Before viewing the specimen under the electron microscope, there are different techniques done as sample preparation. The techniques to be done for a specific specimen would depend on the type of specimen and the type of analysis desired.
Some of the techniques commonly done for electron microscope specimen are cryofixation, fixation, embedding, dehydration, sectioning, staining, freeze fracture or freeze etch, ion beam milling, and conductive coating.
The first type of preparation is called cryofixation. Cryofixation is a method that involves freezing a specimen so rapidly, to liquid nitrogen or even liquid helium temperatures. This results to the water forming into a vitreous or non-crystalline ice form. This preserves the specimen in an instant in its solution state. An entire field called cryo-electron microscopy has branched from this technique. It is now very much possible to observe any biological specimen close to its native state upon the development of cryo-electron microscopy of vitreous sections or CEMOVIS.
The second specimen preparation technique is the fixation technique. In this method. Chemicals such as glutaraldehyde and osmium tetroxide are used. The glutaraldehyde helps for hardening the specimen and the osmium tetroxide is responsible for staining the lipids black for viewing purposes and preserving the sample to make it appear more realistic.
Another technique to prepare specimen samples is called dehydration. With this electron specimen preparation technique, chemicals like acetone and ethanol are being used. Either of these two chemicals can be used to replace water to dehydrate the specimen. These organic solvents have dehydrating properties.
In another technique called embedding, resins are being used. In embedding technique, infiltration of the tissue with a resin such as araldite or epoxy for sectioning is done.
A type of electron specimen sample preparation is the sectioning method. With this kind of technique, there is a deliberate production of thin slices of specimen. This thin slice of specimen should be semitransparent to the electrons. These thin slices can be acquired by cutting with the help of an ultramicrotome with a diamond knife. This produces very thin slices of specimen. Glass knives are also used because they can be made in the laboratories. Glass knives are also a lot more cheaper.
Staining is another type of way to prepare electron microscope specimen. In staing, the use of heavy metals such as lead, uranium or tungsten is done. These heavy metals functions to scatter imaging electrons and thus give contrast between different structures. It is done because many materials, especially biological materials, are almost transparent to the electrons.
Freeze etch is a preparation method useful particularly in examining lipid membranes and their incorporated proteins in face on view. The fresh tissue or cell suspension is frozen rapidly or cryofixed, then fractured by breaking or by using a microtome while maintained at liquid nitrogen temperature.
In a technique called Ion Beam Milling, thinning of samples is done until they are transparent to electrons. This is done by firing argon ions at the surface from an angle and sputtering material from the surface. At times, gallium ions are used. Ion beam milling is also being used for cross-section polishing before SEM analysis of materials that are difficult to prepare using mechanical polishing.
Lastly, in a process called Conductive coating, a very thin coating of electrically-conducting material is covered on the specimen sample. This coating of the sample is done by depositing either by high vacuum evaporation or by low vacuum sputter. Its purpose is to prevent the accumulation of static electric fields at the specimen due to the electron irradiation required during imaging. Sample coatings can be in gold, gold/palladium, platinum, tungsten, and graphite. They are especially important for the study of specimens with the scanning electron microscope because they coat the specimen so the electron beam can bounce off and an image can be formed.
