Three-dimensional Confocal Microscopy Visualization and AFM-IR Chemical Mapping of Lung Surfactant Monolayer Collapse Morphologies

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ABSTRACT Neonatal respiratory distress syndrome (NRDS) and acute respiratory distress syndrome (ARDS) afflict upwards of 250,000 Americans every year. NRDS affects premature infants due to their underdeveloped lung’s inability to produce sufficient functional lung surfactant. Animal replacement surfactant treatment options exist for NRDS, although concerns over bio-variability, interspecies disease transmittance, and religious questions over porcine versus bovine derived surfactants remain. ARDS was seen in ~75% of all admitted COVID-19 ICU patients and is currently untreatable leading to a mortality rate of ~40%. ARDS is initiated by lung trauma or disease that leads to inflammation, causing an uncharacteristic increase of the surface tension within the lungs, leading to atelectasis. A deeper understanding of the fundamental structure and limiting behavior of lung surfactant monolayers may be the avenue to suggest new synthetic replacement surfactant treatments that could mitigate the biological concerns in NRDS as well as develop treatment options for patients with ARDS. My group has previously identified that the “collapse” of a monolayer determines the lower surface tension limit during the alveolar area compression accompanying exhalation. The physical and chemical factors that govern collapse may be altered in patients who develop dysfunctional, high surface tension lung surfactant during ARDS. Monolayers of healthy lung surfactant phase separate into domains of a semi-crystalline ordered phase and a disordered liquid phase of varied composition. We hypothesize that this phase separation dictates many of the dynamic and rheological properties of the monolayer that influence collapse. However, there is little direct information on the composition of the different domains in multicomponent lung surfactants. I will address monolayer collapse and phase separation in the following two aims. In Aim 1, I will visualize monolayer collapse structures using the 3-D serial sectioning capabilities of the confocal fluorescence microscope to determine how monolayer domains alter collapse behavior and the minimum surface tension. I have also recently found that collapse behavior changes on curved, alveolar-size interfaces compared to the flat surfaces in a Langmuir trough, and I will use confocal imaging to determine the relationship between collapse morphology and interfacial curvature. Aim 2 is to pioneer infrared-coupled atomic force microscopy (AFM-IR) methods to map the lateral distribution of the chemical species and their local ordering in multicomponent lung surfactant monolayers. I will use AFM-IR to examine the hypothesis that cholesterol concentrates at domain boundaries to lower the line tension while palmitic acid and hexadecanol promote crystallization of dipalmitoylphosphatidylcholine, increasing the fraction of solid phase in the monolayer. Successful completion of this project will provide a detailed description of the two-dimensional chemical distribution in laterally phase separated lung surfactant monolayers and how this phase separation influences the minimum surface tension at monolayer collapse.