Transmission electron microscopy thin section preparation

III. Transmission electron microscopy of radish seeds Transmission electron microscopy (TEM) of radish seeds was done as listed below For TEM preparations, the specimens after fixation and dehydration, were embedded in Epon 812 resin (Luft, 1961). Thick sections (ca. 1mm each) were stained with 0.1% toluidine blue and observed with a Zeiss light photomicroscope. Thin sections, obtained with a diamond knife on a Supernova microtome, were sequentially stained at room temperature with 2% uranyle acetate (aqueous) for 5 min and by lead citrate for 10 min (Reynolds, 1963). Ultrastructural studies were made using a Philips CM12 transmission electrone microscope (TEM) operated at 80 KV. 

If the sample is thin enough, for example a specially prepared thin section, electrons may go straight through and be detected, as well as elastically and inelastically scattered electrons which are scattered in a forward direction. These form the basis of transmission electron microscopy (TEM). 

When transmission electron microscopy is used, the specimen has to be extremely thin (on the order of 0.1 to 10 pm) for the highly absorbable electrons to penetrate the solid and form an image. Preparing such a thin solid specimen with minimal artifacts is a very complicated problem that makes sample preparation a crucial step in the use of this technique. Therefore, a substantial part of this chapter (Section 9.3) is devoted to specimen thinning issues in TEM. 

Demonstier-Champagne et al. used atomic force microscopy (AFM) to observe microphase separation within cast films of PS-PMPS-PS/ PS-PMPS block copolymer mixtnre [43] that were nsed to compatibilize a blend of PMPS and PS. The fractnre snrface of blend films with the block copolymer incorporated show a far finer dispersion of particle sizes than those without. Matyjaszewski et al. studied PMPS-PS thin films by SFM (scanning force microscopy) and TEM (transmission electron microscopy) and Fig. 8 shows a TEM picture of a thin section of a film which was prepared by slow evaporation from THE, which is slightly selective for the polystyrene block [73]. 

Electron microscopy samples were prepared by techniques similar to that of Kato (8). Film samples were fixed with OSO4, potted in polymethyl methacrylate and then microtomed into thin sections ( 100 nm) for transmission examination using a Philips 100B electron microscope. 

Samples for transmission electron microscopy were prepared in the following manner. A 50/50 blend was potted in an acrylic resin (London Resin Co. Ltd.) and subsequently microtomed in thin films of approximately 600 angstrom thickness and transferred onto electron microscope grids. These sections were then doped with iodine vapor (stained) and were ready for transmission electron microscopy. The blends of other compositions were not examined in this manner for reasons discussed later. 

Despite these rapid developments, transmission electron microscopy could not possibly contribute significantly to coatings and polymer research until commercial machines became available. The first Siemens microscope was marketed in 1938 (35). followed the next year by a more advanced unit. In this country, RCA marketed its first unit in 1941 (36). Even with commercial equipment available, many other problems had to be overcome. The main difficulty was that of sample preparation. Sections had to be extremely thin to be penetrated sufficiently by the electron beam. Often contrast was not sufficient to form a suitable image. In 1939, the shadowing technique was developed to enhance contrast... 

When higher spatial resolution examination of specimens is needed, transmission electron microscopy (TEM) is utilized. Here a specimen thin enough to transmit the electron beam is required. This can be achieved by thinning the specimen directly or mounting it and thinning a cross-section. If small precipitates are the feature of interest, they can sometimes be examined by preparing carbon extraction replicas from the specimen. 

In the emulsion prepared composites, most of the polymer was observed (3) to deposit in untreated leather (shown schematically in Fig. 4, insert a) in the space around individual fibers, largely within the confines of fiber bundles (insert f). In this way, experimental panels or even full sheepskins (38) increased in thickness (insert b) without much change in area. The coarse polymer domains so formed (2 to 50 pm) were in marked contrast to the appearance of polymer depositing within the individual fibers of cotton (7,14) and wool (9), by a variety of polymerization techniques (1,3,7,9,14). In these systems domains as small as 20 A (9) but usually 0.1 to 3 pm (3,14) were routinely observed by transmission electron microscopy (TEM). Removal of soluble polymer by benzene extraction from the cattlehide composites (insert c) reduced the density (Table 1) while retaining the expanded volumes (1). A special feature of emulsion polymer deposition in cattle-hide (not found for sheepskins or other thinner, looser leathers) (38,40) is shown in insert e, where the polymer deposited only in layers near the outer surfaces (comprising 25 to 60% of the cross section) (1), leaving the center section polymer free. A thin,... 

Two preparative techniques have been proven to be invaluable for the microscopic examination of bonded pol3nneric materials the "Ebonite" and "Gyro" methods. By using these methods to harden soft and rubbery materials, it is possible to cut thin sections for either optical or transmission electron microscopy. 

Sample preparation is often the most time-consuming aspect of transmission electron microscopy. It is relatively straightforward if the sample is a homogeneous metal or alloy and if a thin foil can be produced from any part of it. Problems arise if areas near to the surface or to a boundary between two dissimilar materials are required to be analyzed. Normally a thin section of material 1 mm in diameter is produced mechanically, which is then polished to the minimum practical thickness before being finally thinned to electron transparency. The final stage is carried out using either electrolytic or ion-thinning methods. When... 

Preparation of Thin Sections of Drosophila for Examination by Transmission Electron Microscopy... 

Figure 3.3. Electron micrograph (replica) of a graft-type high-impact polystyrene with polybutadiene as the rubbery component (Keskkula and Traylor, 1967). In the absence of agitation, phase inversion does not occur, and an interwoven cellular structure results, with polybutadiene remaining as the continuous phase. The specimen was prepared for electron microscopy by exposing a polished surface to isopropanol vapor, which preferentially swells the polystyrene phase a double replication technique was then used. The reader should compare the results obtained by this technique to results obtained using thin-section transmission techniques (see, for example. Figure 3.2).

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