Fracture-flip and fracture-flip cytochemistry to study the macromolecular architecture of membranes

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Fracture-flip easily produces extended, macromolecular resolution views of cell surfaces. Fracture-flip, however, cannot reveal membrane surfaces in instances where the cell body remains attached to the carbon cast. In this project we have developed the use of fracture-flip combined with Triton X-100 extraction to overcome this obstacle. Briefly, we use unfixed cells that, after freeze-fracture and carbon-stabilization of apolar domains, are treated with Triton X-100 to dissolve unfractured areas of the membrane. This new extension of fracture-flip leads to the direct ultrastructural examination of the cytoplasmic surfaces of cell membranes and, in principle, makes it possible to visualize the exoplasmic luminal surfaces of intracellular organelles. In addition, we have demonstrated that the combination of fracture-flip with cytochemical labeling of individual cell membrane molecules, fracture-flip cytochemistry, would provide direct evidence of the chemical nature and topology of the cell membrane components.