Abstract
Biological membranes are highly heterogeneous structures that are thought to use this heterogeneity to organize and modify the function of membrane constituents. Probing membrane organization, structure, and changes therein are crucial for linking structural metrics with function in biological membranes. Here we report the use of single-molecule fluorescence studies to measure membrane structure at the molecular level. Several groups have shown that polarized total internal reflection fluorescence microscopy using p-polarized excitation can reveal single-molecule orientations when spherical aberrations are introduced into the optics train. We use this approach to measure the orientation of fluorescent lipid analogs doped into Langmuir-Blodgett films of DPPC and arachidic acid. We compare two commonly used fluorescent lipid analogs, BODIPY-PC and DiIC18, which have their fluorophores located in the tailgroup and headgroup, respectively. We find the tilt orientation of BODIPY-PC is very sensitive to the surface pressure at which DPPC films are transferred onto the substrate. At low surface pressures, the tailgroups are largely lying in the plane of the filmand evolve to an orientation normal to the surface as pressure is increased. For DiIC18, however, no evolution in orientation with surface pressure is observed, which is consistent with the headgroup located fluorophore being less sensitive to changes in membrane packing. Single-molecule orientation measurements of DiIC18 in multilayer films of arachidic acid are also measured and compared with previous bulk measurements. Finally, single-molecule measurements are utilized to reveal the ordering induced in DPPC monolayers following the addition of cholesterol.