ELECTRON MICROSCOPE OBSERVATIONS ON FROZEN-DRIED CELLS

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Abstract
To explore the problem of artefacts which may be produced during usual fixation, dehydration, and embedding, the authors have examined pancreas, liver, and bone marrow frozen at about180[degree]C, dried, at55 to60[degree]C, embedded in methacrylate, sectioned, and floated on a formol-alcohol mixture. By these treatments the labile structure of living cells can be fixed promptly and embedded in methacrylate avoiding possible artefacts caused by direct exposure to chemical fixatives. Cell structures are ultimately exposed to a fixative when the sections are floated on formol-alcohol, but at this stage artefacts due to chemical fixation are expected to be minimized, as the fixatives act on structures tightly packed in methacrylate polymer. In the central zone of tissue blocks so treated, the cells are severely damaged by ice crystallization but at the periphery of the blocks the cell structure is well preserved. In such peripherally located cells, elements of the endoplasmic reticulum (ER), Palade''S granules, homogeneously dense mitochondria, and nuclear envelopes and pores, can be demonstrated without poststaining with OsO4. The structural organization in the nucleus is distorted by vacuolization. The mito-chondrial membranes and cristae, cellular membrane, and the Golgi apparatus, however, are detected only with difficulty. The Golgi region generally appears as a light zone, in which some ambiguous structures are em countered. After staining the section with OsO4 or Giemsa solution, an inner mitochondrial structure which resembles the cristae seen in conventional OSO4-fixed specimens appears, but the limiting membrane is absent. Treatment with OsO4 or Giemsa solution also renders more distinct the membrane of the ER and Palade''s granules but not the Golgi apparatus and cellular membrane. Treatment with ribpnuclease results in the disappearance of Palade''s granules. On the strength of these observations the authors conclude that OsO4 fixation gives a satisfactory preservation of such cell structures as the nuclear envelope, endoplasmic reticulum, and Palade''s granules, though it may induce slight swelling of these cell components.
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