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 (PTIRF-M) 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 film and evolve to an orientation normal to the surface as pressure is increased. For DiIC18however, 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.

Single-molecule orientations of the fluorescent lipid analogue BODIPY-PC doped into lipid monolayers and bilayers of DPPC are used to characterize the structure present in the films as a function of sterol content. Out-of-focus polarized total internal reflection fluorescence microscopy (PTIRF-M) measurements are used to characterize the single-molecule tilt angles with respect to the membrane normal. Tilt angle histograms for Langmuir-Blodgett monolayers of DPPC reveal bimodal distributions at all surface pressures studied. A linear dependence in the dye population oriented normal to the membrane plane with surface pressure is found and used to characterize the equivalent surface pressure of supported bilayers formed through vesicle fusion. These measurements reveal an equivalent surface pressure of approximately 23 mN/m, which is somewhat lower than the currently accepted value of approximately 30-35 mN/m.(1-7) The effect of cholesterol, ergosterol, and lanosterol on membrane structure is also compared between DPPC bilayers and monolayers transferred at approximately 23 mN/m. The addition of cholesterol leads to dramatic changes in the tilt angle histograms while lanosterol has essentially no effect. The addition of ergosterol has a slight influence at higher concentrations. Using the average tilt angle calculated from the single-molecule histograms, the order parameter S is calculated as a function of cholesterol and compared with previous studies.(8-10).

Single-molecule fluorescence measurements have been used to characterize membrane properties, and recently showed a linear evolution of the fluorescent lipid analog BODIPY-PC towards small tilt angles in Langmuir-Blodgett monolayers as the lateral surface pressure is increased. In this work, we have performed comparative molecular dynamics (MD) simulations of BODIPY-PC in DPPC (dipalmitoylphosphatidylcholine) monolayers and bilayers at three surface pressures (3, 10, and 40 mN/m) to explore 1) the microscopic correspondence between monolayer and bilayer structures, 2) the fluorophore’s position within the membrane, and 3) the microscopic driving forces governing the fluorophore’s tilting. The MD simulations reveal very close agreement between the monolayer and bilayer systems in terms of the fluorophore’s orientation and lipid chain order, suggesting that monolayer experiments can be used to approximate bilayer systems. The simulations capture the trend of reduced tilt angle of the fluorophore with increasing surface pressure as seen in the experimental results, and provide detailed insights into fluorophore location and orientation, not obtainable in the experiments. The simulations also reveal that the enthalpic contribution is dominant at 40 mN/m resulting in smaller tilt angles of the fluorophore, and the entropy contribution is dominant at lower pressures resulting in larger tilt angles.

Single molecule fluorescence measurements have recently been used to probe the orientation of fluorescent lipid analogs doped into lipid films at trace levels. Using defocused polarized total internal reflection fluorescence microscopy (PTIRF-M), these studies have shown that fluorophore orientation responds to changes in membrane surface pressure and composition, providing a molecular level marker of membrane structure. Here we extend those studies by characterizing the single molecule orientations of six related BODIPY probes doped into monolayers of DPPC. Langmuir–Blodgett monolayers transferred at various surface pressures are used to compare the response from fluorescent lipid analogs in which the location of the BODIPY probe is varied along the length of the acyl chain. For each BODIPY probe location along the chain, comparisons are made between analogs containing phosphocholine and smaller fatty acid headgroups. Together these studies show a general propensity of the BODIPY analogs to insert into membranes with the BODIPY probe aligned along the acyl chains or looped back to interact with the headgroups. For all BODIPY probes studied, a bimodal orientation distribution is observed which is sensitive to surface pressure, with the population of BODIPY probes aligned along the acyl chains increasing with elevated surface pressure. Trends in the single molecule orientations for the six analogs reveal a configuration where optimal placement of the BODIPY probe within the acyl chain maximizes its sensitivity to the surrounding membrane structure. These results are discussed in terms of balancing the effects of headgroup association with acyl chain length in designing the optimal placement of the BODIPY probe.

Near-field scanning optical microscopy (NSOM) is an emerging optical technique that enables simultaneous high-resolution fluorescence and topography measurements. Here we discuss selected applications of NSOM to biological systems that help illustrate the utility of its high spatial resolution and simultaneous collection of both fluorescence and topography. For the biological sciences, these attributes seem particularly well suited for addressing ongoing issues in membrane organization, such as those regarding lipid rafts, and protein-protein interactions. Here we highlight a few NSOM measurements on model membranes, isolated biological membranes, and cultured cells that help illustrate some of these capabilities. We finish by highlighting nontraditional applications of NSOM that take advantage of the small probe to create nanometric sensors or new modes of imaging.