Visualizing monolayers with a water-soluble fluorophore to quantify adsorption, desorption, and the double layer

Abstract

Contrast in confocal microscopy of phase-separated monolayers at the air-water interface can be generated by the selective adsorption of water-soluble fluorescent dyes to disordered monolayer phases. Optical sectioning minimizes the fluorescence signal from the subphase, whereas convolution of the measured point spread function with a simple box model of the interface provides quantitative assessment of the excess dye concentration associated with the monolayer. Coexisting liquid-expanded, liquid-condensed, and gas phases could be visualized due to differential dye adsorption in the liquid-expanded and gas phases. Dye preferentially adsorbed to the liquid-disordered phase during immiscible liquid-liquid phase coexistence, and the contrast persisted through the critical point as shown by characteristic circle-to-stripe shape transitions. The measured dye concentration in the disordered phase depended on the phase composition and surface pressure, and the dye was expelled from the film at the end of coexistence. The excess concentration of a cationic dye within the double layer adjacent to an anionic phospholipid monolayer was quantified as a function of subphase ionic strength, and the changes in measured excess agreed with those predicted by the mean-field Gouy-Chapman equations. This provided a rapid and noninvasive optical method of measuring the fractional dissociation of lipid headgroups and the monolayer surface potential.lipid domains | lung surfactants | surface potential | phase behavior L ipid monolayers have long been used as a simplified model for cell membranes (1-4), especially for the study of 2D phase separation that may underlie the raft hypothesis of cell membrane organization (5-8). Monolayers are also of interest in the physics of ordering and dynamic processes in two dimensions such as the structure and flow of hexatic phases (9-11). Additionally, all air-breathing mammals have a lipid-protein monolayer lining the lung alveoli to minimize surface tension effects on breathing; deficiency or disruption of this monolayer leads to potentially fatal respiratory distress syndrome in infants and adults (12, 13). The general acceptance of complex monolayer and bilayer phase behavior, especially phase coexistence (14-16) and critical phenomena (7,8,17,18), has relied on the visualization of fluorescently tagged lipids segregated between phases in the monolayer. This segregation, due to differences in local molecular organization, provides the necessary contrast to visualize even subtle differences in packing density, molecular tilt, and short-and long-range ordering. Since the technique was introduced (14,(19)(20)(21), visualization of the distribution of labeled lipids has settled numerous debates over the molecular organization of monolayers (2, 4, 22, 23) and bilayers (8, 24).However, fluorescently tagged 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 ...

Protons at the speed of sound: Predicting specific biological signaling from physics

“…With this numbers and the known dissociation degree of 88% of the carboxyl groups at pH 7 ( α = 0.88) [ Fig. 3 ] the head group potential ψ follows from the Gouy-Chapman theory (note: the negatively charged phosphate group and the positively charged amino groups compensate each other) 34 35 :…”

Interfacial Stress in the Development of Biologics: Fundamental Understanding, Current Practice, and Future Perspective

“…These techniques typically require an extrinsic fluorophore to label the protein or molecule of interest, but carefully choosing the dye and limiting the labeling can allow for excellent visualization while not compromising the interfacial behavior of the molecule. Confocal microscopy, which provides good axial resolution for selectively observing interfacial phenomena, can be used to quantify the amount of protein adsorbed to an interface either in a Langmuir trough ( 113 ) or in a microtrough requiring less than 100 μL of solution ( 10 ). Total internal reflection fluorescence (TIRF) microscopy utilizes an evanescent wave to selectively visualize molecules within a few hundred nanometers from a solid–liquid or liquid–liquid interface.…”

Evolution and mechanics of mixed phospholipid fibrinogen monolayers

“…In pure DPPC, LE -LC coexistence occurs at higher surface pressure as temperature is increased, and is suppressed entirely for T 418C, beyond which LE DPPC exists at all P [44,[46][47][48]. Hermans & Vermant [44] demonstrated the importance of temperature in determining the rheological response of a DPPC monolayer, although viscous-dominated behaviour is observed up to 458C and P ¼ 45 mN m 21 .…”

“…Upon compression, the surface pressure increases until a plateau is reached, representing coexistence between liquid expanded and liquid condensed (LC) DPPC. The surface pressure of LE -LC coexistence increases with temperature[44,[46][47][48], but its onset occurs at approximately 4 mN m 21 at 208C. In this region domains of LC DPPC nucleate and grow, until the entire monolayer is in the LC state at P % 10 mN m 21 .…”