Theory of freezing tissue

Freezing for morphological studies must be done very rapidly to minimize the size of the ice crystals. Any ice crystals that occur in the cells will break open the cellular organelles and the plasma membrane and destroy the morphology. 

There are four basic ways of freezing tissue or cells (Fig. 4.1). The most rapid freezing, vitrification, which holds each water molecule in place without the presents of ice crystals (Fig. 4.1, pathway No. 1). This method is difficult to perform and involves slamming the tissue onto a silver block at liquid helium (-214 C) temperature. For light microscopic immunocytochemistry, this method is not practical. 

The third method, dry ice (Fig. 4.1, pathway No. 3), is readily available in most labs and does not require much additional equipment. Dry ice melts at -56°C. Freezing done at this temperature is warmer than isopentane which has slower heat transfer out of the tissue. Dry ice is used as block or as pulverized with a hammer. The tissue contacts the dry ice and freezes in several seconds, which is relatively slow freezing. As such, the process generates ice crystals in the cells and can reduce the quality of the cellular detail seen in the microscope (Rosene et al., 1986). For 

Vitrified water, no ice crystais generated in cells, and no damage to cellular organelles. 

Isopentane cell, very small ice crystals in cells. Minimal damage to cellular organelles. 

---

The ideal solution to microanalysis would be simply to freeze the plant material rapidly to the temperature of liquid nitrogen and then section it while it is still frozen on a cryotome. The frozen sections would then be transferred to a cold stage in a TEM and analyzed. In theory, no ion movement will take place and analysis at the high resolution of TEM should be possible. Indeed, this is a useful technique for liver, kidney, and soft animal tissues, but unfortunately it is almost impossible to cut tough plant material, and maintain the sections in a reasonable state for analysis (2). Even if this problem could be overcome unstained tissues will be difficult to visualize in TEM. 

---