Probing single molecule orientations in model lipid membranes with near-field scanning optical microscopy

Abstract

Single molecule near-field fluorescence measurements are utilized to characterize the molecular level structure in Langmuir–Blodgett monolayers of L-α-dipalmitoylphosphatidylcholine (DPPC). Monolayers incorporating 3×10−4 mol % of the fluorescent lipid analog N-(6-tetramethylrhodaminethiocarbamoyl)-1,2-dihexadecanoyl-sn- glycero-3-phosphoethanolamine, triethylammonium salt (TRITC–DHPE) are transferred onto a freshly cleaved mica surface at low (π=8 mN/m) and high (π=30 mN/m) surface pressures. The near-field fluorescence images exhibit shapes in the single molecule images that are indicative of the lipid analog probe orientation within the films. Modeling the fluorescence patterns yields the single molecule tilt angle distribution in the monolayers which indicates that the majority of the molecules are aligned with their absorption dipole moment pointed approximately normal to the membrane plane. Histograms of the data indicate that the average orientation of the absorption dipole moment is 2.2° (σ=4.8°) in monolayers transferred at π=8 mN/m and 2.4° (σ=5.0°) for monolayers transferred at π=30 mN/m. There is no statistical difference in the mean tilt angle or distribution for the two monolayer conditions studied. The insensitivity of tilt angle to film surface pressure may arise from small chromophore doped domains of trapped liquid-expanded lipid phase remaining at high surface pressure. There is no evidence in the near-field fluorescence images for probe molecules oriented with their dipole moment aligned parallel with the membrane plane. We do, however, find a small but significant population of probe molecules (∼13%) with tilt angles greater than 16°. Comparison of the simultaneously collected near-field fluorescence and force images suggests that these large angle orientations are not the result of significant defects in the films. Instead, this small population may represent a secondary insertion geometry for the probe molecule into the lipid monolayer.