Shieh Ian C, Zasadzinski Joseph A
Department of Chemical Engineering, University of California, Santa Barbara, CA 93106; and.
Chemical Engineering and Materials Science, University of Minnesota, Minneapolis, MN 55455
Proc Natl Acad Sci U S A. 2015 Feb 24;112(8):E826-35. doi: 10.1073/pnas.1419033112. Epub 2015 Feb 9.
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.
在空气-水界面处相分离单层的共聚焦显微镜成像中，对比度可通过水溶性荧光染料选择性吸附到无序单层相来产生。光学切片可将来自亚相的荧光信号降至最低，而将测量的点扩散函数与界面的简单盒式模型进行卷积，可对与单层相关的过量染料浓度进行定量评估。由于液体扩张相和气相中染料吸附的差异，共存的液体扩张相、液体凝聚相和气相得以可视化。在不混溶的液-液相共存期间，染料优先吸附到液体无序相，并且如特征性的圆形到条纹形状转变所示，对比度在临界点处持续存在。在无序相中测量的染料浓度取决于相组成和表面压力，并且在共存结束时染料从薄膜中排出。与阴离子磷脂单层相邻的双层内阳离子染料的过量浓度作为亚相离子强度的函数进行了定量，测量的过量变化与平均场古伊-查普曼方程预测的变化一致。这提供了一种快速且非侵入性的光学方法来测量脂质头基的分数解离和单层表面电位。
