CRYO-SEM & CRYO-FIB/SEM PREPARATION EDITION III Techniques and Applications


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.


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 multi-


phase 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 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.


Freeze & transfer onto preparation chamber cold stage


Pipette liquid sample into tube


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.


Alternative rivet mounting method


Pipette liquid sample into hole in sample stub


Place metal rivet or small piece of plastic tubing on top of hole (containing liquid sample).


Note: Small drop of “Super Glue” can be used to hold tube to stub.


Result: clean surface fracture


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