Staining of Thin Sections

The mode of action of EM is based on the interaction of an electron beam with the atoms of the material under study, with contrast generally arising from the production of secondary or back-scattered electrons (in SEM) or the scattering of electrons by the spedmen (in TEM). Thus, it is necessary to have a sufifident intrinsic atomic mass contrast and an appropriate surface topography for TEM and SEM investigations, respectively. 

Polymers are comprised mainly of low-molar-mass elements such as carbon, hydrogen, and nitrogen, and consequently the atomic mass contrast is very low. 

Therefore, when applying conventional TEM to heterophase polymers, the selective staining of one or more of the phases present in the polymer blends is required, using heavy-metal compounds (e.g., osmium tetroxide, ruthenium tetroxide, uranyl acetate) [45-48]. Such staining can be performed either on the bulk material prior to sectioning with an ultramicrotome, or on the films. As noted above, one advantage of staining the bulk sample is that it becomes hardened and can then be sectioned at room temperature. 

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Roland JC, Vian B. General preparation and staining of thin sections, in Electron Microscopy of Plant Cells (Hall JL, Hawes C, eds.), Academic Press, San Diego, CA, 1991, pp. 2-66. 

Sato T. A modified method for lead staining of thin sections. J Electron Microsc 1967 16 133. 

RAPID STAINING OF THIN RESIN SECTIONS IN MICROWAVE OVEN... 

Conventional bright-field TEM observations of polyolefins often require contrast enhancement, usually by staining with Ru04 or other suitable markers [17]. These accumulate in the amorphous phase, at lamellar surfaces and in cavities, and differential staining can reveal the phase distribution in blends. Staining also hardens the specimens, facilitating preparation of thin sections at room temperature (cryo-sectioning is required for unfixed polyolefins). 

Embedding and staining of tissue sections Previously stained animal tissues may be embedded and sectioned for microscopic examination of single tumor cells in 4-10 [xm thin sections. Alternatively, tissues can be embedded first such that 6-galactosidase and alkaline phosphatase activities are preserved, and subsequently stained for expression of the lacZ, ALP, or both reporter genes (see above) (Lin et al., 1990a, b). 

Microscopical examination should start in the quarry where samples of each of the varieties of limestone, sandstone, shale, etc., are collected, layer by layer, by a geologist or someone with an adequate knowledge of the quarry. An assumption of the mineralogy of most quarried materials, as well as many of the industrial byproducts, is commonly questionable. Representative portions of each rock variety are sent off, if necessary, for thin sectioning (see partial list of professional companies in Table 11-3). Some of these companies will also stain the thin sections as directed. Another portion of the rock is crushed in the plant laboratory with a mortar and pestle or other suitable crushing device, and sieved to produce a 45- to 75- im fraction for examination in a powder mount, using, at first, a liquid with a refractive index of approximately 1.542. Samples of nondeposit materials, such as slag, fly ash, bottom ash, rice husk ash, clay catalyst, etc., are examined similarly. 

In electron micrographs of thin-sections of organisms the cell wall appears as a triple-layered structure, 60-100 angstroms thick (outer membrane) separated by 1 or more layers of variable electron density from the plasma membrane, but showing in addition 1 or more layers of definite order of structure extracellular to the outer membrane. Electron micrographs of negatively stained, freeze-etched or shadowed organisms show a smooth surface structure. 

Characterization of the microstructure of high speed spun polyester fibers has been demonstrated using combined SEM of bulk peeled fibers and fiber surfaces, OM of thin sections and TEM of sections both stained and unstained. 

Walters and Keyte [82] first observed dispersed particles in blends of rubber polymers by phase contrast optical microscopy. Marsh et al. [83] studied elastomer blends by both optical phase contrast and TEM. Electron microscopy was applied to study blends of natural rubber, styrene-butadiene rubber (SBR), cis-polybutadiene (PB) and chlorobutyl rubber [84]. It became obvious that both hardening of the rubber and staining were necessary for producing sections with contrast for TEM. Today, the most common methods of observing multiphase polymers are by phase contrast OM of thin sections, TEM of stained ultrathin sections and SEM of etched or fractured surfaces. 

Pathology is employed in biomedicine to identify and characterize the disease state of human tissue, using well-established optical microscopy techniques [79]. Often, disease-state determination involves the analysis of thinly sectioned, stained biopsied tissue in order to visualize various disease-specific pathologic markers. Although these examinations are typically made by highly trained practitioners, the potential exists to make the determinations more quantitative and less reliant on subjective observations by integrating the efficacy of vibrational spectroscopy with optical microscopy. This section describes... 

Fig. 4.13. Sample of LDPE, having a crystallinity

The preparation of thin sections by ultramicrotomy, generally after special fixation and staining procedures has been performed. Investigations are carried out by conventional TEM, HEM, or AFM. 

The ultrastructural features of chloroplasts in leaves of Columbia wild-type Arabidopsis thaliana (L) Heyn and mutant strains JB67 and LK3 isolated therefrom have been examined by thin section electron microscopy. Leaves from 3 week old plants grown at 22°C under standard conditions were processed in tendem. Tissue was fixed in 4% gluterdi-aldehyde, sectioned and stained with uranyl acetate and lead citrate. The sections were examined by transmission electron microscopy and morphometric features of thin sections through chloroplasts were analysed (see Table 1). 

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