Scanning ELectron Microscopy

Specimens were air-dried or freeze dried. For freeze drying specimens were rapidly frozen on a liquid nitrogen precooled polished copper block, immediately transferred to a liquid nitrogen precooled exsiccator and freeze dried under vacuum (5 × 10⁻² mbar) with subsequent warming to room temperature. Membrane fracturing was performed on rapidly frozen samples under liquid nitrogen with a pair of precooled forceps. Specimens were mounted on SEM stubs with adhesive carbon tape (Plano, Wetzlar, Germany) and coated with a carbon layer of ~15–20 nm (Leica SCD500, Leica Microsystems, Wetzlar Germany). Specimens were viewed in a field-emission SEM (Zeiss Merlin VP compact, Carl Zeiss Microscopy, Oberkochen, Germany) equipped with HE-SE and in-lens-Duo detectors. Images with a size of 1024 × 768 pixels were recorded at different steps of magnification. Measurements of distances were performed using the SmartSEM measurement tools (Carl Zeiss Microscopy).