Freeze fracture-etching Freezing methods

The formation of micelles, or colloidal particles, by block copolymers in organic solvents has been described and reviewed by Price [2881. The molecular weight of polystyrene was estimated from specimens prepared by spraying and evaporation for TEM. Freeze etching a drop of solution rapidly frozen with liquid nitrogen [289] was described (Section 4.9.4) where the solvent was allowed to evaporate and a replica produced of the fracture surface. Another method [290]... 

After quick freezing in liquid nitrogen and fracture, another etching method was used at — 90 °C for less than 1 min in a 1.33 x 10-4 Pa chamber. The etched sample was cooled to — 130°C and coated with platinum and carbon in the same vacuum chamber and transferred to the scanning electron microscope at — 130 °C. The observed structure was closer to reality than that obtained by the method previously described. This is called the cryo-SEM technique [32]. CryoSEM images are shown in Fig. 4 which presents the structural change by stretching [16]. 

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. 

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... 

The freeze fracture method has been used to study the structure of colloidal particles in water-oil mixtures stabilized by polymer emulsifiers. Microemulsions consisting of water, toluene and graft copolymer composed of a polystyrene backbone and a poly(ethylene oxide) graft were deposited onto a small gold plate, quenched in liquid nitrogen in equilibrium with its own solid phase [436]. Replicas of the fractured surfaces were washed with tetrahydrofuran, which showed the micellar structure of the copolymers. A similar method was used for the preparation of polystyrene polymer latexes for TEM study of the size distribution [437]. In this case, the frozen droplet was microtomed, with a cold knife at -100 to -120°C, etched for up to 90 s and then a platinum-carbon replica was prepared. Etching was found to be unnecessary and a potential cause of error. The remaining latex was dissolved away before examination of the replica. Such replicas can reveal the size distribution and structure of the latex particles. 

An excellent review of freeze fracturing is found in a chapter by McNutt [362]. The method has been described for preparation of biological membranes. An older, improved version of the method was described by Steere [363] and Moor et al. [364], while a review of the method and application to membranes was described by Branton [365]. The method involves cementing a 1-2 mm piece of the material onto a copper disk with gum arabic dissolved in 20% glycerin and then transferring the material to liquid Freon 22 (chlorodifluoromethane). The specimen is fractured with a cold knife in a vacuum evaporator, and it may be etched prior to replication. A replica can be cast on the surface of the hydrated... 

The most powerful method for the identification of the different phases and for the determination of their microstmctures is the transmission electron microscopy. This method requires a sample preparation using the freeze fracture and etching technique. With these measurements the microstructures of the various phases can be visualized directly. This is shown for three examples in Fig. 11.12 (Section 11.4.2) and in Figs. 11.30a and 11.30b [13,41, 97]. 

An alternative method is the freeze-etching technique [24, 25], which consists of producing platinum shadowed replicas of fractures created in rapidly frozen biological systems or lyotropic liquid crystals. The fracture orientation is somewhat haphazard, but occurs preferentially within the paraffinic level of the bilayers. These methods offer the possibility of viewing bilayers directly and preparing stereoviews. Beautiful pictures of liquid crystalline DNA, both cholesteric and hexagonal, have been obtained, but individual molecules are not easily resolved. However, the director distribution can be deduced from the images [26],... 

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