Em ace600 coating device

Samples were dehydrated in a graded ethanol series at RT. For further chemical drying, the samples were first incubated in a 1:1 mixture of 100% ethanol and hexamethyldisilazane (HMDS) for 30min at RT and then transferred to 100% HMDS for 1h at RT. Access HMDS was removed with a pipette followed by overnight evaporation at RT. The cover slips with the dried cells were coated with platinum to a thickness of 5nm using an EM ACE600 coating device (Leica Microsystems). The samples were observed with a FE-SEM Merlin compact VP scanning electron microscope (Zeiss) at 5kV using a secondary electron detector.

Embedded samples were trimmed with an Ultratrim diamond knife (Diatome) to a rectangle (70μm x ∼1mm) with a slanted side for orientation using an EM UC7 ultramicrotome (Leica Microsystems). Prior to serial sectioning, a 25 x 25mm silicon wafer was plasma treated using an ELMO glow discharge cleaning system (Agar Scientific) for increasing the hydrophilicity of the wafer. Serial sections were cut at a thickness of 80 nm using a 4mm Leica AT-4 35° diamond knife (Diatome). Section ribbons were collected onto a plasma treated wafer using the water drain device of the knife. Then the wafer with the serial sections was coated with carbon to a thickness of 5nm using an EM ACE600 coating device (Leica Microsystems) to ensure conductivity. The serial sections were observed under a FE-SEM Merlin compact VP scanning electron microscope (Zeiss) equipped with the Atlas 5.3.2.9 Array Tomography software (Zeiss). The images were acquired using a backscattered (5nm pixel size) and a secondary electron detector (7nm pixel size) at 5kV.