human myocardium was collected from human donors with tissue unable to be used for heart transplant. Failing human donor specimens were collected from explanted hearts at the time of heart transplant. Specimens were taken directly at the time of collection in the operating room and fixed in 4 % glutaraldehyde in a 0.1 M phosphate buffer solution and stored at 4 °C. Tissues were macerated in 10 % aqueous NaOH for 10 days to maintain fibrous structure of cardiac tissue ECM while removing non-fibrous elements. Fixation, critical point drying, and sputter coating were performed in the Vanderbilt Cell Imaging Shared Resource Core. Images were acquired using the FEI Quanta 250 field emission gun scanning electron microscope (SEM), which was operated in high vacuum mode, at an accelerating voltage of 8 or 10 kV.