Correlated atomic force and transmission electron microscopy of nanotubular structures in pulmonary surfactant

Pulmonary surfactant stabilizes the lung by reducing surface tension at the air-water interface of the alveoli. Surfactant is present in the lung in a number of morphological forms, including tubular myelin (TM). TM is composed of unusual 40 x 40 nm square elongated proteolipid tubes. Atomic force microscopy (AFM) was performed on polymer-embedded Lowicryl and London Resin-White (LR-White) unstained thin sections. AFM was used in imaging regions of the sections where TM was detected by transmission electron microscopy (EM) of corresponding stained sections. Tapping- and contact-mode AFM imaging of the unstained sections containing TM indicated a highly heterogeneous surface topography with height variations ranging from 10 to 100 nm. In tapping-mode AFM, tubular myelin was seen as hemispherical protrusions of 30-70 nm in diameter, with vertical dimensions of 5-8 nm. In contact-mode AFM and with phase imaging using a sharper (>10 nm nominal radius) probe, square open-ended tubes which resembled typical electron micrographs of such regions were observed. The cross-hatch structures observed inside the tubes using EM were not observed using AFM, although certain multilobe structures and topographic heterogeneity were detected inside some tubes. Other regions of multilamellar bodies and some regions where such bilayer lamella appear to fuse with the tubes were found in association with TM using AFM. EM of acetone-delipidated tubes in LR-White revealed rectangular tubular cores containing cross-hatched structures, presumably protein skeletons. AFM surface topography of these regions showed hollow depressions at positions at which the protein was anticipated instead of the protrusions seen in the lipid-containing sections. Gold-labeled antibody to surfactant protein A was found associated somewhat randomly within the regions containing the protein skeletons. The topography of the gold particles was observed as sharp peaks in contact-mode AFM. This study suggests a method for unambiguous detection of three-dimensional nanotubes present in low abundance in a biological macromolecular complex. Only limited detection of proteins and lipids in surfaces of embedded tubular myelin was possible. EM and AFM imaging of such unusual biological structures may suggest unique lipid-protein associations and arrangements in three dimensions.