ULTRASTRUCTURE OF TRACHEAL SURFACE LIQUID - LOW-TEMPERATURE SCANNING ELECTRON-MICROSCOPY

A layer of liquid lines the airways in the lung. Previous microscopic studies have suggested that it is in two phases, with a mucous gel lyi ng above a periciliary sol. However, shrinkage artifacts due to chemic al fixation, dehydration, and drying have prevented reliable estimates of the depth of these layers. To avoid such problems, we have studied the surface liquid of bovine trachea by low-temperature scanning elec tron microscopy (LTSEM). A polished copper probe cooled to liquid nitr ogen temperature was applied to the mucosal surface of sheets of excis ed tracheal epithelium to effect rapid freezing of surface liquid. Tis sue sheets were then mounted in an LTSEM (AMRay 1000A with Biochamber) which maintains samples at -180 degrees C with a Joule-Thompson refri gerator built into the stage. Tissues were fractured at right angles t o the epithelial surface, coated with gold, and viewed, all at 10(-5) to 10(-6) ton: without transfer through air. The sample was stable und er the electron beam at accelerating voltages up to 20 kV. Epithelial features (nuclei, cilia, microvilli, mucous granules) were well preser ved. The mucosal surface of the cells was covered with material on the order of 8 mu m in depth. The mucous gel and periciliary sol could be seen as distinct layers and could be distinguished by the size and pa ttern of ice crystal voids generated by radiant-etching of the fractur ed surface of the sample.