Preparation of specimens for microscopy

Phase contrast also appears in electron microscopy. The passage of the wavefront through a specimen causes a phase shift which can be converted into image intensity by interference between the retarded wave with another wave. Components which cause phase contrast have been tried in the transmission electron microscope and do work, but they are impractical. 

A general rule in microscopy is that the preparation of a specimen takes a much longer time than the actual examination. The difficulty in preparing good samples is substantial. 

Polymer specimens to be examined by SEM are coated with a thin layer of electrically conductive material. 

Treatment of polydienes with osmium tetroxide (OSO4) was introduced by Andrews (1964) and Andrews and Stubbs (1964). A later development by Kato (1965 1966 1967) showed that OSO4 was able to stain the rubber component in polymer blends. Osmium tetroxide adds to the double bonds and the density of rubber phase is increased. Since it crosslinks the rubber polymer by reacting with two double bonds located in two different polymer chains (Fig. 11.10), the rubber is fixed and sectioning can be carried out without smearing. Osmium tetroxide 

Ruthenium tetroxide is a stronger oxidizing staining agent than OsO, which enables RUO4 to react also with phenyl groups, e.g. in polystyrene. Furthermore, ruthenium tetroxide has been used to stain polyethylene and polypropylene in addition to the list of polymers stained by OSO4. 

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Aluminium is widely applied for decorative and protective requirements, while cadmium , zinc and titanium have been applied to ferrous materials chiefly for their protective value. The method finds particular application in the plating of high-tensile steels used in aviation and rocketry, car fittings and lamp reflectors, and gramophone record master discs, as well as in the preparation of specimens for electron microscopy and in rendering insulated surfaces electrically conducting, e.g. metallising of capacitors and resistors. 

Finally, photomicrography is the culmination of the preparation of specimens for optical microscopy. This is a very technical area requiring proper illumination (41-43), focusing, choice of films, as well as exposure and appropriate film development. This critical area of microscopy should see continued technological innovations as much of photomicrography is being computerized (44,45). This effort is witnessing the concomitant... 

J. Kruse did not elaborate on adhesive systems or the preparation of specimens for the study of finished polymeric products. The reason for this, he stated, was that there were too many objects for this type of investigation. Indeed there are, but perhaps some principles and guidelines for the application of microscopy to adhesive systems would be of value to the adhesive chemist who would like to know more about his product. 

Protocol 13.5 Preparation of Specimens for Scanning Electron Microscopy, 240... 

Protocol 13.6 Preparation of Specimens for Transmission Electron Microscopy, 242... 

In lesions not treated with collagenase, the matrix appeared to be unaffected, since no differences were observed between the matrix in the lesions and the underlying dentin, when the latter had been demineralized during the preparation of fhe specimens for microscopy. 

Ultramicrotomy is basically the same type of method as microtomy for preparing soft specimens for light microscopy. However, ultramicrotomy can be used to section a specimen to the 100 nm scale. It is commonly used to prepare polymeric or biological TEM specimens. 

Preparation of samples for the observation under TEM is more tedious and exacting than that used for SEM. The specimens have to be hardened and stained with Br, OsO, or RuO, microtomed into ca. 20 slices, mounted on a grid and polymeric film support and measured. Frequently, the SEM and TEM methods are being used in parallel. In all cases microscopy is considered but one method of characterization of polymer blends [Karger-Kocsis and JGss, 1987 Kyotani and Kanetsuna, 1987 Hsu andGeil, 1987]. 

Preparation of samples for analysis resembles that used to prepare samples for electron microscopy. In general, preparation consists of polishing small specimens and mounting them on a planchet. A common planchet consists of a cylinder of vitreous carbon with polished ends. The planchet must provide a conductive pathway to drain the charge imparted by the impact of the primary ion beam. Without some means to dissipate the charge, the sample will become charged, which can deflect the primary ion beam and can interfere with the extraction of secondary ions. 

Electron microscopy employs completely different techniques for preparation of specimens and allows observation of fine cell and tissue structures under very high magnification. A glass or diamond knife is used with an ultramicrotome to produce ultra-thin sections less than 0.001 mm thick. In some situations, extracted tissues can be frozen and sliced immediately, then stained and analyzed without lengthy tissue processing. Such tissue sections can deteriorate rapidly but are used to quickly analyze biopsies during surgery or in applications that do not require a detailed structural analysis. 

The specimen preparation methods used for microscopy of polymers involves the use of many toxic chemicals as well as the use of instruments which can be radiation hazards. It is well beyond the scope of this text to provide the information required for the proper and safe handling of such chemicals and instruments and the researcher is... 

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