Specimen preparation method freezing methods

Figure 4 Schematic representation of the TEM specimen preparation by freeze-fracture replica method.

Cellulose nitrate and cellulose acetate (CA) were among the first asymmetric, reverse osmosis membranes to be produced [121]. Plummer et al. [122] described 13 specimen preparation methods for observation of CA membrane structures. They pointed out the lack of contrast in epoxy embedded sections and that one of the best stains, osmium tetroxide, reacts with the polymer. Freeze fractured membranes were found by these authors to be of questionable value. In our experience, if care is taken, SEM study of fractured membranes can provide an informative view of the structure even though some structures collapse, and their sizes cannot be accurately determined. A method found acceptable was ultrathin sectioning of gelatin embedded wet membranes (TEM). The structure of CA membranes was shown by replication [123] and SEM [124]. 

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. 

In this chapter, we summarize the basic principle of TEM and three types of the typical specimen preparation techniques negative staining, freeze-fracture, and cryogenic methods. Then, TEM tomography showing the... 

There are some simple freezing methods that provide adequate preparation for some polymers. Cold stage microscopy of colloidal suspensions, microemulsions and liquids is possible by fast freezing and examination of the thin, frozen specimen in an EM. Talmon et ah [404] developed a rather interesting technique in which a thin sample is trapped between two polyimide films. The liquid layer is about 100 nm thick, while the films are about 40 nm thick. Film selection is quite important as the polyimide is more radia-... 

Manual methods of freeze fracture are often useful in providing specimens for study in the SEM. An example of a freeze shattering method was described by Stoffer and Bone [406] for comparison with microtomy results. Polymers immersed in liquid nitrogen were mechanically shattered with a hammer, mounted, vacuum pumped and sputter coated for observation. This simple method is useful if the materials cannot be sectioned. However, fine structural details are not conclusive when specimens are prepared by such methods. 

Walter and Bryant [414] described a method for freeze drying latex specimens in a home made vacuum system rather than a commercially available device (as was typical of the state of the art at that time). Later, a freeze drying/image analysis method using commercially available equipment was described [413]. Important details of that method included specimen preparation, placement onto a TEM (or SEM) grid, the hardware for the experiment and the metal coating. 

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 mean bulk densities of foams were determined by measuring the weight and volume of specimens that had a length of 50 mm prepared by freeze-cutting. The diameters were measured by a video camera. The volumes of some samples were estimated by a water displacement method. 

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]... 

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... 

TTHIS CHAPTER IS FOR INDIVIDUALS who have not done electron microscopy (EM) before, but it also contains some new information about specimen preparation that can benefit experienced electron microscopists. It covers the materials and methods necessary to do routine thin sections of embedded specimens however, it does not cover specialized EM techniques such as freeze-fracture, rotary shadowing, negative staining, scanning EM, and serial sectioning. Likewise, this chapter focuses mainly on embryos because a description of the fixation methods for all the various types of fly tissues would require an entire book. This chapter is divided into three main sections ... 

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