Drosophila larva
PROTOCOL
Drosophila larvae were sandwiched between two 3 mm aluminum slit carriers with the 100 µm cavities facing each other and high-pressure
frozen with a Leica EM HPM100. No ethanol as synchronization media was used, 1-hexadecene was used as filler. The wholes of the slit
carriers were filled with filter tips dipped in 1-hexadecene to keep the carrier sandwich complete after freezing.
The carrier sandwich was mounted on a self-made 3mm holder in the VCT100 loading station and transferred into an Leica EM ACE900
prototype using the Leica EM VCT100 shuttle and the Leica EM VCT500 dock mounted on the Leica EM ACE900 (connected by the adapter
plate). The cryo-stage was set to -120°C and the holder was sitting there for 15min to reach equilibrium (since the sample came from LN
2
).
Samples were fractured by pushing off the top carrier with the fracturing knife, followed by partial freeze-drying at -100°C for 5 minutes.
Sample was coated (e-beam evaporation) with a layer of 2,5 nm platinum/carbon at an angle of 45° followed by 2,5 nm of C by tilting the
stage between 0° and 90° (rocking) and rotating the stage. Samples were transferred to the cryo-stage if an 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 slit sample carriers
Freeze Fracture -120°C (after 15 minutes waiting period)
Freeze Etching -100°C (5 minutes hold time)
Coating 2,5 nm C and 2,5 C rocking and rotation
Transfer Protected Leica 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 of the group of Prof. Damian Brunner, University Zurich, Institute of Molecular Life Science, Zurich,
Switzerland.
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