SEM Imaging of Plankton Samples

Part of the sample collected as described above was fixed with 2% paraformaldehyde (Electron Microscopy Sciences) and 0.5% glutaraldehyde (Electron Microscopy Sciences) in 0.1 M marPHEM (0.1 M PHEM with the addition of 9% sucrose) (Montanaro et al., 2016 (link)) for 6 h at 4°C. The sample was then transferred to 0.1 M PHEM (60 mM PIPES, 25 mM HEPES, 2 mM MgCl₂, 10 mM EGTA, pH 6.9) containing 1% paraformaldehyde and preserved at 4°C until further processing. The sample was then rinsed once using 0.1 M PHEM at 4°C. The sample was then dehydrated at 4°C using the following (v/v) acetone/water series: 30%, 50%, 70%, 80%, 90%, followed by two pure acetone steps. Samples were left to sediment for a duration of 3 to 12 h before each exchange to avoid loss of material. The sample was then critically point dried (CPD; CPD300, Leica Microsystems) in small containers (1–1.6 µm pore size, Vitrapore ROBU, Hattert, Germany). In the CPD program, 30 slow exchange steps were used. CPD plankton were then distributed on carbon tape placed on an SEM stub (Agar Scientific) before further gold sputtering (Quorum, Q150RS). SEM imaging was performed using a Zeiss Crossbeam 540 with an acceleration voltage of 1.5 kV and a current of 700 pA and a secondary electron secondary ion (SESI) detector.