Contribution of scanning electron microscopy to viewing internal cell structure

Freeze-fracture provides a way of opening up cells and tissues for an internal view of cytoplasm and nucleus, and can give an internal view of a cytoplasmic organelle if the plane of fracture cuts through the organelle. Selective removal of soluble or other components is necessary for a deep view of structure at the fracture face. This may be achieved by osmium digestion, or by glycerol extraction, or by delaying fixation until after freeze-fracture and thawing, or by prior treatment with detergent to remove cell membranes and wash out soluble components. Cells may also be ruptured at room temperature, or in certain cases prepared to expose the inner surface of the plasma membrane for scanning electron microscopy (SEM) viewing. The problems and potential of SEM viewing of the cell interior are in certain respects similar to those encountered and derived from TEM replica study of freeze-fractured cells after deep etching. TEM replicas give better resolution, while SEM offers advantages in study of surfaces with considerable depth of structure. Study of fresh material, without fixation or alcohol dehydration, by rapid freezing and deep etching, is increasing our understanding of the artifacts that can be produced by these two preparative steps which are essential to critical point drying, whether used as a preparative step for SEM or for whole-mount high-voltage TEM microscopy.