SCANNING ELECTRON-MICROSCOPY OF MUSCLE MYOFIBRILS AFTER HIGH-PRESSUREFREEZING AND FREEZE-SUBSTITUTION-STAINING

A novel approach to study the three dimensional ultrastructure of orga nelles and cells by means of scanning electron microscopy is described . Muscle myofibrils have been used in the development of the technique s since their structure is well characterized using conventional elect ron microscopic methods. Myofibrils in rigor buffer (with no cryo-prot ectants or pressure sealants) were frozen at high pressure (2300 bar) within specially designed chambers. The frozen specimens were then fre eze-substituted-stained with methanol containing tungsten and iron sal ts and finally critical point dried. These methods allowed scanning el ectron microscopic observations of the organization of individual fila ments within whole myofibrils over several sarcomeres. Images obtained showed excellent structural preservation with three dimensional infor mation which is not available with other electron microscopic techniqu es. Success in these approaches was ascribed to (a) rapid and uniform freezing at high pressure without ice segregation patterns, (b) unifor m electro-conductivity of the specimen closely attached to the polishe d carbon piston/carrier, and (c) good electron emission (secondary and back-scattered) from the metal incorporated into the myofibril struct ure without additional coating.