Ultrastructure of Tracheal Surface Liquid: Low‐Temperature Scanning Electron Microscopy

Abstract

Summary: A layer of liquid lines the airways in the lung. Revious microscopic studies have suggested that it is in two phases, with a mucous gel lying above a periciliary sol. However, shrinkage artifacts due to chemical 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 electron microscopy (LTSEM). A polished copper probe cooled to liquid nitrogen temperature was applied to the mucosal surface of sheets of excised tracheal epithelium to effect rapid freezing of surface liquid. Tissue sheets were then mounted in an LTSEM (AMRay l OOOA with Biochamber) which maintains samples at -1 80 "C with a Joule-Thompson refrigerator built into the stage. Tissues were fractured at right angles to the epithelial surface, coated with gold, and viewed, all at to 10-6 torr without transfer through air. The sample was stable under the electron beam at accelerating voltages up to 20 kV. Epithelial f e a m s (nuclei, cilia, microvilli, mucous granules) were well preserved. The mucosal surface of the cells was covered with material on the order of 8 p in depth. The mucous gel and periciliary sol could be seen as distinct layers and could be distinguished by the size and pattern of ice crystal voids generated by radiant-etching of the fractured surface of the sample.

Augmentation of Respiratory Tract Lining Fluid Ascorbate Concentrations Through Supplementation with Vitamin C

“…This aqueous layer extending from the nasal cavity to the alveolar region of the lung (Bastacky et al, 1995;Wu et al, 1996) is derived from cellular secretions and pericellular transport from the blood, and represents the first physical interface with which inhaled toxins interact. Due to its structural and compositional heterogeneity throughout the lung, the RTLF is best thought of as a series of interconnected compartments, simplistically viewed as a twolayer mucus and sol phase compartment in the upper airways, characterized by high urate concentrations (Cross et al, 1998;Peden et al, 1993), and a mucus free, surfactant containing and glutathione enriched compartment in the peripheral airways and alveoli (Cantin et al, 1987;Cross et al, 1998;Hatch, 1992).…”

Airway surface liquid depth imaged by surface laser reflectance microscopy

“…Measurement of ASL depth is technically challenging because it can be as low as 5–15 µm. Various methods have been used to estimate ASL depth, including electron microscopy of fixed frozen tissues or cells (Yager et al, 1994; Wu et al, 1996), microelectrode scanning (Rahmoune and Shephard, 1995), and z-scanning confocal microscopy (Jayaraman et al, 2001; Tarran et al, 2001; Song et al, 2009). Confocal fluorescence microscopy is at present probably the most reliable method to measure ASL depth.…”

Cryo-SEM method for the observation of entrapped bubbles and degree of water filling in large wet powder compacts

“…The samples were too large and too hard for cryo‐microtomy, which is commonly employed with organic materials (De Carvalho et al ., 1999). Two types of techniques have been reported in the literature for size reduction of cryogenic sample by machining; trimming with a high‐speed circular dental saw (Wu et al ., 1996) and cryo‐milling (Nijsse & Van Aelst, 1999). On one hand, it is difficult to achieve a controlled flat surface on a sphere in a bath of liquid nitrogen manually with a circular saw.…”