Giardia lamblia
PROTOCOL
A 100 mesh copper grid (12 um thickness) was dipped into a concentrated Giardia suspension and sandwiched between two flat 3 mm
aluminum specimen carriers with scratched surfaces. Subsequently, the sandwich was transferred to the widened hole of a middle plate
(3.1 mm diameter). A 50 um spacer ring was added on top and the specimen immediately frozen with an HPM100 high-pressure freezing
machine without using alcohol as synchronization fluid.
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.
SUMMARY
Procedure Parameters
High Pressure Freezing 3 mm aluminum flat sample carriers
Freeze Fracture -120°C / -150°C
Freeze Etching -105°C (5 minutes hold time) / none
Coating 2,5 nm Pt/C and 4nm C / 2,5 nm Pt/C and 4 nm C
Transfer Protected EM VCT100 Shuttle
Imaging Cryo-SEM at -115°C
RESULTS
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 a several hours.
Samples prepared and imaged September 2015 by Andres Kaech Ph.D., University of Zurich, Center for Microscopy and Image Analysis,
Zurich, Switzerland. Specimen courtesy Jon Paulin Zumthor, Institute of Parasitology, University of Zurich, Switzerland.
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