Cryo-fixation

Negata T. Electron microscope radioautography with cryo-fixation and dry mounting procedure. Acta Histochim Cytochem 1994 27 471 —489. 

Menco BP. 1984. Ciliated and microvillous structures of rat olfactory and nasal respiratory epithelia. A study using ultra-rapid cryo-fixation foUowed by freeze-substitution or freeze-etching. CeU Tissue Res 235 225-241. 

Micelles made of soaps break up and reform within milliseconds. The individual lifetime of a charged micelle is very short. Nevertheless, eicosane sulfate micelles have been trapped by rapid cryo-fixation at liquid nitrogen temperature and freeze etching. They can then be seen under the transmission electron microscope (TEM) (Figure 2.5.3). 

Figure 2.5.3 Electron micrograph of a cryo-fixated eicosane sulfate micelle.

When investigating diffusible ions it is necessary to consider that treatment of the specimen before cryo-fixation may affect the results. For example, dicing tissue into small pieces before cryofixation is not suitable for highly metabolically active tissues such as the heart. It has been shown that in this tissue redistribution of ions can occur in times as short as 10 s. Similarly, in other tissues any delay between harvesting and fixation gives the possibility of element redistribution. In addition it has been shown that treatments such as the use of local anesthetics when taking skin biopsy samples can also affect elemental content. 

Cryo-fixation followed by cryo-sectioning to a thickness of 5-10 pm and freeze-drying on thin (<1 pm) plastic films Small particles on filters analysis in situ on filter membrane. 

Laman ID, Kors N, Heeney JL, et al. Fixation of cryo-sections under HIV-1 inactivating conditions integrity of antigen binding sites and cell surface antigens. Histochemistry 1991 96 177-183. 

Although studies on potato structure had been carried out previously using conventional SEM, van Marie et al [70] used cryo-SEM to advantage in this high moisture material. The fracture planes of cooked and uncooked samples were used to help characterize cell wall adhesion in the four potato cultivars. In particular, differences in cell wall contact area and surface detail were used to explain the mealy versus firm textural attributes in the cultivars. By determining the parameters which contributed to the texture of potatoes, processing conditions and selection of suitable raw materials could be facilitated. Such information would be difficult to obtain with conventional, chemically fixed material due to the high moisture content and the inability of standard chemical fixation to retain carbohydrate-based structures. 

Physical fixation. Using cryo-ultramicrotomy, polymeric samples are cooled below their glass transition temperature and cut at these temperatures. Additional selective chemical staining of the ultrathin sections is helpful to enhance contrast. 

Figure 7 Preparation of Cryo-TEM specimen (a) fixation of the grid using a pair of tweezers, (b) removal of excel solvent, (c) close-up of the specimen grid, and (d) freezing of the specimen in a coolant.

It is well known that the particle shape, size, and distribution of a latex or emulsion control the properties and end-use applications. Many types of latex are manufactured with a controlled and sometimes monodisperse distribution of particle sizes. These polymer liquids are wet and sticky, making specimen preparation for microscopy very difficult. Because particle size and shape are so important to properties, the preparation must focus on not changing the particles as found in the fluid state. Preparation includes simple methods (see Section 4.1) such as dropping a solution onto a specimen holder, staining/fixation (see Section 4.4), microtomy (see Section 4.3), and special cryo methods (see Section 4.9). All microscopy techniques can be used for these studies. This section is meant to provide a brief survey of the types of microscopy applications that have been found useful in the evaluation of emulsions, latexes, and their use as coatings and adhesives. 

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