Shadowed carbon replica

Fig. 4. Transmission electron micrograph of shadowed carbon replica of freeze-fractured cells of A. eutrophus showing plastic deformation of PHB...

Question (3). A shadowed carbon replica of an etched sample contains little or no polymer. If it contains the information required, it is an excellent way of producing a less sensitive sample. Stains by definition increase the contrast of the specimen, but do not necessarily increase the stability of the image. Some stains, such as iodine, are rapidly driven off by irradiation in vacuum while others are completely stable. 

Fig. 4.21 Portion of a banded spherulite of linear polyethylene, also showing a spherulite boundary, as revealed by one hour etching using 2% w/w potassium permanganate in concentrated sulfuric acid as described by Naylor and Phillips [275]. The micrograph is of a chromium shadowed carbon replica studied in the TEM where each disk-like object in the bands is composed of lamellae nucleated at that point by a screw dislocation. (From Phillips and Philpot [274] used with permission.)...

Fig. 11.6 Examples of silver halide emulsions. [Note a and c are scanning electron micrographs of AgX crystals b is a transmission electron micrograph of gold-shadowed carbon replicas of octahedral AgX crystals with the substrate fixed out]. Micrographs reproduced with courtesy of Eastman Kodak Company...

Figure 10. Electron micrograph of a platinum shadowed carbon replica of a tin oxid film rubbed with 1 jum diamond paste (after reference [41], [42]).

Fig 1. Electron micrograph of a platinum/carbon replica prepared by the fast-freeze, deep-etch, rotary-shadow replica technique printed in reverse contrast. Cell walls of onion parenchyma have an elaborate structure with many thin fibres bridging between thicker cellulosic microfibrils. Scale bar represents 200nm. 

Ghadially EN, Lock CJL, Lalonde JMA, Ghadially R. Platinosomes produced in synovial membrane by platinum coordination complexes. Virchows Arch., B, Cell Pathol. 1981 35 123-131. Beretta GL, Righetti SC, Lombardi L, Zunino F, Perego P. Electron microscopy analysis of early localization of cisplatin in ovarian carcinoma cells. Ultrastruct. Pathol. 2002 26 331-334. Ruben GC. Ultrathin (Inm) vertically shadowed platinum-carbon replicas for imaging individual molecules in freeze-etched biological DNA and material science metal and plastic specimens. J. Electron. Microsc. Tech. 1989 13 335-354. 

ELECTRON MICROSCOPY. The samples were prepared for electron microscopy as follows. The test materials with the adsorbed protein were placed in a vacugm evaporator (2 ). The specimens were dehydrated in vacuo (10 Torr) for 60 min., shadowed with tungsten, and carbon coated. The plastic substrates were dissolved in an appropriate solvent, either 1,2-dichloroethane (EDC), or N,N-dimethylacetamide (DMAC). The carbon replica, mounted on an microscope grid, was examined in the electron microscope. For observation, we used a Philips 300 electron microscope operated at 80 kV with a 50 micron objective aperture. 

The removal of direct carbon replicas is dependent upon the polymer. Boiling xylene vapor was used to remove drawn PE from replicas [296] in work on drawn polymer morphology. Hobbs and Pratt [297] described a direct carbon replica method for replication of a PBT impact fracture surface by evaporation of platinum at 20° and PBT removal in hexafluor-oisopropanol (HFIP). Latex film coalescence in poly(vinyl acrylate) homopolymer and vinyl acrylic copolymer latexes was studied using direct replicas [298]. As the latex films have a low glass transition temperature, they were cooled by liquid nitrogen to about -150°C in the vacuum evaporator and shadowed with Pt/ Pd at 45° followed by deposition of a carbon support film at 90° to the specimen surface. The latex films were dissolved in methyl acetate/ methanol. TEM micrographs of the latex films show the difference between films aged for various times (Section 5.5.2). 

Carbon replicas [427] are formed by the evaporation of a thin layer of carbon in a vacuum evaporator. Metal shadowing, at an angle of 20 to 45° to the specimen surface is performed while the specimen is in the evaporator. The highest resolution direct replica material is carbon/platinum (C/Pt). After evap-... 

Shadowed platinum Platinum carbon replica carbon replica preparation removing in solvent... 

Materials used for replication, such as carbon, are usually so transparent to electrons that small thickness variations produce no observable contrast. It is customary, therefore, to enhance contrast by shadowing the replica with a heavy metal, which produces marked variations in contrast. In addition, shadows help accentuate height differences in the specimen and open the way to obtaining quantitative information about the surface topography via stereomicrometry. 

The samples of BR-reconstituted vesicle (100 pg BR/1.5 mM lipid) were quick-frozen using the technique of Heuser [23], and fractured in a Balzers BAF 400D freeze-fracture apparatus (Balzers, Liechtenstein). The replicas were obtained by rotary shadowing with platinum/carbon of ca. 7 nm thick and carbon of ca. 25 nm, and then examined in a Philips CM200 Ultra Twin electron microscope at 200 kV. 

Three different techniques, namely FFEM [20, 22], Cryo-Direct Imaging (Cryo-DI) [104] and freeze-fracture direct imaging (FFDI) [105], can be used to visualise the structure of micro emulsions. In FFEM the samples are prepared in a protected fashion in a sandwich. They are then rapidly frozen, fractured, shadowed with metal, and replicated with a thin carbon film. The replica of the fractured surface, the morphology of which is controlled by the sample s microstructure, is then studied by a TEM. In contrast to FFEM, in Cryo-DI thin films of the sample are rapidly frozen but immediately, without replication, trans-... 

---