Scanning Electron Microscopy of Muscle Myofibrils After High Pressure Freezing and Freeze-Substitution-Staining

A novel approach to study the three dimensional ultrastructure of organelles and cells by means of scanning electron microscopy is described. Muscle myofibrils have been used in the development of the techniques since their structure is well characterized using conventional electron microscopic methods. Myofibrils in rigor buffer (with no cryo-protectants or pressure sealants) were frozen at high pressure (2300 bar) within specially designed chambers. The frozen specimens were then freeze-substituted-stained with methanol containing tungsten and iron salts and finally critical point dried. These methods allowed scanning electron microscopic observations of the organization of individual filaments within whole myofibrils over several sarcomeres. Images obtained showed excellent structural preservation with three dimensional information which is not available with other electron microscopic techniques. Success in these approaches was ascribed to (a) rapid and uniform freezing at high pressure without ice segregation patterns, (b) uniform electro-conductivity of the specimen closely attached to the polished carbon piston/carrier, and (c) good electron emission (secondary and back-scattered) from the metal incorporated into the myofibril structure without additional coating