Auriga 405

The ultrastructural analysis of C. vulgaris treated with different concentrations of Ag ions and AgNPs-G for one day and one week was performed by TEM (Hitachi HT 7700 transmission electron microscopy) analysis.
Algae were centrifuged to remove culture media and then fixed with glutaraldehyde (2.5% in sodium cacodilate buffer 0.1 M, pH 7.2) for 2 h at 4 °C. Then, samples were washed twice for 15 min in sodium cacodilate buffer, postfixed in osmium tetraoxide (1% in sodium cacodilate buffer 0.1 M, pH 7.2) and washed twice for 30 min in deionised H₂O. Samples were stained with 0.5% uranyl acetate o.n. (over night) at 4 °C. Samples were dehydrated in a graded series of ethanol, from 30% to 100%. After dehydration, samples were embedded in Spurr resin (TAAB, Berks, UK).
Ultrathin sections of 50 nm in thickness were then cut using an ultramicrotome PowerTome PT-PC (RMC, Tucson, AZ, USA). Sections were picked up in 200 mesh copper grids and examined under a Hitachi HT7700 transmission electron microscope (Tokyo, Japan) at 75 kV.
Samples were analyzed by energy-dispersive X-ray spectroscopy (EDX) microanalysis with the TEM module of the Auriga 405 microscope (Carl Zeiss AG, Oberkochen, Germany) for the elemental analysis of the electron-dense particles inside the cells.

Scanning electron microscope (SEM) analysis was performed with a high-resolution field emission SEM (HR-FESEM; model Auriga 405; Carl Zeiss Microscopy GmbH, Jena, Germany) equipped with an energy dispersive spectrometer for X-ray microanalysis (XEDS, model Quantax; Bruker, Berlin, Germany). Before analysis, a small portion of the powder sample was fixed to specific support and coated with a thin layer of C by a sputtering machine (Q150T Turbo-Pumped Sputter Coater/Carbon Coater; Quorum Technologies Ltd., East Sussex, United Kingdom) to make its surface conductive. HR-FESEM XEDS acquisitions were performed under a high vacuum (10⁻⁶ hPa) at 20 keV accelerating voltage. Micrographs were acquired by secondary electron detector (SED) at a working distance (WD), magnification, spot size, and tilt angle conditions properly adjusted to optimize image resolution. The microanalysis was performed at WD and magnification ranging from 3.0 to 7.8 mm and 100× to 45,000×, respectively.

Scanning Electron Microscopy (SEM) measurements were carried out at CNIS Laboratory of Sapienza University Rome, with a field emission Zeiss Auriga 405 instrument, with a resolution of 1 nm at maximum magnification, by using a beam energy of 19 keV with a working distance in the 1.5–3.5 mm range.