Drosophila larvae - Sample Preparation for Cryo-SEM

Application Note for Leica EM ACE900 - Life Science Research

January 30, 2017

Drosophila larvae were sandwiched between two 3 mm aluminum slit carriers with the 100 µm cavities facin 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 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 15min to reach equilibrium (since the sample came from LN2). 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 2,5nm platinum/carbon at an angle of 45° followed by 2,5nm 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

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.

Fig.1: Fracture plane through multiple cells, Nucleus (N), Endoplasmatic reticulum (Er)
Fig.2: Fractured intestinal cells forming the intestine with brush border: (Mv) Microvilli forming the brush border, (Mi) Mitochondria (cross-fractured and fractured through membrane), (Pm) Plasma membrane between two adjacent cells (cross fractured).
Fig.3: Fractured cells at higher magnification: (Mv) Microvilli fractured between the lipid bilayer showing membrane proteins.
Fig.4
Fig.5: Muscle tissue with actin (Ac) filaments and mitochondria.
Fig.6: Endoplasmic reticulum (ER), cross fractured.
Fig.7: Outer and inner membrane (o and i) of the nucleus (N) and nucleus pores (Np)s of a membrane and cross fracutred nucleus.
Fig.8: nucleopores with baskets (B) at high magnification.

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