Giardia lamblia - Sample Preparation for Cryo-SEM

Application Note for Leica EM ACE900 - Life Science Research

January 30, 2017

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

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 several hours.

Fig.1: Sublimation, fracture face through one cell of giardia lamblia, both nuclei (N) are visible. Plasma membrane (Pm) and adhesive discs (Ad).
Fig.2: Visible are proteins of the fractured plasma membrane (Pm). Due to sublimation the outer plasma membrane surface (Pm*) is visible.
Fig.3: Cross fractured cilia (C) with microtubule organisation.
Fig.4: Visible are peripheral vesicles (Pv), glycogen granules (Gi), plasma membrane (Pm) and longitudinally fractured cilium (C).
Fig.5: No Sublimation, details of the nucleus and nucleus pores (Np) visible.
 

Comments