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Application Note
CRYO-SEM WORKFLOW OF OIL BASED SAMPLES
related instruments: EM ICE, EM ACE900, EM VCT500 & EM VCM
2
CRYO-SEM WORKFLOW OF OIL BASED
SAMPLES
Application Note for High Pressure Freezer EM ICE, Freeze Fracture System EM ACE900
and Vacuum Cryo Transfer System EM VCT500 with Vacuum Cryo Manipulation System
EM VCM
Julia König, Leica Microsystems, Vienna, Austria
Roland Fleck, Dr., Kings College London, Centre for ultrastructural imaging
Freeze fracture is a powerful method to investigate the internal structures of different sample types like cells and tissues but also
suspensions and emulsions in their native, watery state. To keep the samples as native as possible, a small portion of the sample is
high pressure frozen in between two specimen carriers. To “open” the sample for further investigation in the cryo SEM, the carriers are
subsequently forced apart in a freeze fracture machine. The fractured surface is coated with platinum and carbon before imaging to
avoid any sample charging during SEM investigation. Advantage of that workow is the high sample throughput and resolution of the
internal structure of the sample. Because the samples are still in their native state, the distribution of the different components like oil
and water can be easily analysed. This makes this technique very interesting for quality analysis in the pharmaceutical or food industry.
In this article we will show the power of this technique by using classic food mayonnaise as a sample.
Specimen: Mayonnaise
Desired preparation: Cryo-SEM workow
Objective of observation: Freezing quality of oil sample
Procedure
High pressure freezing
Samples were added to the 3 mm interlocking carriers for freeze fracture. Sample carriers were slightly overloaded to prevent any air
inclusion inside the carrier. Subsequently samples were directly frozen with the EM ICE and stored in liquid nitrogen for further proces-
sing.
Sample transfer to EM ACE900 via EM VCT500
To prepare the samples for cryo SEM investigation, the samples need to be freeze fractured and freeze etched. To do that under cryoge-
nic conditions, the samples must be loaded into a pre-cooled EM ACE900. This is done with the EM VCT500/ EM VCM.
After liquid nitrogen storage the samples are transferred to the EM VCM and loaded onto a VCT freeze fracture holder. For loading into
the EM ACE900, the sample holder is transferred with the EM VCT500 shuttle. For this purpose, the EM ACE900 has to be equipped with
an EM VCT500 dock.
Freeze fracture and etching
The samples were loaded in the EM ACE900 at a stage temperature of -160 °C. The samples were fractured at a temperature of -145°C
after reaching an equilibrium (wait 10-15 minutes). The freeze etching was processed for 1 min at -110°C. The last step, the coating,
was performed with electron beam evaporation and a mixture of platinum and carbon (Pt/C coating with multi angle rotary shadowing).
In this way a 4 nm thick layer was coated on top of the samples.
LNT Application Note - CRYO-SEM WORKFLOW OF OIL BASED SAMPLES 3
Sample transfer to cryo-SEM
To keep the samples under optimal conditions the samples were directly transferred with the EM VCT500 shuttle from the EM ACE900
to the JEOL JSM 7800F Prime equipped with the EM VCT500 cryo stage.
Results
The imaging proves the nicely frozen quality of the mayonnaise samples. A very well fractured plane is visible. The large “round” struc-
tures can be identied as fat globules held in a water system as an emulsion. The fat globules have a well-dened round shape as well
as no visible structure inside, showing exactly what is expected. Also in the surrounding emulsion, no ice crystal formation is visible.
Furthermore, an additional regular structure in between the fat globules can be detected. This may be some additional phase separati-
on, occurring due to air expose of the mayonnaise over a period of time.
4
LNT Application Note - CRYO-SEM WORKFLOW OF OIL BASED SAMPLES 5
Sample preparation process time with Leica solution
EM ICE: 1hr total process time. User interaction time 30 minutes (high pressure freezing).
EM ACE900: 1hr total process time. User interaction time 30 minutes (freeze fracturing and etching).
EM VCT500: 15 minutes total process time. User interaction time 10minutes (sample transfer).
Instruments used
Related articles
https://www.leica-microsystems.com/science-lab/drosophila-larvae-sample-preparation-for-cryo-sem/
https://www.leica-microsystems.com/science-lab/giardia-lamblia-sample-preparation-for-cryo-sem/
https://www.leica-microsystems.com/science-lab/brief-introduction-to-freeze-fracture-and-etching/
EM ICE
EM ACE900
EM VCT500 with
EM VCM
© 2018 by Leica Microsystems GmbH.
Subject to modications. LEICA and the Leica Logo are registered trademarks of Leica Microsystems IR GmbH.
Leica Mikrosysteme GmbH | Vienna, Austria
T +43 1 486 8050-0 | F +43 1 486 8050-30
www.leica-microsystems.com
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