Sem stub

The photonic chip with minimally embedded cells and landmarks on the surface was mounted on a SEM stub (6 mm length, Agar Scientific) using a conductive carbon sticker (12 mm, Plano GmbH, Germany). To reduce the amount of charging, the samples were surrounded by silver paint and gold coated for 180 s at 30 mA in a sputter coater (Quorum, Q150RS). The samples were introduced into the Crossbeam 540 (Carl Zeiss Microscopy, Germany). Light microscopy images of the landmarks were used to target the correct cell inside the FIB-SEM. The FIB was used at 15 nA to mill a trench and expose a cross-section through the cell. A current of 3 nA was used for polishing the cross-section before imaging. For imaging, the FIB milling was operated with 1.5 nA, the SEM imaging and the FIB milling operating simultaneously⁴¹ . The SEM images were acquired at 1.5 kV with the Energy selective Backscattered (EsB) detector with a grid voltage of 1100 V, analytical mode at a 700 pA current, setting the dwell time and line average to add up to 1.5 min per image. The final dataset was acquired with 5 × 5 nm² pixel size and a slice thickness of 8 nm.

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.