Leo 1525

The tensile-fractured surface morphology of the virgin PECoVA, PECoVA/DOL composite and PECoVA/OMCD composite was examined through field emission scanning electron microscopy (FESEM), with the equipment model number of ZEISS LEO 1525 (ZEISS, Oberkochen, Germany) by referring ASTM F1877-98(2003). A thin layer of platinum was employed as the coating layer to allow the capture of good images. The tensile fracture end of the sample was mounted rigidly on aluminum pin stubs then sputter-coated with a thin layer of palladium. This was to avoid electrostatic charging during characterization. The magnifications used were 500×, 1000× and 2500×.

For documentation of morphology using SEM, three radulae were carefully extracted and cleaned by a short ultrasonic bath in 70% EtOH. Subsequently they were mounted on SEM specimen holders by double‐sided adhesive carbon tape and sputter‐coated with platinum (5 nm layer). For visualization, we used a SEM Zeiss LEO 1525 (One Zeiss Drive). Only mature teeth from the working zone, which can be identified by lack of covering epithelia, were studied. To document the wear on the teeth, radulae were rewetted by 70% EtOH afterwards, cleaned by a short ultrasonic bath, rearranged on SEM sample holders and visualized again in the SEM. Nomenclature of teeth was adapted from Ong et al. (2017 ).
For the 3D visualization, mature radular teeth of the working zone of two radulae were extracted manually with forceps. Each tooth was mounted on SEM specimen holders by double‐sided adhesive carbon tape, sputter‐coated with platinum (5 nm layer), and visualized under the SEM from all sides. Using the 3D software Blender v2.83 (Blender Foundation), the teeth were then modeled by hand constantly comparing the 3D visualization with the SEM images taken from different sides (see also protocol in Krings, Marcé‐Nogué, et al., 2020 (link); Krings, Marcé‐Nogué, & Gorb, 2021 (link)). In the same manner, the position and embedment of the teeth within the membrane were reconstructed.

Sample morphology was analysed using a FE-SEM model LEO 1525 (Carl Zeiss SMTAG; Oberkochen, Germany). The EVs enriched in exosomes were fixed with 2% v/v p-formaldehyde and 1% v/v glutaraldehyde (Sigma-Aldrich; Saint Louis, MO, USA) in PBS. Next, a drop of the suspension was spread on a carbon tab placed on an aluminium stub (Agar Scientific; Stansted, UK) and left to dry in a stream of nitrogen for 25 min. Then, the dried samples were coated with gold (layer thickness 250 Å) using a sputter coater (model 108 A, Agar Scientific; Stansted, UK). Each analysis was performed in triplicate.

The morphology and the microstructural features of samples produced by GM E. coli strains were investigated using the field-emission scanning electron microscope (FESEM) LEO 1525 (Zeiss, Germany) operating at 5 kV. Prior to analysis, samples were dried until constant weight and placed onto double-sided carbon tape mounted onto an aluminum stub. They were gold coated for 2 min at 20 mA using an Emitech K575X Peltier (Ashford, UK) cooled sputter coater.

The morphology of the fracture surfaces of the composites was examined using a Leo 1525 (Carl Zeiss, Germany) scanning electron microscope equipped with a field emission gun (FEG-SEM). An accelerating voltage of 5 kV was used, with the samples being sputter-coated with a thin layer of chromium prior to examination.

For scanning electron microscopy, samples of a salt-induced C. merolae culture were fixated in formaldehyde (1%), dehydrated through an ascending series of ethanol (30, 50, 70, 90, 100%), and dried at the critical point with Balzers CPD 030 Critical Point Dryer (BALTEC, Pfäffikon, Switzerland). After coating samples with gold using a sputter coater (SCD 050, BAL-TEC), scanning electron micrographs were taken with a LEO 1525 (Zeiss, Jena, Germany).