excerpt from Microcosmos by Jeremy Burgess, Michael Marten and Rosemary Taylor
© Copyright 1987, Cambridge University Press, reproduced by permission.
Biological SpecimensElectron microscopy places many restraints on the specimen to be examined. First, it has to withstand a high vacuum. This precludes the examination of living materials at ordinary temperatures without prior treatment, except in very special circumstances. In the case of the TEM, the specimen must also be thin. If it is an organic material, it should not exceed 100 nanometers in thickness if good resolution is required. Mineral and metal specimens containing elements of high atomic number must be even thinner because heavy atoms absorb the electrons more readily.
SEM specimens do not need to be thin, but they have other requirements. They must be capable of dissipating the energy of the focused electron beam without building up a local surface electric charge. They must also be a good source of secondary electrons if that is the imaging mode to be used. In practice this last requirement means that the specimen is usually coated with a thin layer of a conducting metal such as gold.
Finally, there is a size limitation on specimens in both types of instrument. This is nor very stringent for SEM specimens, but TEM specimens are almost invariably mounted on a fine mesh grid which is 3 millimeters in diameter. This is because of space restraints around the TEM's objective lens.
Biological specimens for the TEM range from single molecules (proteins, nucleic acids, polysaccharides, antibodies) through micro-organisms (viruses, bacteria) to tiny sections of much larger creatures (animals, plants). In general, specimens up to the size of whole bacteria can be studied with no prior treatment other than a staining procedure to enhance contrast.
The most rapid and simple staining technique is 'negative staining'. A suspension of the specimen is mixed with a solution of a heavy metal salt on a specimen grid which has been previously coated with a thin film of carbon. After removing the excess liquid by blotting, the specimen can be examined. What the operator sees is a uniform gray background - the heavy metal salt- with The bright, unstained specimen embedded in it. Negatively stained specimens are naturally very thin, and the technique therefore gives very high-resolution images. It has been used to elucidate the structure of viruses. It is also very quick - the specimen is ready literally within seconds. This makes it particularly useful when a large number of specimens needs to be examined. In plant pathology, for example, the leaves of a large number of plants may need to be checked for a virus infection; it is a simple matter to negatively stain a drop of sap from each and then examine these specimens for virus particles.
In order to study the structure of cells, and how they combine into plant and animal tissues, other methods have to be adopted. The most common of these involves several stages. First, a small piece of tissue is 'fixed' in an organic aldehyde solution, usually glutaraldehyde. This kills the cells rapidly and stabilizes their chemical structure. The fixed material is then placed in a solution of osmic acid, which completes the fixing process and stabilizes lipids within the cells, as well as adding electron contrast in the form of metallic osmium. The water in the specimen is next removed, a process called dehydration, and replaced by alcohol. The piece of tissue is then embedded in a liquid plastic such as Araldite. After curing in an oven, the tissue is at last fully stabilized and set in a hard plastic matrix. Slices of the embedded tissue can now be made with an ultramicrotome, placed on specimen grids, and subjected to staining procedures in order to enhance image contrast.
For simple examinations of structure, staining usually consists of immersing the specimen in solutions of uranium salts or lead salts, or both. Alternatively, specialized stains may be used. For example, if a specimen is floated on a solution containing a hydrolysable phosphate compound and a soluble lead salt, hydrolytic enzymes within the specimen will reveal their position through deposits of insoluble lead phosphate. Proteins which are not enzymes, and other antigenic molecules, can be localized by the use of antibodies tagged with colloidal gold particles. In both cases, the image consists of a view of the structure of the tissue overlaid with localized deposits of heavy metal (lead phosphate, gold) which show the precise position of a particular molecule under investigation. This is a very powerful technique in biology.
There is always doubt whether structures seen after chemical fixation are an accurate reflection of the corresponding structure in vivo. This has led to methods of preparing biological specimens by freezing. In so-called freeze substation, the object is rapidly frozen in liquid nitrogen or liquid propane (at about -200 degrees centigrade). This results in the water within it becoming a solid without the formation of ice crystals-it is 'vitrified'. The vitrified water can then be replaced, at low temperature, by an organic solvent such as acetone. The dehydrated specimen is then brought up to room temperature and processed normally. In cryopreservation, the object is frozen as before, but it is then sectioned at low temperature with a special microtome. The advantage of this is that the ability of antigens within the tissue to bind to applied antibodies is not impaired. Its disadvantage is that structural preservation may be poor, even if the material is chemically fixed after sectioning and staining.
