Cryogenic Specimen Preparation Principles

EMS Catalog PP3000T Cryo-SEM Preparation System

What is Cryogenic Specimen Preparation?

In this instance we are referring to Frozen Hydrated Bulk Specimens for Scanning Electron Microscopy, commonly termed L.T.S.E.M. (Low Temperature Scanning Electron Microscopy). When Biological specimens are prepared by conventional methods, they may collapse and distort. In addition to which, there may be a loss of the diffusible elements that they may normally contain, and therefore affecting the validity of the X-Ray Microanalysis.

The use of L.T.S.E.M. offers a solution to this, and in addition allows viewing and analysis of ‘liquid’ specimens such as emulsions and suspensions, which was not previously practical. In preparing the specimen, we first want to freeze it as quickly as possible. This will reduce the morphological distortion important for structural observations, and also minimize any redistribution of solutes which is essential for X-Ray Microanalysis.

The aim of fast freezing is to reduce the size of ice crystals by reaching as quickly as possible the point at which recrystallization takes place, which is on the order of -130°C (for pure water), and maintaining the specimen below this temperature. Rapid freezing is commonly obtained by plunging the specimen into a cryogenic fluid. This is commonly liquid nitrogen, usually in the form of ‘slushy’ nitrogen at -210°C, and for the types of specimen we are considering, this has proved to be an effective cryogen.

Having frozen the specimen, we need to maintain it below -130°C (the recrystallization temperature) and prevent sublimation (below -130°C the rate of sublimation of ice is very low, of the order of 0.001nm/sec). We should, therefore, maintain the specimen with cryoprotection in a good clean vacuum to avoid contamination. Frozen specimens can subsequently be fractured, etched, coated with gold or carbon, using a preparation system, and then viewed and analyzed on the cold stage of the S.E.M.

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 surfac

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 lowvacuum techniques)
• Extra information obtained by lowtemperature 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)

Zoological	Botanical	Foodstuffs
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.	Pollen of cactus Zygocactus truncatus Germinating pollen grains of Zygocactus truncatus.	Chocolate Bar

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 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 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.

Alternative rivet mounting method

Geological	Cryo-DualBeam	Polymers
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.	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.	Stable emulsion of a hydrophobic polymer This image illustrates a stable emulsion of a synthetic liquid polymer dispersed in an aqueous continuous phase.

Online Ordering

EMS PP3000T Cryo-SEM Preparation System is available online from the EMS Catalog. For ordering or product information, click here.