Freeze-dried embedded specimens for biological microanalysis.

The main problems associated with freeze-drying of biological material for electron microscopy concern the freeze-drying temperatures and times necessary to minimize artifacts. Due to the many parameters involved these problems have to be resolved experimentally. It can be shown that good morphological preservation of chemically unfixed material is possible when freeze-drying is done exclusively in a temperature range between -80 degrees C and -50 degrees C. OsO4 vapour fixation of the freeze-dried tissue is not necessary and should be avoided because it may cause ion redistribution artifacts. Embedding at low temperature of properly freeze-dried material does not seem to disturb structure and ion distribution of the freeze-dried material. Hence, sections of such freeze-dried material and embedded biological material seem to be suitable for microanalysis. Preliminary micro-analytical results obtained from sections of freeze-dried and Lowicryl K11M embedded muscle reveal an uneven distribution of potassium in the sarcomeres similar to the visualized uneven distribution of the electron dense thallium (potassium surrogate) in frozen hydrated cryosections. A comparison of different cryomethods shows that freeze-drying and embedding is the simplest way to obtain stable thin sections of chemically unfixed biological material which, for instance, may be used for future microanalytical investigation of the interaction of proteins with physiological and non-physiological ions.