Leica em scd500

To inspect if the nanostructures’ morphology is preserved during the two-step replica molding transfer into OrmoStamp and PDMS, scanning electron microscopy (SEM) images were taken. To reduce charging effects during SEM imaging, a 15 nm chromium metal layer was sputtered on the OrmoStamp and the PDMS replica mold (Leica EM SCD 500, Leica Microsystems, 35578 Wetzlar, Germany, sputtering rate 0.1 nm s⁻¹). The conductive silicon master was not specially pretreated. The wafers were imaged by a Zeiss Supra 55 VP SEM (Carl Zeiss AG, Jena, Germany) using the following imaging parameters: silicon master, EHT 10 kV, InLens, WD 6 mm; OrmoStamp master, EHT 1 kV, InLens, WD 5 mm; PDMS, EHT 3 kV, SE2, WD 16 mm.

Neat PLA and nanocomposite films were cryo-fractured, glued on an aluminum sample holder with conductive carbon tape (Leit-C, Neubauer Chemikalien, Berlin, Germany), and sputter-coated with ~10 nm Tungsten (Leica EM SCD500, Leica Microsystems, Amsterdam, The Netherlands). The surfaces were observed with FESEM (FEI Magellan 400, FEI Electron Optics B.V., Eindhoven, The Netherlands) at room temperature at working distance of 4 mm, with SE detection at 2 kV and 13 pA.

Hind legs from ethanol-preserved specimens were removed from the body, dissected with a razor blade in sagittal plane, air dried at a room temperature for at least 24 h. Then dissected samples of femurs and tibiae were glued onto aluminium SEM stubs, coated with gold–palladium using a Leica EM SCD500 sputter coater (Leica Microsystems GmbH, Wetzlar, Germany), and examined with a Hitachi S4800 (Hitachi High-Technologies Corp., Japan) scanning electron microscope at 3 kV.

Biofilm for scanning electron microscopy (SEM) analysis was performed on 6-well polystyrene plates (PPHU Genos s.c., Strońsko, Poland) following the method described at point 4.4. After 24 h of incubation with phages or antibiotics, the wells were washed with sterile PBS to remove all planktonic cells. After washing, adhering bacterial cells were fixed with 3% buffered glutaraldehyde (Poch S.A., Gliwice, Poland) overnight at 4 °C as described before [36 (link)]. The samples were dehydrated through a graded ethanol (Poch S.A., Gliwice, Poland) series (50%, 70%, 80%, and 90%) for 10 min each and two times with 96% for 30 min at room temperature. The bottom of each well was cut to a length suitable for electron microscopy. After coating with a 10-nm-thick conductive layer of gold deposited by a high-vacuum sputter coater (LEICA EM SCD 500, Leica Microsystems, Wetzlar, Germany), the samples were examined with a scanning electron microscope (QUANTA FEG 250, FEI, Hillsboro, OR, USA) using a secondary electron detector (ETD).

After growing periods of 48 h (Methanosarcina mazei, Methanosphaera stadtmanae) or 72 h (Methanobrevibacter smithii) cultures were prepared as described above and mica plates were fixed on the aluminum stubs with double-sided carbon conductive tapes (Plano, Wetzlar, Germany). Subsequently, samples were air dried in a desiccator with silica gel (Merck KGaA, Darmstadt, Germany) for a period of 72 h. After coating with a 10 nm thick layer of gold-palladium in a sputter coater (Leica EM SCD500, Leica Microsystems GmbH, Wetzlar, Germany), samples were examined in SEM Hitachi S-4800 (Hitachi High-Technologies Corp., Tokyo, Japan) at an accelerating voltage of 3 kV.

The mean size and the formation process of nanoparticles were assessed continuously using dynamic light scattering (DLS) (Nanosizer, Malvern, UK), on temperature steps (i.e., 0.5 °C/step, 10 min step duration) ranging from 22 °C to 37 °C. HA-L-PNIPAM 0.5, HA-L-PNIPAM 0.1, and HA-PNIPAM were formulated at three concentrations (0.1%, 0.01%, and 0.1% w/v) in PBS buffer. The morphology of nanoparticles was observed using scanning electron microscopy (SEM) (JEOL microscope, JSM-7001TA, Tokyo, Japan). The samples were prepared by vacuum drying a drop of formulation (0.001% w/v) heated at 37 °C on carbon tape. The SEM samples were sputter-coated with a 20 nm gold layer (Leica EM SCD 500, Leica Microsystems GmbH, Wetzlar, Germany), then observed with a scanning electron microscope at 15 kV.