Hexamethyldisilazane

Algal cells were pelleted by centrifugation (1500 g, 10 min), resuspended in distilled water and pipetted on coverslips coated with poly-L-lysine (Sigma-Aldrich, Darmstadt, Germany). After 20 min, the cells were centrifuged on the coverslips (1000 g, 10 min), and then dehydrated in a graded series of ethanol (50%, 96%, 100%; 5 min each step), transferred to hexamethyldisilazane (Carl Roth, Karlsruhe, Germany) and incubated for 10 min. After an exchange of hexamethyldisilazane the samples were air-dried, sputter-coated with gold and imaged with a ZEISS Neon 40 scanning electron microscope (secondary electron detector, 2.5 kV acceleration voltage; Carl Zeiss, Oberkochen, Germany).

Cell pellets were fixed with a 1.6% glutaraldehyde solution in 1:1 mixture of 0.2 M sodium cacodylate buffer (pH 7.4) and artificial sea water at room temperature and then stored at 4 °C. After three washes in distilled water, cells were filtered on a 0.2 μm isopore filter (Merck Millipore, Carrigtwohill, Ireland). Samples on filters were subsequently dehydrated in a series of ethanol baths (70%, 96%, 100% three times, 15 min each). After a final bath in hexamethyldisilazane (Carl Roth GmbH, Karlruhe, Germany) (HMDS, 5 min), samples were left to dry overnight. Samples on filters were mounted on SEM stubs with silver paint and coated with platinum (3 nm) prior to observation. SEM observations were performed with a JSM-6700F SEM (JEOL, Tokyo, Japan) at an accelerating voltage of 3 kV.

Scanning electron microscopy (SEM): For SEM analysis, cells were fixed with 1.6% glutaraldehyde in 0.1 M phosphate buffer for several times before washing with distilled water, and cryoprotection in a solution of 30% glycerol for 1 h. The samples were then dehydrated in a graded ethanol series, and finally immersed in hexamethyldisilazane (Carl Roth, Karlsruhe, Germany), and dried at room temperature. Sample were then mounted on aluminium stubs and sputter-coated with a 3-nm gold–palladium coating (Cressington 308EM, UK) prior to analysis with a Field Emission Scanning Electron Microscope (FESEM JEOL 6700F, Japan). Transmission Electron Microscopy (TEM): For TEM, the samples were fixed by the same procedure as for SEM. After an adequate fixation, the samples were rinsed in 0.1 M cacodylate buffer, and post-fixed for 1 h in 1% osmium tetroxide in 0.1 M cacodylate buffer. Samples were then rinsed in distilled water, dehydrated in alcohols and lastly embedded in epoxy resin. Contrasted ultrathin sections (70 nm) were analyzed under a JEOL 1400 transmission electron microscope mounted with a Morada Olympus CCD camera.