Another major technique for preparing biological specimens is replica formation, and it is also applicable to a range of materials in metallurgy, engineering and mineralogy. The specimen which goes into the microscope is neither the whole object (as in negative staining) nor a section of it, but a thin film of carbon which has been evaporated onto the specimen in a vacuum. Two applications of replica techniques are worth detailed consideration.
The replica may be of an external surface of an object, for example a group of virus particles. The particles are dried onto a suitable substrate, such as clean glass or freshly cleaved mica. This preparation is placed inside a vacuum evaporator and coated with a film of carbon. The carbon forms a coherent layer over the substrate and the virus particles, and the specimen is then produced by floating the carbon layer off the substrate and dissolving away the virus particles underneath. Such a replica, because of its thinness, will yield a very high-resolution image of the surface topography of the replicated particles. Its value as a specimen can be increased by evaporating a layer of a heavy metal such as platinum onto it at a low angle of incidence. This strengthens the replica film: more important, it Creases contrast by the production of 'shadows' from high-points in the replica.
Alternatively, replicas may be made of internal surfaces of cells and tissues. This procedure requires much more elaborate equipment, and is called freeze-fracturing or freeze-etching, depending on what happens to the object before it is replicated. The object is first frozen rapidly by plunging it into a mixture of liquid and solid nitrogen, or by propelling it onto the surface of a copper disc cooled by liquid helium ('freeze- slamming'), so that the water within the superficial layers of the object is vitrified. The frozen object is then transferred to the main apparatus, which consists of a vacuum evaporator fitted with sophisticated temperature controls, a microtome and electrodes for the evaporation of carbon and heavy metals. Once inside the machine, the object is sliced at low temperature to reveal internal surfaces. A shadowed replica may then be taken of these surfaces by the evaporation of a thin carbon film followed by a coating of platinum. The microscope specimen is produced by removing the object from the machine and dissolving away organic material with strong acids.
Freeze-etching introduces the additional stage of raising the temperature of the sliced and frozen object to about -100 degrees centigrade for a short period. This causes water within the surface of the object to sublime away, leaving nonvolatile components set in sharper relief. Replication and shadowing then follow as before.
These techniques are particularly valuable in the study of biological membranes. Frozen membranes fracture down the middle of their two layered structure. Freeze- fracture techniques thus make it possible to see the 'internal' surfaces of these membranes. This has been of great value in understanding the functioning of membranes such as those involved in photosynthesis.
It should be emphasized that no one method of specimen preparation can be considered 'correct' for a particular object. Different techniques yield different information about the same structure. For example, a thin section of a mitochondrion within a cell will show its position, outline shape and the way in which the membranes are folded; a freeze-fracture replica will show the distribution of particles within and between those membranes; and a negatively stained preparation of isolated membranes will show details of the particles associated with the surface of the membranes.
With the exception of negative staining, most techniques are time-consuming and require expensive ancillary apparatus. It may take several days to produce a viewable specimen from fresh tissue. Fortunately, both replicas and sections are more or less permanent, and can be retained for examination at a later date.
Turning to biological specimens for the SEM, it used to be necessary first to fix and dehydrate them, and then dry them either in air or by the use of liquid carbon dioxide ('critical point drying'). The only exceptions to this were objects robust enough to withstand a vacuum without such treatment - pollen grains, for example, and insects with hard exoskeletons.
The favored technique for modern SEM work is cryopreservation. The object is rapidly frozen by being plunged into a mixture of liquid and solid nitrogen at about-200 degrees centigrade. This results in the almost instantaneous freezing of the surface of the object and prevents its distortion. The object is then transferred directly to a special specimen stage, inside the SEM, which is also cooled by liquid nitrogen. This technique is successful even with the most fragile objects.
In order to achieve good emission of secondary electrons from organic materials and to prevent the build-up of surface electric charge during examination, the specimen is usually coaxed with a very thin layer of gold or gold-palladium alloy. With dried specimens, this coating can be deposited in a vacuum evaporator. Frozen specimens are usually coated in a special chamber attached to the microscope column and linked to its cooling system.
Cryopreservation does have a few disadvantages compared to conventional chemical fixation and drying. The freezing equipment is costly to buy and run. The specimen stage within the microscope has to be cooled to a very low temperature, and this reduces its mechanical stability. Movements of the specimen stage may also be restricted compared with operation at ambient temperatures. Finally, although the technique is rapid - it typically takes no more than 10 minutes - the resulting specimen is not permanent; once removed from the microscope it is of no further use. The overriding advantage of cryopreservation is that it allows objects to be examined in a fully hydrated state, and without any chemical extraction having taken place.
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Page authored by Paul Perkes and the ACEPT W3 Group
Department of Physics and Astronomy, Arizona State University, Tempe, AZ 85287-1504
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