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.;Single-molecule fluorescence studies were used to measure membrane structure at the molecular level. Several groups have shown that polarized total internal reflection fluorescence microscopy (PTIRF-M) using p-polarized excitation can reveal single-molecule orientations when spherical aberrations are introduced into the optics train. This approach was used here to measure the orientation of fluorescent lipid analogs doped into Langmuir-Blodgett and bilayer films of DPPC and DPPC/sterol mixed monolayers.;Two commonly used fluorescent lipid analogs, BODIPY-PC and DiIC 18 which have their fluorophores located in the tailgroup and headgroup, respectively were used and a variety of other probes are currently being studied. It was found that the tilt orientation of BODIPY-PC is very sensitive to the surface pressure at which the DPPC films are transferred onto the substrate. At low surface pressures, the tailgroups are largely lying in the plane of the film and 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.;The monolayer/bilayer "equivalent surface pressure" was also found to be ∼23 mN/m by directly comparing the molecular structure in the two films. Using this information, the condensing affect of cholesterol and other biologically relevant sterols on monolayers and bilayers at the equivalent surface pressure was studied. Molecular dynamics simulations were also compared with the experimental results to probe the insertion of BODIPY-PC into membrane lipids.
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