Cryo ultramicrotomy

Specimens for (S)TEM have to be transparent to the electron beam. In order to get good contrast and resolution, they have to be thin enough to minimize inelastic scattering. The required thin sections of organic materials can be obtained by ultramicrotomy eitlier after embedding into suitable resms (mostly epoxy- or methacrylate resins [H]) or directly at low temperatures by cryo-ultramicrotomy [12]. 

FIGURE 20.4 (a, b) Height and phase images of rubber blend of natural rubber (NR) and butylene rubber (BR) (50 50), which were taken immediately after cryo-ultramicrotomy. (c, d) Height and phase images of the same sample location as in (a, b) taken 12 h later. 

A second approach to postembedding electron microscopic immunocytochemistry is the use of special aqueous resins, while these resins are still hydrophobic, they tolerate some aqueous material in the tissue. Examples of these resins are LR White or Lowicryl K4M. Another approach with aqueous embedding is to freeze tissue in buffer very rapidly, and to section with a cryo-ultramicrotome. Cryo-ultramicrotomy is a technique requiring considerable training and cannot be used by the novice. 

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. 

Ultramicrotomy (including cryo-ultramicrotomy) is a standard method for the preparation of ultrathin or semithin sections, as well as very flat surfaces for various microscopic investigations. Improvements in preparation techniques over the last few decades have demonstrated that thin sections of different materials that are free from artifacts can be successfully prepared for EM investigations. Therefore, successful sectioning now depends primarily on the experience of the experimentalist rather than on the instrumentation used [14]. 

The second and main disadvantage of the TEM is that it can be used only on thin samples, less than 1 pm thick and preferably 50-100 nm thick. These ultrathin samples can be cut from bulk material using (cryo-)ultramicrotomy with special glass or diamond knives. 

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