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 (PP2000) or by a specially designed turbomolecular pumping system (PP2000T). 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.

Why choose cryo-SEM?

The limitations of conventional ‘wet processing’ include:
* Shrinkage and distortion
* Extraction of soluble materials
* Relocation of highly diffusible elements
* Mechanical damage (fragile specimens can be damaged during conventional processing)
* Slow (24 hours or longer)
* Toxic reagents are required (fixatives, buffers etc)


Advantages of cryo-SEM:
* Specimen viewed in fully hydrated state
* Soluble materials are retained
* Less relocation of highly diffusible elements
* Little or no mechanical damage
* Time lapse experiments and evaluating industrial processes at timed intervals
* Usually no exposure to toxic reagents
* Rapid process
* High resolution capability (compared to low-vacuum techniques)
* Extra information obtained by low-temperature fracturing (compared with conventional and low-vacuum methods)
* Good for liquid, semi-liquids and beam sensitive specimens
* Ability to selectively etch (sublimate to reveal information)
* Ability to ‘rework’ specimen (eg re-fracture and coat)


Some applications of cryo-SEM:
* Botanical
* Zoological
* Food stuffs
* Resins, polymers and paints
* Beam sensitive materials (eg photographic emulsions)
* Geological (muds, slurries, oil bearing rocks etc)
* Liquid and semi-liquid specimens
* Foams (eg foods, beverages such as beer, industrial foams)
* Cryo-DualBeam (FIB/SEM)

Some specimen mounting techniques for cryo-SEM

Surface 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 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
This 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 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.

There are a range of methods for handling liquid or semi-liquid specimens - please contact us for more information.