Figure 2.

Lamellarity analysis and a comparison of the lamellarity of liposomes prepared by the inverted emulsion and hydration methods. (A) The membrane fluorescence intensities of individual liposomes prepared by the inverted emulsion method were plotted against their diameter. The lowest black solid line indicates the theoretical curve I(r) fitted to the lowest fluorescence intensity group of the plots. The upper four black solid lines are two-, three-, four-, and fivefold larger than the I(r). The black dashed lines are 1.5-, 2.5-, 3.5-, 4.5-, and 5.5-fold larger than the I(r). We determined that the liposomes with intensities that are under the lowest dashed line were unilamellar, whereas the liposomes that are between the lowest and second lowest dashed lines were bilamellar, and so on. Egg PC (1 mM) containing 0.1% (mol/mol) rhodamine PE dissolved in oil was used to prepare the liposomes. (B) A schematic illustration of the side view of a liposome under an epifluorescence microscope. The red circle represents the membrane of the liposome with radius r. The horizontal axis represents the focal plane. O: center of mass. δ: pixel size of the image. The fluorescence signal of the membrane out of the focal plane, illustrated as the green area, may contribute to the apparent fluorescence intensity measured in the focal plane. (C) Plots of the membrane fluorescence intensities of individual liposomes prepared by the hydration method. Egg PC containing 0.1% (mol/mol) rhodamine PE was used to prepare the liposomes. (D) Lamellarity distributions of the liposomes prepared by the inverted emulsion and hydration methods. Several independent experiments were performed and the total counts were plotted. The proportion of unilamellar liposomes prepared by the two methods were compared to each other with the χ²test and Fisher’s exact test. ^∗∗∗p < 0.001.