Em med020

Electrosprayed samples were coated with a gold layer using a sputter coating (EM MED020, Leica, Wetzlar, Germany) and their morphology was observed by SEM (JSM6300, JEOL, Peabody, MA, USA), with an accelerating voltage of 10 kV. Then, the average diameter of approximately 550 microspheres was measured with the ImageJ Software using the SEM images (https://imagej.nih.gov/ij/).
FTIR was performed at room temperature in a Thermo Nicolet Nexus apparatus in Attenuated Total Reflectance (ATR) mode (GMI, Ramsey, MN, USA). The spectrum was obtained from 4000 to 400 cm⁻¹, using 128 scans at a resolution of 8 cm⁻¹.
DSC measurements were performed in a PerkinElmer DSC 8000 (PerkinElmer, Villepinte, France) apparatus using a heating rate of 20 °C/min under nitrogen purge.

Energy-dispersive X-ray microanalysis (EDX) was performed with an AMETEK EDAX Octane Plus detector attached to a JEOL IT300 scanning electron microscope equipped with a LaB₆-cathode. Semithin sections of HP- frozen, rapidly freeze-substituted embedding of ovisacs, 2.5 µm in thickness, were placed on silicon wafers, mounted with carbon tabs onto aluminum stubs and coated with carbon by using a Leica EM MED020 vacuum coating system. Areas containing cellular inclusions were identified with SE-and BSE detectors and selected for measurement. Spectra and dot-mappings were recorded at 20 kV, at a working distance of 11 mm and under 35° -positioning of the EDAX detector. The dead-time of the detector was set between 22 and 25 s, and its lifetime at 30 s. Data collection, automated background subtraction and analysis were performed using the EDAX TEAM Software Version (V4.20) AMETEK GmbH, EDAX Division.

Laminin matrices attached to coverslips were fixed in Karnowsky reagent (4% PA and 0.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2) for 2 hours, washed three times with sodium cacodylate buffer 0.1 M, pH 7.2, dehydrated through increasing concentrations of ethanol and dried in E300 (Polaron, Quorum Technologies Ltd, Laughton, United Kingdom) critical point. The samples were then coated with a thin layer of gold sputter (Leica EM MED020) and viewed under a scanning electron microscope Jeol JSM6300.

The morphology
of the hydrogels
was analyzed by field emission scanning electron microscope (FESEM)
(Zeiss ULTRA 55, Carl Zeiss Microscopy) with an accelerating voltage
of 1.5 kV. The samples were lyophilized after swelling in liquid water
at 37 °C for 24 h to constant weight and freezing at −80
°C overnight. The cross-section was observed in the lyophilized
samples, which were previously immersed in liquid nitrogen and cryofractured
for FESEM observation. Finally, the samples were coated with a carbon
layer using a sputter coating (EM MED020, Leica). The percentages
of Ca and Zn ions were obtained with an Energy Dispersive X-ray Spectrometry
(EDX, X-Max N, Oxford Instruments) mounted on the Zeiss ULTRA 55 FESEM
(accelerating voltage 15 kV).

Si/ZnO layers and electrospun polymer mats were covered with a thin layer of sputtered gold (ca. 90 nm) using PVD equipment from Leica (model EM MED020). Au target was obtained from Mennica Metale Szlachetne, Warsaw, Poland. During this procedure a vacuum level of 10⁻² mbar and current of 25 mA were applied.

Fresh tomato leaves or stems (three biological replicates) collected from wild-type (WT) and transgenic plants were fixed for 24 h in a solution of 2.5% paraformaldehyde, 2.5% glutaraldehyde buffered with 0.1 M sodium cacodylate, pH 7.4 (Electron Microscopy Sciences, Hatfield, PA, USA). Samples were dehydrated in the following ethanol concentrations (30, 50, 70, 80, 90, 100, and 100%), each treatment lasting 10 min. Samples were then critical point dried with liquid CO₂ (Quarum K850 CPD), mounted, and sputter coated with a 5 nm thin layer of platinum (LEICA EM MED 020). Images were acquired with a scanning electron microscope (SEM Quanta 250 FEG FEI) at 5 kV, spot size 3 or 2 with a working distance of 1 cm.

The surface morphology of the scaffolds was analysed by means of a Zeiss Ultra 55 field emission scanning electron microscope (Oberkochen, Germany). The samples were cut into small pieces and dried at 30 °C in a vacuum oven for 24 h and then kept in a desiccator during 48 h. Afterwards, the specimens were mounted on metal studs and sputter-coated with a platinum layer during 10 s using a Leica EM MED020. The testing was performed at room temperature with a 2 kV voltage. The fibre diameters were measured from the FE-SEM micrographs at random locations (n = 100) with the aid of the ImageJ software (National Institutes of Health, Bethesda, MD, USA).