S 4700 sem

We used two-photon polymerization (TPP) nanolithography to fabricate the designed engineering prototypes (flat, prism and foil) for experimental investigations. The 3D laser lithography system (Photonic Professional GT, Nanoscribe GmbH, Germany) utilized a dip-in configuration with a 63×, 1.4 N.A. oil immersion objective lens (Zeiss, Germany) to focus the laser beam. An acrylic-based monomer liquid photoresist optimized for TPP applications (n_r = 1.52, IP-Dip, Nanoscribe GmbH) was drop-casted on a silicon wafer (500 μm thick with an oxidation layer of 3000 Å) and the objective lens immersed directly in the photoresist. A femtosecond laser (centre wavelength of 780 nm, pulse width of 100 fs, repetition rate of 80 MHz, and maximum power of 150 mW) was used as the irradiation source. A laser power of 25 mW was used in the TPP process and was controlled by an acousto-optic modulator. 50 mm/s writing speed was controlled by a galvo-mirror scanner^{43 (link)}. Each design was fabricated to a 135 × 135 μm² area. The fabricated structures were then characterized using a Hitachi S-4700 SEM (Hitachi High-Technologies Corp., Tokyo, Japan), sputter-coated with 5 nm of chromium. The imaging voltage was kept low (<10 kV) to avoid damaging the structures.

For histology and immunolabeling studies, multilayers were fixed in 2% formaldehyde for 10 minutes at 4°C, then washed with PBS. Wells were then incubated with X-Gal buffer [55] (link) containing X-Gal (1 mg/ml) for 48 h at 37°C then washed with PBS before photomicroscopy was performed. For standard histological staining, membranes, removed from the transwell insert using a scalpel, were embedded in paraffin and then sectioned (6 um) and stained with hematoxylin and eosin before being imaged [36] (link). Immunofluorescent labeling for specific proteins was performed using an anti-human CD4 antibody (BD). Labeled sections were cover slipped with hard set containing DAPI (Vector Labs, Burlingame, CA) before imaging on a Nikon Eclipse Ti, Nikon. Scanning electron microscopy was completed as previously described [36] (link). Briefly, fixed cultures were dehydrated in ethanol and processed through hexamethyldisalazane followed by air-drying before mounting onto metal stubs and sputter-coated with iridium in an Emitech K575x Sputter Coater (Emitech, Houston, TX) at 20 mA for 20 sec. The filters were examined in a Hitachi S4700 SEM (Hitachi High Technologies, Schaumburg, IL) at 2 kV.

Sterile, NMB communities- or methicillin-resistant Staphylococcus aureus (MRSA)-colonized NEC cultures in 24 well transwell cups (n = 3–4 for each condition) were fixed 48 h after bacterial application in primary fixative solution (1.25% paraformaldehyde, 2.5% glutaraldehyde, 0.03% CaCl₂,50 mM cacodylate buffer) and stored at 4°C. The MRSA strain USA300 isolated in our lab from a pediatric nasal sample was cultivated in Tryptic Soy broth overnight at 37°C with agitation, and 5 × 10² bacteria were added to the NEC culture. Each sample was processed by rinsing in 0.1 M cacodylate buffer, postfixed in 1% OsO₄ in 0.1 M cacodylate buffer and then dehydrated in absolute ethanol and hexamethyldisalazane. After air drying, the membrane was removed, mounted for sputter-coating with iridium in an Emitech K575 × Sputter Coater (Emitech, Houston, TX, USA) at 20 mA for 20 s and then examined as described previously (Rose et al., 2012 (link)). Imaging was completed on a Hitachi S4700 SEM (Hitachi High Technologies, Schaumburg, IL, USA) with indicated magnifications.