(611c) Quantitative Measurement of Fluorescent Molecule Adsorption Via Confocal Microscopy
Contrast in confocal microscope images of phase-separated monolayers at the air-water interface can be generated by the selective adsorption of water-soluble fluorescent dyes from the subphase. The optical sectioning capability of confocal imaging minimizes the fluorescence signal from the subphase as well as allowing for quantitative measurement of the dye concentration in the monolayer. The partitioning of the water-soluble dyes depends strongly on the surface pressure or area/molecule measured from the isotherms. The extent of adsorption is consistent with a free area model previously used to model lateral diffusion and surface viscosity. The rates of adsorption and desorption are governed by Langmuir-type kinetics. Unlike fluorescent lipids, the water-soluble dye is in exchange equilibrium between the interface and the subphase so that the dye is not concentrated as the proportions of the two phases change at coexistence. This approach may be extended to study the adsorption of a variety of subphase-soluble molecules and interfacial films, as well as allow multi-color imaging of the monolayers during dynamic processes.
General acceptance of complex monolayer and bilayer phase behavior, especially phase coexistence and critical phenomena, has relied on the visualization of fluorescently tagged lipids preferentially segregated between phases. This segregation, which is due to differences in local molecular organization corresponding to the different phases, provides the necessary contrast to visualize even very subtle differences in packing density, molecular tilt, and short and long-range ordering. Since the technique was introduced, fluorescence microscopy has settled numerous debates over the molecular scale organization of monolayers and bilayers. However, fluorescent lipids, like many of the other lipids in monolayers and bilayers, are effectively insoluble in the surrounding aqueous subphase and are trapped in the monolayer or bilayer. As a result, expelling the fluorescent lipid from one phase means concentrating the fluorescent lipid in another phase, which can result in fluorescence quenching, alterations in the structures of both the less ordered (dye-rich) and more ordered (dye-poor) domains, or perturbations of the mechanical properties of the monolayer. Improvements in the sensitivity of cameras and other recording devices, and improved quantum efficiencies of fluorescent dyes, have led to a gradual decrease in the overall mole fraction of fluorescent lipid required for good imaging, from 2 mol% in early experiments to 0.05 - .1 mol% in recent work. An alternative to fluorescently labeled lipids is Brewster Angle Microscopy (BAM), in which the contrast in the image is developed by small variations in refractive index between different areas in the monolayer, due to different phases, molecular density, or molecular tilt. Resolution limitations and ease of use have limited the general acceptance of BAM.
Here we show that the selective adsorption of a fluorescent water-soluble dye from the subphase to the monolayer provides similar contrast in confocal images of monolayer phase separation and critical phenomena to insoluble lipid dyes. However, unlike insoluble lipid dyes, the water-soluble dye remains at a concentration at a given surface pressure or area per molecule that is set by a dynamic equilibrium between the monolayer and subphase. The optical sectioning of the confocal microscope allows the interfacial fluorescence of the monolayer-adsorbed dye to be visualized and rejects the fluorescence from the subphase, which is not possible with conventional wide-field microscopy. By proper de-convolution of the confocal image, the total adsorption and the adsorption/desorption rates of the dye can be quantified. Dye adsorption can modeled using a simple, free area model previously used to describe diffusion in condensed phases. The amount of dye adsorption depends non-linearly on the free area which is the difference between the area per molecule at a given surface pressure measured by the isotherm, and the minimum, close packed area of the lipids making up the monolayer. The rates of dye adsorption and desorption can be described with classic Langmuir adsorption kinetics. These methods might be extended to quantify monolayer adsorption of a variety of labeled proteins, polymers, and other materials of interest.