Freeze etching

Riehie U and Hdchii M 1973 The theory and technique of high pressure freezing Freeze-Etching Technique and Appiications ed E L Benedetti and P Favard (Paris Societe Frangaise de Microscopie Eiectronique) pp 31-61... 

FIG. 1 Freeze-etching image of a bacterial cell of (a) Desulfotomaculum nigrificans (bar, 100 nm). Atomic force micrographs of the S-layer proteins of (b) Bacillus sphaericus CCM 2177 and (c) Bacillus stearothermophilus PV72/p2 recrystallized in monolayers on silicon wafers. Bars, 50 nm. The insets in (b) and (c) show the corresponding computer-image reconstructions. 

Electron micrographs of freeze-etched preparations clearly demonstrated that S-layers completely cover the cell surface during all stages of cell growth and division [5,56,57]. 

FIG. 8 Electron micrographs of freeze-etched preparations of whole cells from (a, b) Bacillus sphaericus CCM 2120 exhibiting a square S-layer lattice or from (c, d) Thermoanaerobacter ther-mohydrosulfuricus Llll-69 carrying a hexagonally ordered S-layer lattice, (a, c) Native S-layer lattices (b, d) S-layer lattices after covalent binding of ferritin to carbodiknide-activated carboxylic acid groups of the S-layer protein. Bars, 100 nm. 

FIG. 18 Schematic drawing of a liposome with entrapped functional molecules, coated with an S-layer lattice, that can be used as immobilization matrix for functional molecules. Alternatively, liposomes can be coated with genetically modified S-layer subunits incorporating functional domains. (Modified from Ref. 59.) (b) Electron micrograph of a freeze-etched preparation of an S-layer-coated liposome (bar, 100 nm). 

Mirgall F., Breipohl W. and Bhatnagar K. (1979). Ultra-structural investigation on the cell membranes of the vomeronasal organ in the rat — a freeze-etching study. Cell Tiss Res 200, 397-408. 

Figure 5.14 Freeze-etch electron micrograph of glycolipid nanotubes from 24 1 nonhydroxy galactocerebrosides (21) cryofixed from room temperature in water. Bar = 250 nm. Reprinted from Ref. 64 with permission of the Biophysical Society.

Conventional electron microscopy (Devine et al 1972) and freeze-etch (Somlyo Franzini-Armstrong 1985) of VSMCs reveals that the jSR is separated from overlying PL by a 12—15nm cytosolic space that is traversed by electron-dense structures. These structures appear similar to the foot processes of cardiac and skeletal muscle (Franzini-Armstrong et al 1998). Indeed, there is striking structural similarity between these PL—jSR regions in VSMC and the diads and triads of cardiac and skeletal muscle (Franzini-Armstrong et al 1998). Moreover,... 

Verkleij, A.J., Zwaal, R.F., Roelofsen, B., Comfurius, P., KasteUjn, D. and van Deenen, L.L.M., 1973, The asymmetric distribution ofphosphoUpids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy. 

For freeze-fracture, a drop of the formulation containing 30% glycerol was deposited on a thin copper planchet and rapidly frozen in liquid propane. Fracturing and shadowing using Pt-C were performed in a Balzers BAF 310 freeze-etch unit. Other samples were simply deposited on a freshly cleaved mica plate and air-dried before shadowing as above. Replicas were examined with a Philips 410 electron microscope. 

These results were confirmed by an electron microscopy study using a freeze-etching replication technique (1 ). The aim of this technique was to conserve the real gel structure by blocking any diffusion processes in the gel sample by the freezing action of liquid nitrogen. The three-dimensional network is then recovered... 

Figure 3. Sclid-like gel network. Freeze-etching replication technique. (Reproduced with permission from Ref. 17. Copyright 1985 Academic Press.)...

Witchett CE Exposure of dog erythrocytes in vivo to phenylhydrazine and monomethylhy-drazine—a freeze-etch study of erythrocyte damage, p 33. Springfield, VA, US Department of Commerce, NTIS, 1975... 

