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

Single molecule near-field fluorescence measurements are utilized to charac terize the molecular level structure in Langmuir-Blodgett monolayers of L-a lpha-dipalmitoylphosphatidylcholine (DPPC). Monolayers incorporating 3x10(- 4) mol % of the fluorescent lipid analog N-(6-tetramethylrhodaminethiocarba moyl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine, triethylammonium salt (TRITC-DHPE) are transferred onto a freshly cleaved mica surface at l ow (pi=8 mN/m) and high (pi=30 mN/m) surface pressures. The near-field fluo rescence images exhibit shapes in the single molecule images that are indic ative 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 alig ned with their absorption dipole moment pointed approximately normal to the membrane plane. Histograms of the data indicate that the average orientati on of the absorption dipole moment is 2.2 degrees (sigma=4.8 degrees) in mo nolayers transferred at pi=8 mN/m and 2.4 degrees (sigma=5.0 degrees) for m onolayers transferred at pi=30 mN/m. There is no statistical difference in the mean tilt angle or distribution for the two monolayer conditions studie d. The insensitivity of tilt angle to film surface pressure may arise from small chromophore doped domains of trapped liquid-expanded lipid phase rema ining at high surface pressure. There is no evidence in the near-field fluo rescence images for probe molecules oriented with their dipole moment align ed parallel with the membrane plane. We do, however, find a small but signi ficant population of probe molecules (similar to 13%) with tilt angles grea ter than 16 degrees. 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 mole cule into the lipid monolayer. (C) 2000 American Institute of Physics. [S00 21-9606(00)70616-7].