EQUIPMENT & ACCESSORIES CATALOG EDITION IV CRYO-SEM & CRYO-FIB/SEM PREPARATION SYSTEMS


What is... Cryogenic Specimen Preparation?


In this instance we are referring to frozen hydrated bulk specimens for SEM, commonly termed Cryo-SEM. When biological specimens are prepared by alternative methods, such as critical point drying, they may collapse and distort due to the removal of their water content. In addition diffusible elements are often removed or relocated, affecting the validity of subsequent X-ray microanalysis.


Cryo-SEM offers the best solution to this and in addition allows observation and analysis of liquid, semi-liquid and beam-sensitive specimens, such as emulsions, suspensions and foams. Cryo preparation is increasingly being used with FIB/SEM instruments for a wide range of specimen types, including some materials where low temperature milling conditions are desirable.


For biological and other “wet” specimens rapid freezing is essential in order to reduce morphological distortion, a key consideration for structural observation.


The aim of fast freezing is to reduce the size of ice crystals within specimens by reaching as quickly as possible the point at which recrystallization takes place (for pure water this is in the order of -130°C) and maintaining the specimen below this temperature during transfer, preparation and observation.


For larger specimens commonly used in SEM and FIB/SEM rapid freezing is normally done by plunging into liquid nitrogen in its ‘slushy’ form at -210°C. This is the standard method supplied with the PP3010, but it is also fitted with an “advanced specimen handling” system which allows specimens that have been frozen by alternative (faster) freezing methods to be manipulated and loaded under liquid nitrogen and then transferred under vacuum into the PP3010 for subsequent processing and observation.


Zoological


Techniques and Applications 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 98% of some animal and plant tissues it represents a most formidable specimen problem to most Scanning Electron 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 “higher 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 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:


• 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)


Botanical


Fungi


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.


58


Pollen of cactus Zygocactus truncatus Germinating pollen grains of Zygocactus truncatus.


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.


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