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

断裂翻转很容易产生扩展的、大分子分辨率的细胞表面视图。然而，在细胞体仍然附着在碳铸件上的情况下，断裂翻转不能显示膜表面。在这个项目中，我们开发了裂缝翻转与Triton X-100提取相结合的方法来克服这一障碍。简而言之，我们使用未固定的细胞，在冷冻断裂和极性区碳稳定后，用Triton X-100处理以溶解膜的未断裂区域。这种断裂翻转的新扩展导致了对细胞膜细胞质表面的直接超微结构检查，原则上，使胞内细胞器的外质腔表面可视化成为可能。此外，我们已经证明，将断裂-翻转与单个细胞膜分子的细胞化学标记（断裂-翻转细胞化学）相结合，将为细胞膜组分的化学性质和拓扑结构提供直接证据。

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.