The carrier sandwich was mounted on a self-made 3 mm holder in the VCT100 loading station and transferred into an ACE900 prototype using the VCT100 shuttle and the VCT500 dock mounted on the ACE900 (connected by the adapter plate). The cryo-stage was set to -120°C and the holder was sitting there for 10min to reach equilibrium coming from liquid nitrogen. Sample was fractured by pushing off the top carrier with the fracturing knife, followed by partial freeze-drying at -105°C for 5 minutes. Sample was coated (e-beam evaporation) with a layer of 2,5 nm platinum/carbon at an angle of 45°, no rotation followed by 4nm of C under 90° followed.
On a second sample no freeze etching was performed, it was fractured at -150°C and immediately coated with 2,5 nm Pt/C and 4nm of C, same angles as before, no rotation. The sample was transferred to the cryo-stage in a Zeiss Auriga 40 scanning electron microscope using the Leica EM VCT100 shuttle keeping the specimen under high-vacuum conditions surrounded by a cooling shield. Images were acquired at -115°C using the Inlens secondary electron detector.
Large, clean fracture faces throughout the whole organism were produced using the described equipment. Ultrastructural details such as organelles and cytoskeletal components were exposed by partial freeze-drying after fracturing. Samples were stable in the microscope and were imaged for several hours.