A correlative study of the freezing patterns in rat myocardium using scanning and transmission electron microscopes

Abstract

Rat myocardial tissue, both fresh and chemically fixed, was quench frozen in melting Freon 22 at rates that resulted in in the formation of large ice-crystals. The material was studied with both scanning and transmission electron microscopy. Particular attention was paid to the characteristic freezing patterns within the myofibrils and mitochondria, and to features exposed by such freezing that might otherwise remain obscured. The freezing patterns found in tissue subjected to different processes were compared with the ultrastructure of unfrozen, chemically fixed counterparts. Ice-crystal cavities in the contractile material varied considerably along the length of a given myofiber in which the freezing front had progressed in parallel with the long axis of the myofibers. It is suggested that the intercalated disc functioned as a barrier to the freezing process. Ice generally compressed and distorted the contractile material beyond recognition, although the positions of the Z-bands remained evident and in register. Most single-fixed mitochondria were devoid of visible ice-crystal cavities, and the cristae generally remained intact. On the other hand, ice-crystal cavities were commonly seen between cristae of double-fixed mitochondria. No signs of perforations were seen in lipid bilayer membranes. Numerous strand-like connections between mitochondria as well as between mitochondria and the myofibrillar surface, which were believed to be sarcotubules, were made visible by the slow freezing process. Other strands, transverse to the myofibrils, connecting adjacent Z-bands and joining Z-bands to the sarcolemma, were interpreted as cytoskeletal elements. These strands, together with what apparently were SR tubules, exhibited high resistance to the stresses associated with the formation of ice-crystals.