Specimen preparation method staining

Specimen preparation and staining methods as described previously (15). 

Cellulose nitrate and cellulose acetate (CA) were among the first asymmetric, reverse osmosis membranes to be produced [150]. Plummer et al [1511 described 13 specimen preparation methods for the observation of CA membrane structures. They pointed out the lack of contrast in epoxy embedded sections and that one of the best stains, osmium tetroxide, reacts with the... 

There are multiphase polymers where OM and SEM techniques cannot fully describe the microstructure due to a combination of small particle size (less than 0.5 /xm) and good adhesion between the dispersed phase and the matrix. Additionally, broad particle size distributions are often encountered, and in these cases a combination of techniques is required to describe the microstructure. TEM requires ultrathin specimens, about 50-500 nm or less in thickness, which are prepared by film casting or ultrathin sectioning. Films formed by casting or dipping methods provide a much easier specimen preparation method than ultrathin sectioning of bulk plastics. However, a major question in such studies is always whether the microstructure is the same as in bulk polymers of industrial interest. Specific stains are often required to provide contrast between the dispersed phase and the matrix pol)m[ier. 

Cellulose nitrate and cellulose acetate (CA) were among the first asymmetric, reverse osmosis membranes to be produced [121]. Plummer et al. [122] described 13 specimen preparation methods for observation of CA membrane structures. They pointed out the lack of contrast in epoxy embedded sections and that one of the best stains, osmium tetroxide, reacts with the polymer. Freeze fractured membranes were found by these authors to be of questionable value. In our experience, if care is taken, SEM study of fractured membranes can provide an informative view of the structure even though some structures collapse, and their sizes cannot be accurately determined. A method found acceptable was ultrathin sectioning of gelatin embedded wet membranes (TEM). The structure of CA membranes was shown by replication [123] and SEM [124]. 

Despite the high power of the electron microscope, which achieves resolutions of the order of 3 A, i.e. molecular resolution, the reliability of the method has been questioned in the past. These doubts especially involve the possibility of electron-beam damage and the shortcomings of the preparation techniques. The latter involve chemical fixation, dehydration and staining which might interfere or even disturb the native structure. The development of adequate preparation methods is therefore of utmost importance for high resolution electron microscopy of biological specimens. This has been realized from the early days of electron microscopy. 

Conventional methods used for the preparation of biological material both for light microscopy and EM consist of fixation, dehydration, and embedding. These procedures result in increased permeability of the cell membrane with resultant loss or redistribution of mobile intracellular elements. Sections are then stained, resulting in the deposition of elements onto the specimen so that the resultant spectrum is not representative of the elemental composition in vivo (Figure 2). In consequence, such preparation methods are rarely used when the specimen is intended from the outset for microanalysis. 

In this chapter, we summarize the basic principle of TEM and three types of the typical specimen preparation techniques negative staining, freeze-fracture, and cryogenic methods. Then, TEM tomography showing the... 

There are two methods for the specimen preparation by negative staining. The researchers are recommended to test both methods to diminish the artifact that arises from the specimen preparation. It is also better to test different stain methods to evaluate the interactions between the object and stain. 

Specimen preparation for TEM generally in-voles the formation of a thin film of the material less than 100 nm thick. The methods used for this preparation depend upon the nature of the polymer and its physical form. In the case of thick or bulk specimens, microtomy is generally used. In the case of solutions, powders or particulates, simpler methods can provide a thin, dispersed form of the material. Three types of simple preparations will be described later in this section dispersion, disintegration and film casting. The more complex methods such as microtomy, replication, etching and staining will be described in other sections of this chapter. 

Table 4.4 includes functional groups and polymers and their respective etchants. Chemical etching, such as with solvents and acids, and ion and plasma etching are conducted in order to reveal selectively structures in polymers that may not be observed directly. In all these methods, interpretation of the structures formed can be more difficult than specimen preparation. Accordingly, the etching methods are best used to complement other methods, such as microtomy, fractography and staining. Controls are essential to any experiment of this type, but, with care, the structures of semicrystalline polymers and polymer blends may be observed. 

The major structural unit of interest in emulsions, microemulsions, colloids and latexes is the particle. It is well known that the particle shape, size and distribution of a latex controls the properties and end use applications. Many latexes are manufactured with a controlled and sometimes monodisperse distribution of particle sizes. Polymer liquids, in the form of emulsions and adhesives, are wet and sticky, and therefore specimen preparation for electron microscopy is very difficxilt. As a result of the importance of the determination of particle size distribution, microscopy techniques have focused on specimen preparations which do not alter this distribution or which alter it as little as possible. Methods have included special cryotechniques (Section 4.9), staining-fixation methods (Section 4.4), microtomy (Section 4.3) and some simple methods (Section 4.1) such as dropping a solution onto a specimen holder. This section is meant to provide a brief survey of the types of microscopy applications which have been foimd useful in the evaluation of emulsions and latexes. 

The purpose of the sample preparation is to obtain a contrast-rich representation of the True structure. If contrast is not naturally present in the specimen, it may be created by staining, i.e. adding a substance with a characteristic colour or high (electron) density selectively to a specific phase. The contrast may also be achieved by selective etching, leaving a topography indicative of the phase structure. It is possible, however, that the preparation method used may produce artificial structures. 

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