Correlated Atomic Force and Transmission Electron Microscopy of Nanotubular Structures in Pulmonary Surfactant

Abstract

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 ؋ 40 nm square elongated proteolipid tubes. Atomic force microscopy (AFM) was performed on polymerembedded Lowicryl and London Resin-White (LRWhite) 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 (G10 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 threedimensional 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. Academic Press

Human SP‐A and a pharmacy‐grade porcine lung surfactant extract can be reconstituted into tubular myelin – a comparative structural study of alveolar surfactants using cryo‐transmission electron microscopy

“…There is other evidence indicating that SP-A is localized in this mode; textures of ·-shaped (Ôcross-hatchedÕ) electron-dense linear formations in the tubular cross-sections have been observed, cf. Nag et al (1999). It seems likely, therefore, that the electron dense material along the tubuli shown in Fig.…”

Disparate effects of two phosphatidylcholine binding proteins, C-reactive protein and surfactant protein A, on pulmonary surfactant structure and function

“…Small amounts of the BLES plus protein (SP-A and CRP; ϳ500 g) emulsions were removed from the diluted stocks in buffer A just before performing captive bubble studies for morphometry using transmission electron microscopy (TEM). The samples were immediately fixed using glutaraldehyde, pelleted, and processed using methods similar to those described previously (27). The pellets were postfixed with potassium ferrocyanide-reduced osmium tetroxide, embedded in London Resin-White (Polyscience, Warrington, PA), and dehydrated in an acetone series by progressive lowering of temperature.…”

“…Ultrathin sections (ϳ90 nm) of these embedded samples were cut using diamond knives and stained with uranyl acetate and lead citrate. The grids were imaged using a Philips 410 transmission electron microscope, and micrographs were obtained at ϫ14,700 magnification (27).…”

Atomic Force Microscopy in Mechanobiology: Measuring Microelastic Heterogeneity of Living Cells

“…Classical AFM contact mode imaging involves raster scanning the probe over the sample in the plane orthogonal to the indentation axis (z-axis) while applying a constant force onto the sample by keeping the cantilever deflection constant (103). Contact mode imaging provides exceptional detail and is still used extensively in cell biology for various applications (104)(105)(106)(107)(108). Other modalities such as tapping mode (109), jumping mode (110), or phase imaging (111) are also used in investigation of cellular processes (104,112).…”

“…Contact mode imaging provides exceptional detail and is still used extensively in cell biology for various applications (104)(105)(106)(107)(108). Other modalities such as tapping mode (109), jumping mode (110), or phase imaging (111) are also used in investigation of cellular processes (104,112). Even though the working principles of these techniques may differ, an inherent attribute of the AFM that translates through all these imaging modalities is the fact that none of the images are pure topographical information; instead they are convolutions of material height and the underlying mechanical properties.…”