Quanta 650 feg sem

Cleaned frustules were drop‐cast onto silicon wafers and left to dry at 60 °C, followed by 7 nm gold deposition with a multi‐target confocal sputtering tool (Kenosistec, Binasco, ITA). The cover slip was mounted on a microscope stub and grounded with Electrodag silver paint. Frustule surface structures of ten C. granii individual specimen of four strains (K‐1831, K‐1832, K‐1833, and strain K‐1834) were observed in nontilted samples with a Quanta 650 FEG SEM (FEI, Oregon, USA), or with a dual beam focused ion beam SEM (FEI, Oregon, USA). The gold‐covered frustules were shattered by pressing a glass cover slip sharply on top of the silicon wafer. By this, girdle fragments align normal to the stub with the X‐ and Y‐direction, facilitating observation of surface features in nontilted mode. At the side of fracture, the internal morphology could be studied. Structures were analyzed on SEM micrographs using Fiji.^[38] Dimensions were aligned with a Pelcotec Critical Dimension Standard (AISthesis Products, Inc., Clyde, USA). Periods a₁ and a₂ and pore diameter d were measured over 20 surface micropores per girdle micrograph. In total, ten individual girdles were measured for all C. granii strains, while one exemplary girdle was studied for each of the other centric diatom species. Measurements of internal structures were performed over five individual girdles for the species C. granii.

Scanning electron microscopy (SEM) imaging was used to localize areas of interest with cells and mineral, using a FEI Quanta 650 FEG SEM with a 15 kV beam. The SEM image acquisition was kept to large areas to reduce beam damage to the cells as much as possible. The wafers were stored under anoxic conditions between analyses.

Samples of soft tissue were mounted on double-sided carbon tape and sputter-coated with gold. Scanning electron microscopy (SEM) was performed with an environmental FEI Quanta 200 SEM and a FEI Quanta 650 FEG-SEM, using a working distance of 8.6–13 mm, accelerating voltage of 10–30 kV and a probe current of 1.5–3.0.

To observe adherent cells on electrospun scaffolds, samples were fixed in 10% neutral buffered formalin, then permeabilized in 0.05% Triton X-100 and stained with Acti-stain 555 phalloidin (Cytoskeleton Inc, Denver, CO) and 4',6-diamidino-2-phenylindole (DAPI). Stained samples were imaged on a Nikon A1 confocal microscope (Melville, NY). Additionally, cells were imaged using electron microscopy. Samples of cell-laden electrospun matrices were prepared for Scanning Electron Microscopy (SEM) by fixing in 10% neutral buffered formalin and chemically dehydrated via serial dilutions in ethanol followed by serial dilutions of hexamethyldisilazane before drying overnight in a vacuum desiccator. After drying, the samples were sputter-coated with gold-palladium and imaged using a Quanta 650 FEG SEM (FEI, Hillsboro, OR) with an accelerating voltage of 10kV.

Light images were obtained either at sea, as described above, or in the laboratory using a Leica M205 C stereomicroscope equipped with a Leica DFC 450 C camera. Cytoplasm extracted from Psammina aff. limbata was examined using scanning electron microscopy (SEM). Fragments of the granellare were air-dried onto an SEM stub and examined with a FEI Quanta 650 FEG SEM in low vacuum under the following conditions: Accelerating voltage of 10 kV, BSE detector, chamber pressure 40 Pa.