Fig. 57a. A typical multilayered cloth made cf potassium tartaric amide 31b at pH 5. Its observed physical shape is fortuitous. (Negative stain, uranyl acetate 1%). b Freeze-etching erf a similar multilayer made of the sodium salt 31a. At higher magnification (below), the bilayer profiles become visible (Pt/C shadowed), c Fiber pattern of 31a. (Negative stain, uranyl acetate 1%). d Digitized area of the fiber bundle taken from (c). e Fourier transform of the input image from (d), as obtained by calculating the reciprocal space frequencies (x-y exchanged). Two intense spots yield a periodical pattern of 38.78 A [376]...

Fig. 8 Different views of T. foetus hydrogenosomes ( ) after field-emission scanning electron microscopy (FESEM) (a) and freeze-etching (b,c). An isolated hydrogenosome obtained from T. foetus observed by FESEM, where details of its surface can be seen, b A calcium deposit in the peripheral vesicle (asterisk) c shows that the peripheral vesicle (arrow) presents a smooth surface, distinct from the organelle body. Bars = 50 nm. (From Benchimol 2000)...

Fig. 17 Association of hydrogenosomes and microtubules in T. foetus, a Thin section of hydrogenosomes (H) in close proximity with microtubules (asterisks), b Freeze-etching after quick-freezing and rotatory shadowing showing a hydrogenosome in close association with cytoskeletal structures, probably microtubules. Bars = 200 nm. (Benchimol, unpublished)...

The interpretation of electron micrographs is often hampered by artefacts introduced during the sample preparation and etching. The technique of freeze etching is almost imperative for the study of swollen networks, where the structure depends often on the presence of diluent (see Chapter II, Section 3 and 4). 

Various electron microscopy techniques have been used to study the structures of whippable emulsions such as normal and cryo-scanning electron microscopy or transmission electron microscopy using various preparation methods such as freeze fracturing, freeze etching, etc. The literature is quite extensive, and only a few important papers will be discussed in this chapter. 

Raspanti, M., Alessandrini, A., Gobbi, P., and Ruggeri, A. (1996). Collagen fibril surface TMAFM, FEG-SEM and freeze-etching observations. Microscopy Res. Tech. 35, 87-93. 

Figure 14.20 TEM image of a freeze-etching preparation of a bacterial cell exhibiting an S layer with square (PA) lattice symmetry (scale bar = 100 nm) (reproduced by permission of Blackwell Publishing Ltd).

Further morphological and ultrastructural changes could be determined using thin sectioning and freeze etching ... 

The most thorough study of the formation of artificial casein micelles is that of Schmidt and co-workers (1977 1979 Schmidt and Koops, 1977 Schmidt and Both, 1982 Schmidt and Poll, 1989), who not only studied the properties of the casein aggregates but also attempted to relate them to the solution conditions under which they were formed. In the precipitation of calcium phosphate from solution, the means by which solutions are mixed together is of crucial importance Schmidt et al. (1977) described a method in which four solutions were pumped simultaneously into a reaction vessel while keeping the pH constant. As a result of careful, slow mixing, the reproducibility of the size distributions of particles, measured by electron microscopy on freeze-fractured and freeze-etched specimens, was very good. In the first series of experiments, the objective was to produce milk like concentrations of the most important ions while... 

Fig. 2.5. Taenia taeniaeformis (strobilocercus) freeze-etch preparation, x 23 800. The tegument contains numerous oblong vesicles (OV) and round, irregularly shaped inclusions. Microtriches have short, broad bases (B) and long, thin tips (T). Note also the double-bound intrusions in the base of the tegument (arrow) and channel (double arrows) within the tegument. The arrow at top right shows the direction of the external surface. (After Conder et al., 1983.)...

Freeze-etch characterization of the teguments of three metacestodes Echinococcus granulosus, Taenia crassiceps and Taenia taeniaeformis. Journal of Parasitology, 69 539-48. 

The surface layer of the fat globule may act as a catalytic impurity (e.g., when it contains mono-glycerides or di-glycerides with long-chain fatty acid residues) however, there is still some uncertainty as to whether this process actually occurs (see Walstra, 1995). Although concentric layers of apparently crystalline fat have been observed in electron micrographs of freeze-etched or freeze-fractured milk or cream samples (Buchheim, 1970 Henson et al., 1971), these observations could not be confirmed by other microscopy techniques. Noda and Yamamoto (1994) reported that it is thermodynamically more favorable for fat crystals to be located at the oil/water interface, rather than in the interior of the droplet, which may explain the presence of fat crystals at the membrane. 

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