Leica Microsystems EM ACE900 Application Note

Brand: Leica Microsystems

Model: EM ACE900

Type: Application Note

Leica Microsystems EM ACE900 is a versatile cryo-electron microscope that enables high-resolution imaging of biological samples at cryogenic temperatures. With its advanced features, the EM ACE900 offers exceptional image quality, ease of use, and a wide range of applications in structural biology, cell biology, and materials science.

Key capabilities of the EM ACE900 include:

High-resolution imaging: The EM ACE900 is equipped with a high-brightness electron source and a state-of-the-art detection system, enabling the acquisition of high-resolution images with excellent signal-to-noise ratio.

Key capabilities of the EM ACE900 include:

High-resolution imaging: The EM ACE900 is equipped with a high-brightness electron source and a state-of-the-art detection system, enabling the acquisition of high-resolution images with excellent signal-to-noise ratio.

Application Note

Cryo-SEM / Giardia lamblia

related instruments: Leica EM ACE900, Leica EM VCT100,

Leica EM HPM100

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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.

Sublimation, fracture face through one cell of giardia lamblia, both nuclei (N) are visible. Plasma membrane

(Pm) and adhesive discs (Ad).

Visible are proteins of the fractured plasma membrane (Pm). Due to sublimation the outer plasma membrane

surface (Pm*) is visible.

LNT Application Note - GIARDIA LAMBLIA

Cross fractured cilia (C) with microtubule organisation.

Visible are peripheral vesicles (Pv), glycogen granules (Gi), plasma membrane (Pm) and longitudinally fractured

cilium (C).

No sublimation, details of the nucleus and nucleus pores (Np) visible.

LNT Application Note - GIARDIA LAMBLIA

Leica Microsystems EM ACE900 Application Note

Brand: Leica Microsystems

Model: EM ACE900

Type: Application Note

Key capabilities of the EM ACE900 include:

High-resolution imaging: The EM ACE900 is equipped with a high-brightness electron source and a state-of-the-art detection system, enabling the acquisition of high-resolution images with excellent signal-to-noise ratio.

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Leica Microsystems EM ACE900 Application Note

Leica Microsystems EM ACE900 Application Note

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