Investigating lipid membrane structure with near-field scanning optical microscopy

A combination of several high resolution techniques including confocal microscopy, atomic force microscopy (AFM), and near-field scanning optical microscopy (NSOM) are used to study the structure of model lipid membranes. NSOM is particularly well suited for this type of analysis due to the combination of simultaneously collected high resolution optical and topographical information. This combined information provides a more complete view of the lipid phase structure of monolayer and multilayer films. In this research, monolayer and bilayer lipid membranes containing a small mole percentage of fluorescent lipid analog probes are fabricated using a combination of Langmuir-Blodgett and Langmuir-Schaefer techniques. Results from studies using the NSOM technique indicate the presence of coexisting lipid phases on the submicron level in both the monolayer and bilayer model systems. For model lipid bilayers, this assignment is shown to be complicated based solely on topographical information available to AFM and is limited by resolution constraints associated with conventional optical techniques such as confocal microscopy. In addition, the single molecule fluorescence sensitivity and unique properties of the electric fields near the NSOM aperture can be used to characterize the three-dimensional orientation of individual fluorescent lipid analogs in the lipid films. Using these attributes, single molecule near-field fluorescence measurements are employed to analyze the molecular level structure in model membranes. The results of these studies reveal no change in orientation of the lipid analog probe doped into lipid monolayers fabricated at high and low surface pressures. These results indicate the presence of a less ordered liquid-expanded phase that persists at high surface pressures where the majority of the film exists in a highly ordered solid-condensed phase. This single molecule orientation analysis is also used in the determination of the structure of the host lipid matrix. The results of this analysis reveal that the lipid tailgroups in the membrane are tilted 41° from the normal of the membrane which is in agreement with previous results. Finally, the extension of NSOM to living systems to investigate similar submicron coexisting lipid phase behavior is discussed.