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.

Whispering gallery mode resonators are small, radially symmetric dielectrics that trap light through continuous total internal reflection. The resonant condition at which light is efficiently confined within the structure is linked with refractive index, which has led to the development of sensitive label-free sensing schemes based on whispering gallery mode resonators. One resonator design uses inexpensive high index glass microspheres that offer intrinsically superior optical characteristics, but have proven difficult to multiplex and integrate with the fluidics for sample delivery and fluid exchange necessary for assay development. Recently, we introduced a fluorescence imaging approach that enables large scale multiplexing with microsphere resonators, thus removing one obstacle for assay development. Here we report an approach for microsphere immobilization that overcomes limitations arising from their integration with fluidic delivery. The approach is an adaptation of a calcium-assisted glass bonding method originally developed for microfluidic glass chip fabrication. Microspheres bonded to glass using this technique are shown to be stable with respect to fluid flow and show no detectable loss in optical performance. Measured Q-factors, for example, remain unchanged following sphere bonding to the substrate. The stability of the immobilized resonators is further demonstrated by transferring lipid films onto the immobilized spheres using the Langmuir-Blodgett technique. Bilayers of DOPC doped with GM1 were transferred onto immobilized resonators to detect the binding of cholera toxin to GM1. Binding curves generated from shifts in the whispering gallery mode resonance result in a measured Kd of 1.5 × 10−11 with a limit of detection of 3.3 pM. These results are discussed in terms of future assay development using microsphere resonators.

Single molecule fluorescence measurements are used to probe the effects of GM1 in DPPC monolayers. Langmuir-Blodgett films of GM1 and DPPC were doped with ~10−8 mol% of the fluorescent lipid probe, BODIPY-PC, and transferred onto glass substrates at 23 mN/m. As shown previously, the individual orientation of each BODIPY-PC probe in the membrane can be measured using defocused polarized total internal reflection fluorescence microscopy, revealing changes in film properties at the molecular level. Here, BODIPY-PC tilt angle histograms are used to characterize the effects of GM1 in DPPC films from 0.05 mol% to 100 mol% GM1. At high GM1 levels (>5 mol% GM1), trends in the single molecule measurements agree with previous bulk measurements showing the turnover from condensing to expanding influence of GM1 at ~20 mol%, thus validating the single molecule approach. At biologically relevant, low concentrations of GM1 (<5 mol% GM1), where bulk fluorescence measurements are less informative, the single molecule measurements reveal a marked influence of GM1 on film properties. The addition of trace amounts of GM1 to DPPC films leads to an expansion of the film which continues to 0.10 mol% GM1, above which the trend reverses and the condensing effect previously noted is observed.

The desire to directly probe biological structures on the length scales that they exist has driven the steady development of various high-resolution microscopy techniques. Among these, optical microscopy and, in particular, fluorescence-based approaches continue to occupy dominant roles in biological studies given their favorable attributes. Fluorescence microscopy is both sensitive and specific, is generally noninvasive toward biological samples, has excellent temporal resolution for dynamic studies, and is relatively inexpensive. Light-based microscopies can also exploit a myriad of contrast mechanisms based on spectroscopic signatures, energy transfer, polarization, and lifetimes to further enhance the specificity or information content of a measurement. Historically, however, spatial resolution has been limited to approximately half the wavelength due to the diffraction of light. Near-field scanning optical microscopy (NSOM) is one of several optical approaches currently being developed that combines the favorable attributes of fluorescence microscopy with superior spatial resolution. NSOM is particularly well suited for studies of both model and biological membranes and application to these systems is discussed.