Abstract
Four topics obtained by a cryofixation technique for electron microscopic research are introduced. 1. The cell ultrastructures of high pressure and/or liquid propane frozen twenty species including 2 higher plants, 14 algae, 2 fungi and 2 animals were examined and their definitions on electron micrographs were compared. 2. In Euglena gracilis, the number of chloroplast envelopes has been considered to be three according to the electron microscopic specimens by conventional chemical fixation. However, this idea turned out to be reexamined by high pressure frozen samples preserving membrane structure. By the latter, a lipid bilayer structure of plasma membrane and membrane of Golgi cisterna was clearly visualized. On the other hand, the unit-membrane of mitochondria and chloroplast envelopes was not seen as a bilayer. 3. Among oleaginous microalgae, Botryococcus braunii has attracted attention for obtaining a renewable biofuel, because it accumulates especially large amounts of hydrocarbons around cell surfaces. However, this surrounding oil interfered with electron microscopic preparation using a chemical fixation. In contrast, we optimized high pressure and/or liquid propane freeze-substitution methods for B. braunii that showed the best definitions among examined species (except 2 animals) listed above. As a result, we were able to progress the cytological researches on lipid synthesis and secretion in B. braunii. 4. The COPⅡcoat of coated vesicles and clathrin coated pids at plasma membrane were especially well-defined in the specimens prepared by cryofixation technique.
