Nova nanosem field emission scanning electron microscope

Samples were prepared by placing an aliquot of the material on the surface of a glass cover slip adhered to the specimen stub with a double-sided copper tape. The samples were allowed to air dry prior to being sputter coated with platinum for 60 s. Samples were imaged using an FEI NOVA nanoSEM field emission scanning electron microscope using the Everhart-Thornley detector (ETD) for low-resolution images or the high-resolution through-the-lens detector (TLD) to capture high-resolution images. Samples were imaged at an accelerating voltage of 5 kV with optimal working distances between 3–5 mm and a 30 µm aperture. For the focused ion beam analysis, the microcages were coated with a layer of platinum. The sample was tilted 52° and ablated with a 3 nA gallium ion beam source with a window of 18 × 7 × 5 µm.

Morphology
of ASD particles and ASD films (prepared by the spin-coating method
as described above) before and after incubation in acidic media was
examined by a Nova nanoSEM field emission scanning electron microscope
(FEI Company, Hillsboro, OR). Samples were mounted on an aluminum
stub using double-sided sticky carbon tape and coated with a thin
film of platinum using a sputter coater (Cressington Sputter Coater,
Watford, UK) for 60 s. SEM images were obtained using an Everhart–Thornley
detector with a spot size of 3 nm, beam energy of 5 kV, and working
distance of approximately 5 mm.

Cell morphology was determined using a Leica Microsystems Model DM1000 phase-contrast light microscope. Capsule stains were performed using Maneval’s method (Maneval, 1941 (link)). Motility was determined by microscopic observation and visual assessment of stab inoculations into semi-solid (0.35% agar) sulfide-indole-motility (SIM) medium (Becton, Dickinson and Company, Sparks, MD, United States; Zimbro et al., 2009 ).
For transmission electron microscopy (TEM), samples of late logarithmic/early stationary phase cells grown at 4°C were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate buffer, post-fixed in buffered 1% osmium tetroxide containing 0.8% potassium ferricyanide, and stained in 1% aqueous uranyl acetate. The samples were then dehydrated with a graded series of acetonitrile and embedded in EMbed-812 resin. Thin sections (80 nm) were stained with 2% uranyl acetate and lead citrate and imaged using an FEI/Philips CM-10 transmission electron microscope (FEI Company, Hillsboro, Oregon, United States). Samples for scanning electron microscopy (SEM) were fixed in buffered 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated in a graded ethanol series, and critical-point dried. Specimens were then platinum-coated and cryosamples imaged using an FEI NOVA nanoSEM field emission scanning electron microscope.