EMS Technical Data Sheets

Cryogenic Specimen Preparation Principles

EMS Catalog #PP3010, Cryo-SEM Preparation System

What is Cryogenic Specimen Preparation?

In this instance we are referring to frozen hydrated bulk specimens for Scanning Electron Microscopy (SEM), commonly termed Cryo-SEM.

Cryo-SEM, The Advantages

The Scanning Electron Microscopist is faced with the inescapable fact that liquid is a fundamental part of practically all lifesciences – and many materials – specimens. Since water occupies up to 90% of some animal and plant tissues it represents a most formidable specimen problem to most Microscopists.

Cryo-SEM is a quick, reliable and effective way to over come these not inconsiderable SEM preparation problems. Additionally the technique is widely used for observing 'difficult' samples, such as those with greater beam sensitivity and of an unstable nature. An important application, often overlooked, is the ability to use cryo-SEM to study dynamic processes (industrial or otherwise) by using a series of time resolved samples.

Naturally the advent of various "high pressure" modes, such as VP, LV and ESEM has allowed such samples examined in SEM without resorting to freezing or drying methods. However, cryo-SEM is still by far the most effective method of preventing sample water loss, which will in fact occur at any vacuum level – even with Peltier stages fitted to the SEM and the careful addition of water vapor in the SEM chamber. Cryo-SEM also has a number of a additional advantages, including the ability to fracture and selectively remove surface water (ice) by controlled specimen sublimation.

Why choose cryo-SEM?

The limitations of conventional 'wet processing' include

Advantages of cryo-SEM

A summary of the cryo-SEM preparation technique

Cryo preparation techniques for scanning electron microscopy (SEM) have become essential for the observation of wet or 'beam sensitive' specimens. Using such techniques removes the need for conventional preparation techniques, such as critical point drying or freeze-drying, and allows observation of the specimen in its 'natural' hydrated state.

The specimen is rapidly cooled and transferred under vacuum to the cold stage of the preparation chamber, which is mounted onto the SEM chamber. The preparation chamber is pumped either with a rotary pump (91005) or by a specially designed turbomolecular pumping system. The specimen can be fractured, sublimated ('etched') to reveal greater detail, and coated with metal by sputtering or with carbon by thermal evaporation.

Finally, the specimen can be moved under vacuum into the SEM chamber where it is easily located on a cold stage specifically tailored to the SEM. At all stages of the procedure the specimen is maintained at a 'safe' temperature of typically lower than -140°C.

Typical applications

Biological sciences including botany, mycology, zoology, biotechnology and biomedical – plus economically import agricultural sciences. More recently cryo-SEM is becoming an essential tool for pharmaceutical, cosmetics and healthcare industries, where it is used in basic applied research and for routine QA of many products, such as creams, cosmetics and drug delivery systems.

Cryo-SEM has long been a standard preparation method in the food industry. Of interest are multiphase products, such as ice cream, confectionery and dairy products.

Botanical: Cryo-SEM is the perfect method for highly hydrated botanical material.

Some specimen mounting techniques for cryo-SEM

Surface mountSurface mounting

This technique is used for leaf specimens etc. Roughen stub surface with fine emery paper. Specimen is laid on top of mounting media.

Edge mountEdge mounting

This technique is used for edge observation and fracture. Roughen surface of stub with fine emery paper. Specimen is placed on its edge in a machined slot and secured with mounting media.

Film emulsion mounting

Film mountThis technique is useful when a small specimen would be obscured by the Tissue-Tek mounting media, or when specimens need to be recovered. Specimens need to be slightly damp to use this method (good for nemotode worms).

The specimen is laid on surface so that its dampness slightly dissolves the film emulsion allowing the specimen to adhere to the film surface. Exposed unused film with the emulsion side uppermost is secured to the stub with mounting media. It may be useful to scrape off the protective coating of the film emulsion first to assist conductivity.

Rivet mountingRivet mounting

For liquids and for when specimens need to be frozen off the stub to achieve fast freezing rates. The rivet is placed in the hole and filled with liquid prior to freezing. If the specimen needs to be frozen away from the stub, two liquid-filled rivets are held together and then frozen prior to transfer onto the stub.

Alternative rivet mounting method

Alternate rivet mounting method

Cryo-SEM Micrographs

Dendritic Ice Crystals Face Cream Stamen from Yellow Mum
Dendritic Ice Crystals:
If it is cooled slowly, water forms dendritic ice crystals. These can have a variety of branching patterns – the complexity of which depends upon cooling rate. Arms extend from the main body of the crystal at an angle of 60°. Some, such as the one illustrated, resemble the arms of a snowflake. Bar: 2µm
Face Cream:
Anti-aging face cream. Specimen rapidly frozen in slushy nitrogen, fractured at -140°C and sputter coated with 5nm of platinum
Stamen from Yellow Mum

Cryo-SEM Applications

Pollen of Cactus Zygocactus Truncatus:
Germinating pollen grains of Zygocactus truncatus.Frozen Hydrated Aphid:
In comparison with the critical point dried aphid, this image shows that there is no distortion of the abdomen nor any other parts of the aphid following freeze drying.Arabidopsis Plant:
Cryo-FIB/SEM. Image courtesy of Hannah Edwards and Arabidopsis plants provided by Darren Wells, Centre for Plant Integrative Biology, School of Biosciences, University of Nottingham, UK.




Pollen of Cactus Zygocactus Truncatus Frozen Hydrated Aphid Arabidopsis Plant Arabidopsis plant




Algae Predatory Mite Soya Bean Leaf
Algae Predatory Mite Soya Bean Leaf




Maize Root Starch Granules Hand and Nail Cream Chocolate Bar
Maize Root Starch Granules Hand and Nail Cream Chocolate Bar




Baker's Yeast Baker's Yeast Wax Crystals in Gas Oil Stable Emulsion of a Hydrophobic Polymer
Baker's Yeast (Saccharomyces cerevisiae):
The specimen was rapidly frozen in nitrogen slush, fractured and coated with 4nm of platinium (Pt). 10nm yeast cell transmembrane particles (in hexagonal arrays) can be observed.
Wax Crystals in Gas Oil:
When cooled to a temperature below about 2°C, the waxes in fuel oils such as this tend to crystallize out. Wax crystal size and shape can be varied by altering the rate at which the oil is cooled.
Stable Emulsion of a Hydrophobic Polymer:
This image illustrates a stable emulsion of a synthetic liquid polymer dispersed in an aqueous continuous phase.

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PP3010 Cryo-SEM Preparation System