Phenom xl

The imaging of particle morphology was conducted with scanning electron microscopy (SEM) using Phenom XL (Phenom-World BV, Eindhoven, The Netherlands). To guarantee sample grounding and to minimise charging effects, samples were fixed onto carbon stickers and gold-sputtered with BAL-Tec SCP 050 Sputter Coater (Leica Instruments, Wetzlar, Germany). All images were taken at 1000× or 2500× magnitude with an acceleration voltage of 10 kV and with the use of a backscatter detector.

The material was dissected to separate the labia from the head and cleaned in detergent using an ultrasonic cleaner. Then, the standard procedure was applied [54 (link)]: dehydration with a series of baths in 80%, 90%, and 96% ethanol solutions, for 20 min each, and two baths of 99.8% ethanol solution for 30 min each. The labia were glued with carbon adhesive discs on the aluminium pin stubs, which then were coated with a film of gold (30 nm) using the Q150T ES sputter coater with the rotary planetary stage (Quorum Technologies Ltd., Laughton, UK). SEM micrographs (Figure 2, Figure 3 and Figure 4) were obtained using a Phenom XL field emission scanning electron microscope (Phenom-World B.V., Eindhoven, The Netherlands) at 15 kV accelerating voltage and with a BackScatter Detector (BSD) and Secondary Electron Detector (SED) and Hitachi UHR FE-SEM SU8010 (High Technologies, Tokyo, Japan) with a secondary-electron detector (ESD) at 5, 7, and 10 kV accelerating voltage. To obtain high-quality figures, fragments of labia were imaged at high magnifications and combined using the Image Composite Editor (panoramic image stitcher) and the graphic editor Adobe Photoshop CS6. In a few cases, a series of images at different focal distances were taken and combined using the software mentioned above to attain the appropriate depth of field.

The occurrence of chelation reactions often alters the apparent structure of the peptide. Hence, the scanning electron microscopy (Phenom XL, Phenom-World BV, Eindhoven, Holland) was used to distinguish PSCP-Fe and PSCP by applying 10 mA current and 5 kV voltage. The chelating ability of peptides and Fe²⁺ is closely associated with the types of amino acid residues present. The amino acid composition of PSCP-Fe was characterized by a known method (16 (link)) with minor modifications. 100 mg of PSCP-Fe was hydrolyzed with 6 M HCl at 110°C for 24 h. The mixture was then analyzed using a Sykam amino acid analyzer (S-433D, Sykam Corporation, Munich, Germany). The chelation sites on PSCP and ferrous could be more easily diagnosed using a Fourier transform infrared spectroscopy (FTIR), whose experimental design was as follows: 5.0 mg each of PSCP and PSCP-Fe mixed with potassium bromide were flaked and then analyzed using an FTIR spectrometer (Shimadzu IRTracer-100, JPN) in the wavelength range of 4,000–400 cm^–1 at 30°C.