Freeze-fracture method

Rash JE, Hudson CS (eds) Freeze Fracture Methods, Artifacts and Interpretations. New York, Raven Press, 1979. 

Belkoura, L., Stubenrauch, C. and Strey, R. (2004) Freeze fracture direct imaging A new freeze fracture method for specimen preparation in cryo-transmission electron microscopy. 

Cryogenic SEM See Freeze-Fracture Method, Electron Microscopy. 

Freeze-Fracture Method A sample preparation technique used in electron microscopy in which specimens are quickly frozen in a cryogen and then cleaved to expose interior surfaces. In some techniques, the sample is then observed directly in an electron microscope equipped with a cryogenic stage in other cases, the cleaved sample is coated with a metal coating to produce a replica, which is observed in the electron microscope. See also Electron Microscopy. 

Moreover, removal of a membrane is necessary for observing inside of the cells. By using the sputtering technique, the membrane can be removed and then 3D distribution of each ion can be obtained. Sandwich freeze-fracture method is often employed to remove the membrane before TOF-SIMS measurement [62]. 

The latex beads serve as spacers in the sandwich freeze-fracture method of sample preparation (see later). Latex beads with approximate diameter of 11 xm (Duke Scientific, Palo Alto, CA, USA) were used in the study. 

Fig. 6.2. Simple tools used for fracturing cell cultures grown on the silicon substrate under liquid nitrogen in sandwich freeze-fracture method. Reproduced from ref. (28).

Several polymer studies have been reported where the specimens were prepared by freeze fracture techniques. A modification of the freeze fracture method was used by Singleton et ah [432] in the preparation of plasticized PVC. The sample was notched, cooled and fractured and then immediately replicated with platinum-carbon. Replicas were stripped after warming to room temperature. The authors noted that the preparation was not highly reproducible, perhaps due to nonuniform cooling of large specimens. The results of such a study must be compared with other characterizations for accurate analysis. 

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. 

Several polymer studies have been reported where the specimens were prepared by freeze fracture techniques. A modification of the freeze fracture method was used by Singleton... 

In Figure 6 (a) the fracture surface of HDPE-2, which is characterized by the freeze fracture method, is shown. HDPE-2 has almost 3 times higher ESCR in comparison to HDPE-1. The difference of fibrillation at the crack surface, by observation of cross section of an ES crack, is explained above in terms of the population of fibrillation. This feature is also observed in Figure 6 (b). 

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