K550x sputter coater

From the same selected skin samples, small sections were fixed in paraformaldehyde (4%) for 3 h and then mounted on aluminum stubs and subsequently carbon coated (Sputter Coater K550X, Emitech, Quorum Technologies Ltd., Laughton, UK). Scanning electron microscopy (Quanta250 SEM, FEI, Hillsboro, OR, USA) was performed in secondary electron detection mode. The working distance was adjusted in order to obtain suitable magnification, and the accelerating voltage was 30 kV. The elemental composition was determined using an EDS probe (Quanta250 FEI with EDAX probe, Hillsboro, OR, USA) working in full-frame acquisition.

Bacteria were grown for 2 h on sterile 13 mm diameter coverslips (Sarstedt, Newton, NC, U.S.A.) in plates containing 100 μl of LB (10⁵ CFU) and irradiated with protocols A-low, A-intermediate, and A-high, delivering either blue or infrared laser wavelengths. The plates were washed twice with Phosphate Buffer Saline (PBS), fixed with Paraformaldehyde (PFA) 4% (w/v) in PBS for 1 h, dehydrated using ethanol at increasing concentrations (30% (v/v), 50% (v/v), 70% (v/v), 95% (v/v) twice and 100% (v/v) twice −30 min in each solution) and 1,1,1,3,3,3-hexamethyldisilazane (HMDS; Acros Organics, Springfield, NJ, USA) for 90 min. The coverslips were then mounted on metallic stubs, sputter-coated with gold (Sputter Coater K550X, Emitech, Quorum Technologies Ltd, UK) and high resolution SEM images were obtained at different magnifications (×300, ×2400, ×20,000) using a Quanta250 SEM (FEI, Hillsboro, OR, U.S.A.). The working distance and the accelerating voltage were adjusted to obtain the suitable magnification. The ×300 magnification images taken from quadruplicates were analyzed using the ImageJ software (Rasband, W.S., ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, U.S.A., https://imagej.nih.gov/ij/, 1997–2016) to calculate bacteria density. Every image was segmented and the percentage of white pixels, corresponding to the surface covered by bacteria, was calculated.

Once removed, capsule specimens were immediately placed in a sterile container filled with fixative fluid and prepared for SEM analysis.
The samples were fixed in 2.5% glutaraldehyde in sodium cacodylate buffer and then dehydrated in an ascending series of alcohol rinses.
Specimens were mounted on aluminium stubs covered with conductive double-sided carbon adhesive tape. Next, the samples were sputtered with gold (Sputter Coater K550X, Emitech, Quorum Technologies Ltd, UK) and immediately analysed by means of SEM (Quanta250 SEM, FEI, Oregon, USA) operated in secondary electron detection mode. The working distance was adjusted in order to obtain the suitable magnification, and the accelerating voltage was set to 30 kV. The thickness was assessed with ten measurements taken along the capsulorhexis. The roughness of the cut surface, which included microgrooves, surface pitting, and notches, and overall irregularity of the cut surface, was graded on a scale of 0 to 3 [19 (link)].

To analyze
fiber morphology, membrane samples were placed on aluminum
stubs covered with a double-sided carbon tape and sputter-coated with
gold using a Sputter Coater K550X (Emitech, Quorum Technologies Ltd,
U.K.). They were then analyzed using a scanning electron microscope
(Quanta 250 SEM, FEI, Oregon), working in secondary electron detection
mode. The acceleration voltage was set at 25–30 kV, while the
working distance was set at 10 mm to obtain optimal magnification.
Fiber diameters were calculated using Fiji software,^{72 (link)} by randomly selecting 50 fibers from each sample.

Colonized pine veneers were introduced into plastic tubes with 2% of glutaraldehyde in cacodylate buffer (0.1 M, pH 7.4) and left at 4 °C for 2 h. Part of the solution was discarded avoiding the contact of the samples with atmospheric air and sodium phosphate buffer was added and the samples left for 30 min. Such a step was performed twice. Dehydration was performed with graded ethanol series, graded ethanol:amyl acetate substitution series and CO₂ critical point drying (73 atm, 31.3 °C). Then, the samples were coated with a layer of gold (thickness: 10–20 nm) (K550X Sputter Coater, Emitech, Ashford, UK). A Philips XL30 (SEMTech Solutions, Billerica, MA, USA) scanning electron microscope (SEM) was used to perform the SEM analysis. The applied acceleration voltage was 12kW and the magnifications were 100×, 200×, 500×, 1000×, 1500× and 2500×.

The micromorphology was examined by a light microscope (LM) Leica DMLB (Leica Microsystems GmbH, Wetzlar, Germany) at a magnification of 200×. Lactophenol blue solution (Fluka) was used to observe cyanophilic reaction of fungal hyphae. The images were captured by a video camera Leica DFC490 using calibrated image analysis software Image-Pro plus 6.3 (Media Cybernetics, Inc., Rockville, MD, USA).
For scanning electron microscopy (SEM), the surface of samples was coated with gold plasma using a K550X sputter coater (Emitech, Ashford, UK) and examined with Vega TC (Tescan, Brno-Kohoutovice, Czech Republic) with accelerating voltage of 15 kV, software 2.9.9.21.