High-pressure freezing/freeze substitution of plant cells
Electron microscopy of chemically fixed plant tissues has lead to important insights into the relationship between structure and function of plant cells. However, the slow rate of chemical fixation (seconds to minutes) potentially permits numerous artifacts to be induced. Most of these limitations ofs chemical fixatives can be overcome by the use of cryofixation techniques since cell structure is stabilized rapidly (milliseconds). Several types of cryofixation techniques have been developed such as cold metal block freezing and propane jet freezing. Although application of these techniques has yielded exciting new information, they are limiting in that specimens can be preserved only to a relatively shallow depth (approx. 40 μm). In contrast, under optimal conditions, high pressure freezing (HPF) at 2100 bar can produce excellent freezing of biological samples up to 600 μm in thickness. Since a commercial HPF apparatus has only recently become available, the number of systematic structural studies of biological samples utilizing HPF is still rather limited, and basic questions concerning specimen preparation and processing, HPF artifacts, and interpretation of images need to be addressed.