Study of model lung surfactant: Probing membrane structure from the macroscopic to the single molecule level

The high resolution techniques of confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM) are utilized to probe the structure of model lung surfactant (LS) from the macroscopic scale down to the single molecule level. Research has highlighted the particular importance of a surfactant-associated protein, SP-B, in stabilizing the pulmonary surfactant monolayer during respiration through a mechanism not fully understood. The aforementioned high resolution techniques are used, therefore, to provide insights into this mechanism through the study of Langmuir-Blodgett (LB) films of relevant systems. Background studies on palmitic acid (PA) are presented which provide insight into the phase behavior of the fatty acid and to elucidate factors contributing to the final film structure of supported two-dimensional films. These studies are then extended to model systems containing both PA and a truncated form of natural SP-B (SP-B_1–25). Confocal microscopy studies are conducted on these films to provide a macroscopic scale picture of peptide concentration dependence. Evidence is presented indicating a bimodal dependence in the phase behavior of the films with peptide concentration. Further, the results suggest the addition of 11 wt.% SP-B_1–25 to monolayers of PA induces large density fluctuations in the membrane over macroscopic length scales, a signature of critical behavior. These experiments on the macroscopic scale are extended to the single molecule level by exploiting the unique nature of the electric fields present at the NSOM tip aperture. Single molecule fluorescence NSOM measurements reveal the orientation distribution of single fluorescent probe molecules doped into LB films of DPPC. The data indicate that the environment of the probe molecules becomes more ordered as surface pressure is increased, as would be expected for lipid films of DPPC. Finally, these measurements will be further supplemented through a new technique presented here which combines attributes from NSOM and fluorescence resonance energy transfer (FRET) to increase optical imaging capabilities in both the lateral and axial directions. (Abstract shortened by UMI.)...