Visualization of extracellular lining layer of lung alveoli by freeze-etching

Previous demonstration of a duplex extracellular lining layer of alveoli on fixed and embedded lung tissue suffered from the fallacy that (1) the air-liquid interface was disturbed at one step or another, and (2) the tissue had to be chemically treated. The present study was undertaken to check on the effect of these methodical inadequacies. Air-filled lung tissue was quick-frozen in Freon near liquid N₂ temperature, cleaved in high vacuum, the freeze-etehed fracture surface replicated, and the replicas examined by electron microscopy. Chemical treatment was eliminated in three stages: elimination of (1) embedding and (2) embedding and fixation, whereby glycerol was applied as cryoprotective agent, and (3) freezing of untreated lung tissue. In all three instances the existence of a duplex lining layer of alveoli with a base layer and a surface film could be confirmed. The film appeared to be continuous as evidenced by a smooth surface of alveoli viewed face on. Some evidence for the existence of tubular myelin figures in vivo is presented. These findings further corroborate the view that the alveolar surface is lined by extracellular material topped by a smooth surface film probably related to surfactant.