Atomic Force Microscopy Imaging of Adsorbed Pulmonary Surfactant Films

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.