In the present work, dye-based sum-frequency generation (SFG) imaging using sodium 4-[4-(dibutylamino)phenylazo]benzenesulfonate (butyl orange, BO) as a new non-fluorescent specific azo dye is employed to monitor the morphological evolution of giant vesicles (GVs). After loading BO to the membrane of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) single-component GVs, the outermost membranes were clearly visualized using SFG microscopy, which provided images of the distinct outer and inner faces of the lipid bilayers. In addition, SFG-active vesicles were detected also inside the GVs, depending on the dye concentrations. The dye-based SFG imaging technique provided experimental evidence that these oligolamellar vesicles containing an SFG-active interior had been formed after BO loading. The formation process of the oligolamellar vesicles with inner SFG-active vesicles was successfully monitored, and their formation mechanism was discussed.
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