Quanta 650 feg environmental sem

ESEM micrographs were obtained using a Quanta 650 FEG Environmental SEM with EDXS analysis (Fei Company, Hillsboro, OR, USA) under service acquisition from the International Nanotechnology Laboratory (INL, Braga, Portugal). Before examination, hydrated cuticle located at the mid-body level was sectioned to expose the internal component (cross-section). Then, the cuticle was imaged, namely, the external surface in the longitudinal direction and the exposed cross-section in the longitudinal and transversal directions (Figure 10C). Samples were placed into a drop of water on the Peltier cooling stage at 4 °C, 100 to 760 Pa of water vapor and an acceleration voltage of 10 kV at different magnifications. Chemical analysis was measured on the outer surface and cross-sections at an energy of 10 keV. For SEM (JSM-6010 LV, JEOL, Kyoto, Japan), dried untreated and treated cuticles were sectioned as indicated above, and the external surfaces and the exposed cross-sections were sputter-coated with an electrically conducting layer of gold using the EM Leica ACE600 Sputter coater (Wein, Austria). The cross-section imaging results are presented only for the transversal direction, as the results for the longitudinal direction were similar or less clear.

All chemicals
are of analytical grade and obtained from Sigma-Aldrich, Fluorochem
or Alfa Aesar and used as received. X-ray photoelectron spectroscopy
(XPS) was performed using a Kratos Axis Ultra DLD spectrometer with
a monochromated Al Kα X-ray source (E = 1486.6
eV, 10 mA emission). Scanning electron microscope (SEM) analysis was
carried out using a FEI Quanta 650 FEG environmental SEM. Powder X-ray
diffraction analysis was performed using a Philips X’pert PRO
diffractometer with Cu Kα radiation (λ = 0.154 nm) and
operating at 40 kV and 30 mA.