Auriga 40

Scanning electron microscopy (SEM) was used to examine microtopography and nanotopography of the polyurethane samples. Samples were prepared for SEM by critical point drying and coated with a 10 nm film of gold. Surface imaging was performed with a Zeiss Auriga 40 SEM (Oberkochen, Germany) at 3 keV acceleration voltage using the chamber secondary electron detector.

We observed structures with a digital photonic microscope (Keyence VHX-5000) to determine scale form (lamellar scale vs. piliform scale), scale colour (coloured vs. transparent) and scale insertion (flat vs. erected) on ventral side, which is the side exposed at rest for most of the species in this study. Moreover, we checked that there were not significant differences between ventral and dorsal sides regarding main structural features (see Appendix and Supplementary file 3f,g and h). We defined as scale type the interaction between scale form and scale insertion (erected lamellar scale, flat lamellar scale and piliform scale). Wings were imaged using SEM (Zeiss Auriga 40) to determine nanostructure type and to measure scale density, scale length and width, membrane thickness, and nanostructure density (see SI for more details). We also determined for each species the structural syndrome, defined as the association between micro- and nanostructural features. On a subset of 3 species, we measured 10 specimens per species, each specimen being measured twice for density and five times for scale dimensions. Given that scale structural features were shown to be repeatable (see ‘High-resolution imaging and structure characterisation’ section in SI) within species we retained one specimen per species in structure characterisation.

Cladocopium (C3) cells were collected in 3, 14, and 35 days. To transmission electron microscopy (TEM) analysis, cells were fixed 2.5% glutaraldehyde in 0.1 M cacodylate buffer and 1.75% NaCl, pH 7.2 for 24 h. Scanning electron microscopy (SEM), cells adhered to Poly-L -lysine-coated (mol wt 300,000) glass coverslips and fixed for 1 h in the same solution previously described. After fixation, cells were washed in 0.1 M cacodylate buffer and postfixed for 1 h in 1% OsO4 containing 0.8% potassium ferrocyanide in 0.1 M cacodylate buffer (pH 7.2). Then for TEM analysis, the samples were washed in 0.1 M cacodylate buffer, dehydrated in acetone, and embedded in Epon. Ultrathin sections were stained with uranyl acetate and lead citrate and observed using Hitachi HT 7800 and Fei Tecnai Spirit transmission electron microscope. To SEM after postfixed with OsO4, cells were dehydrated in ethanol and critical point dried with liquid CO2. Finally, cells were coated with a 5 nm-thick layer of platinum and observed using a Zeiss Auriga 40 scanning electron microscope.

One hundred larvae (N = 300) were exposed to 100 mL of blastospore or conidial suspensions at 1 × 10⁷ propagules mL⁻¹. After 24 and 48 h, a pool of hemolymph from 100 surviving larvae per group was transferred to microtubes and fixed in glutaraldehyde (2.5%, pH 7.2) (v/v) at 4 °C overnight. The microtubes were previously treated with Sigmacote^® (Sigma-Aldrich©, US) to avoid the adhesion of hemocytes to the wall. Next, the microtubes were centrifuged (4 °C, 10 min, 2000 rpm) (Centrifuge 5418, Eppendorf^®), the supernatant was discarded, and the pellet was resuspended in 100 µL of sodium cacodylate buffer (0.1 M; pH 7.2). The processes of centrifugation and resuspension were repeated three times. The samples then were fixed in 1% OsO₄ at room temperature for 1 h [46 ]. After fixation, the samples were centrifugated and resuspended as previously described, followed by dehydration in an ethanol series [10 ]. Finally, samples were dried at the critical point in CO₂, embedded with gold, examined, and photographed using a high-resolution SEM Auriga 40^® (ZEISS©, Oberkochen, Germany). Please see the Additional file 1: Fig. S2 for pictures of scanning electron microscopy of hemocytes identified.

The surface morphology of the treated fibers was characterized using a scanning electron microscope with field emission (Auriga 40, Zeiss, Oberko-chen, Germany). To obtain the FTIR spectra of the samples, they were recorded on a Frontier spectrometer (Perkin Elmer, Waltham, MA, USA), with a horizontal attenuated total reflectance (ATR) accessory. The surface wettability of fibers was evaluated by measurement of water and diesel contact angles, using a Tensiometer, model K100C (Krüss, Hamburg, Germany). The thermal stability of the CP, CPNaOH, and CPNaOHT was evaluated by thermogravimetry analyses-(TG) and Derivative Thermogravimetry using a thermogravimetric analyzer from NETZSCH, TG209F1 Libra (Netsch, Selb, Germany). Approximately 7 mg of sample were used in the TG/DTG analyses, heating rate 10 °C.min⁻¹, temperature range from 28 to 600 °C, under a dynamic oxygen atmosphere and flowrate of 20 mL.min⁻¹.

Overnight cultures of E. faecalis OG1RF were diluted as described above in medium containing 10 μg ml⁻¹ of the fatty acid or solvent control. Initial sample preparation and fixations for scanning electron microscopy (SEM) were previously described (5 (link)), with the noted modifications. Following osmium tetroxide fixation, SEM samples were added to 5- by 5-mm silicon chips (Ted Pella, Inc.). Silicon chips were passed through a graded ethanol series (25, 50, 70, 95, and 100%) for 15 min at each interval. Chips then were placed in a LADD critical point dryer for three 10-min cycles. Each silicon chip was coated with gold for 10 s prior to visualization. Samples were viewed using a Zeiss Auriga 40 at the Center for Advanced Microscopy and Imaging at the University of Tennessee at a kEV of 1.0. Biological triplicates were performed for each growth condition, with a minimum of 10 fields examined per replicate.

Giardia interphase and mitotic trophozoites cells were imaged in a Zeiss Auriga 40 FIB/SEM workstation operating under SmartSEM (Carl Zeiss Microscopy GmbH, Oberkochen, Germany). FIB/SEM milling was started right in front of the anterior part the cell. Ion beam currents of 50 pA were used. High-resolution images were obtained with the EsB detector at 1.5 kV at a grid voltage of − 500 V. Dependent on the desired resolution, image pixel sizes between 2 and 10 nm in x/y were chosen. The milling rate was set to 2 nm, which allows the adjustment of the z resolution in 2 nm steps at any time during the FIB/SEM run. The average voxel size achieved was 3 × 3 × 8 nm.