Atomic Force Microscopy Imaging of Adsorbed Pulmonary Surfactant Films

Abstract

Pulmonary surfactant (PS) is a lipid-protein complex that adsorbs to the air-water surface of the lung as a thin film. Previous studies have suggested that the adsorbed PS film is composed of an interfacial monolayer, plus a functionally attached vesicular complex, called the surface-associated surfactant reservoir. However, direct visualization of the lateral structure and morphology of adsorbed PS films using atomic force microscopy (AFM) has been proven to be technically challenging. To date, all AFM studies of the PS film have relied on the model of Langmuir monolayers. Here, we showed the first, to our knowledge, AFM imaging of adsorbed PS films under physiologically relevant conditions using a novel, to our knowledge, experimental methodology called constrained drop surfactometry. In conjunction with a series of methodological innovations, including subphase replacement, in situ Langmuir-Blodgett transfer, and real-time surface tension control using closed-loop axisymmetric drop shape analysis, constrained drop surfactometry allowed the study of lateral structure and topography of animal-derived natural PS films at physiologically relevant low surface tensions. Our data suggested that a nucleation-growth model is responsible for the adsorption-induced squeeze-out of the PS film, which likely results in an interfacial monolayer enriched in dipalmitoylphosphatidylcholine with the attached multilayered surface-associated surfactant reservoir. These findings were further supported by frequency-dependent measurements of surface dilational rheology. Our study provides novel, to our knowledge, biophysical insights into the understanding of the mechanisms by which the PS film attains low surface tensions and stabilizes the alveolar surface.

An adverse outcome pathway for lung surfactant function inhibition leading to decreased lung function

“…A sensor plate measures the variation in surface pressure over compression to yield surface pressure – area isotherms. It should be noted that in addition to the traditional Langmuir trough, the Langmuir-Blodgett technique has been adapted in the constrained drop surfactometer to study adsorbed surfactant films ( Xu et al, 2020 ). The comparison of such isotherms in the presence or absence of the test substance gives insights in the interaction of a substance with lung surfactant at the molecular level.…”

Surfactant-laden film lining an oscillating cap: problem formulation and weakly nonlinear analysis

“…Apart from the parametric investigation of the effect of varying H 0 , to be undertaken next, the values H = 0.3 μm and H 0 = 0.14 μm are kept constant for the rest of the study. The validity of the selected value of H 0 is further supported in § 5, by discussing and interpreting additional experimental evidence (Xu et al 2020).…”

“…. Term H 0 is a key parameter for the problem, and its magnitude will be estimated from direct experimental evidence (Bastacky et al 1995;Xu et al 2020).…”

“…A new boundary condition is formulated for the alveolar rim, by matching the 'large-scale' dynamics of the alveolus to 'small-scale' equilibrium over the finite thickness of the mid-alveolar wall. The complex dynamics of the pulmonary surfactant is described by a recently developed model (Bouchoris & Bontozoglou 2021), which was found to predict with quantitative accuracy the surface tension-surface area hysteresis loops measured independently for various lung surfactant preparations (Saad, Neumann & Acosta 2010;Xu, Yang & Zuo 2020). Small-amplitude oscillations around the equilibrium conditions of the alveolus are considered.…”

Suppression of Lα/Lβ Phase Coexistence in the Lipids of Pulmonary Surfactant

“…Experimental evidence contradicts that prediction (64-67). 2) Multilamellar films: Several groups have shown that surfactant films on modestly curved surfaces collapse slowly when the subphase contains surfactant vesicles (64)(65)(66)(67). Electron micrographs show that at least portions of the alveolar film are multilamellar rather than monomolecular (68,69).…”