Scd 030

Finally, the degraded and perfused membranes were visually assessed using scanning electron microscopy (SEM), in order to evaluate the surface structure alterations due to collagen degradation and sperm cell penetration at the different time periods.
Samples were fixed for 24 h in 2.5% glutaraldehyde solution, then rinsed with PBS and dehydrated twice for 15 min in ascending concentrations of ethanol (50%, 70%, 80%, and 90%, respectively). The membranes were then immersed three times for 15 min in 94% and 60 min in 100% ethanol, respectively. Finally, samples were subjected to critical point drying (Bal-Tec CPD030, Balzers, Liechtenstein). All the samples were then cut in the middle and mounted on SEM mounts (Bal-Tec AG, Blazers, Liechtenstein), with each cut shown on the side of the membrane (i.e., upside-down and vice versa). Samples were gold sputtered (Balzers SCD 030, Balzers Union, Balzers, Liechtenstein) for 60 s in an argon gas atmosphere at a target distance of 50 mm and pressure of 5 Pascal (Pa) at 45 mA. SEM images (Supra 50 VP FESEM, Carl Zeiss, Oberkochen, Germany) were taken at a working distance of 9.2 mm, an acceleration voltage of 10 kV, and a magnification of 5000×.

The biofilms were grown on stainless steel coupons placed in a 12-well polypropylene microplate (Microtech, Naples, Italy). A volume of 1 mL of bacterial suspension at a density of 1 × 10⁸ CFU/mL was added to each selected well and the plate was statically incubated for 24 hours at 37°C. After that, the coupons were washed with 1X PBS, air-dried, and treated with plasma for 0, 3, and 30 min. Samples were fixed in glutaraldehyde (2.5% v/v) for 30 minutes and then dehydrated with cold solutions of ethanol at increasing concentrations (30, 50, 70, 90, 95, and 100% v/v), each for 20 minutes. All samples were dried in a critical point desiccator (Emitech K850, Kent, UK). Then, about 15–20 nm gold spray coating was performed with the Balzers SCD 030 (New York, USA) and the images were achieved using the Supra 40 ZEISS (EHT = 5.00 kV, WD = 22 mm, detector in the lens) (Berlin, Germany) [28 (link)].

Biofilms were grown on stainless steel coupons placed in a 24-well polypropylene microplate (Greiner CELLSTAR Sigma Aldrich). Each well was inoculated with 2 mL of a bacterial suspension in LB medium at a final OD_550nm of 0.1 and the microplate statically incubated for 24 h at 37°C. Then coupons were rinsed with 1 mL of sterile saline, air-dried, and plasma-treated for 0, 3, and 30 min. Samples were fixed in 2.5% (v/v) glutaraldehyde for 30 min and then rinsed in water to remove excess reagent. Dehydration was carried out by placing the coupons in a series of increasing concentration of cold ethanol solutions. The ethanol concentration in the dehydrating solution was 30, 50, 70, 90, and 95% v/v and each iteration lasted 20 min. Two additional increments at 100% ethanol for 20 min each were added to ensure complete ethanol saturation. All samples were critical-point dried in an EMITECH K850 dryer displacing ethanol with liquid CO₂ and further evaporated at 31.1°C and 1072 psi. A ~15–20 nm gold sputter-coating was accomplished in a Balzers SCD 030 apparatus, and images were obtained using a SEM Philips 505 scanning electron microscope.

For SEM, larvae were bathed in hot water for 1.5–2 min. Fixation was carried out in 2.5% glutaraldehyde buffered in 0.05 M Na-cacodylate buffer (pH 7.4, 396 mOsm) at 4°C. After 30 min, approximately the anterior third was cut off and put back in fresh fixative for additional 18 h at 4°C. After fixation, samples were washed three times for 10 min, respectively, in 0.1 M Na-cacodylate (pH 7.4), followed by post-fixation in 1% osmium tetroxide (OsO₄) for 2 h at 4°C. After additional washing steps (3 × 10 min with 0.2 M Na-cacodylate, pH 7.4), specimens were dehydrated in ascending ethanol concentrations and then transferred into a critical point device (Bal-Tec CPD 030, Liechtenstein) and dried via CO₂. After mounting on aluminum stubs with CCC (Conductive Carbon Cement, Plano GmbH, Wetzlar, Germany), specimens were coated in a sputter coater (Balzers SCD 030, Liechtenstein) with 5 nm gold-palladium in order to enhance conductivity. Samples were examined in a FESEM Auriga TM Crossbeam workstation (Zeiss, Jena, Germany). Images were analyzed and processed with Image J software (http://imagej.nih.gov/ij).

To produce gels for SEM imaging the solutions were prepared according to the procedure described in Section 2.2. For gelation, the samples were heated in a water bath at 90 °C for 30 min. After cooling, cubes with an edge length of 1 cm were cut from the gels. To observe the optical effects of the thermally induced gel formation, photos were taken after gelation. For freezing the samples were immersed in liquid nitrogen. The sample 100 mM, 5 wt%, Lev4 did not gel during the heat treatment. Therefore, it was placed in a container made of aluminum foil and frozen therein. Immediately after freezing, the samples were dehydrated by freeze drying (Beta 1–8 LSCplus, Martin Christ Gefriertrocknungsanlagen GmbH, Osterode, Germany). Lyophilized samples were carefully broken into pieces and the breakage site was gold sputtered in a sputter coater SCD 030 (Balzers, Wiesbaden-Nordenstadt, Germany). SEM imaging was carried out at the Center for Electron Microscopy (ZELMI, Technische Universität Berlin, Berlin, Germany) with an S-2700 scanning electron microscope (Hitachi, Tokyo, Japan). Images were recorded at a magnification of 100×, 300× and 1000×. SEM was carried out at least on time for each formulation.

Nodules were soaked with low viscosity epoxy in a vacuum before and between cutting. The most representative areas, up to 25 × 45 mm, were selected and then thin sections (about 200 µm thick) were made for SEM investigation. Backscattered electron (BSE) imaging, secondary electron (SE) imaging and energy dispersive X-ray spectroscopy (EDS) analyses were performed using Tescan scanning electron microscope Mira 3 with analytical equipment of the Oxford Instruments AztecLive Automate with detector Max 80. The SEM was operated at 10 kV for BSE images, 4 kV for SE images and 20 kV for EDS analyses. Sections were subsequently mapped in Si, Mn, Fe, Ca and P X-rays using the Aztec program. Si, Mn, Fe, Ca and P were measured using Kα lines. Investigations of the nodule microstructures were made by SEM analyses with a Mira 3 TESCAN. Polished thin, 150 μm-thick sections were prepared. Fragments of the outer parts and central zone of nodules were selected for the identification of microbial associated structures. The surface of the samples was covered with 15 nm-thick gold with Balzers SCD 030 equipment. Major element concentrations were determined using energy-dispersive X-ray spectroscopy on an X-MAX 80 (EDS, Oxford Instruments, UK). Analyses were conducted at 20 kV using a diaphragm of 60 μm. Data analysis was carried out using the INCA Oxford software package.