Figure 5

Contour plot of microvesicle release superimposed on phase maps of membrane properties observed previously with the fluorescent probes merocyanine 540, laurdan, and diphenylhexatriene. The white contour lines represent increments of 0.08 normalized units of light scatter intensity change (same units as Figure 4). Contour lines were obtained by a two-step nonlinear regression as described previously [1]. Data analogous to those displayed in Figure 4 were first fit to arbitrary functions (e.g. polynomials) with curves for each cholesterol concentration and with temperature as the independent variable. The idealized values obtained from these regressions were then fit again with cholesterol concentration as the independent variable and separate curves for each temperature. The optimized values from the second regression were then used to generate a 3-dimensional surface with the Z-axis (intensity) oriented perpendicular to the plane of the figure. These contour plots were superimposed on phase maps for erythrocytes reported previously [1]. Panel A: Merocyanine 540 fluorescence intensity; increasing brightness indicates increasing intensity (from 0.64 to 0.89 normalized units). Panel B: Laurdan fluorescence generalized polarization (GP, see Ref. [1] for details); increasing brightness indicates decreasing GP (from 0.26 to 0.022 GP units). Panel C: Diphenylhexatriene anisotropy; increasing brightness indicates decreasing anisotropy (from 0.24 to 0.18 anisotropy units). Panels D-F: The values used to generate the phase maps ("probe fluorescence") were plotted against the values used to generate the white contour lines ("microvesicle release") for merocyanine 540 (D), laurdan (E), and diphenylhexatriene (F). The solid lines represent perfect correlation for reference (not a linear regression).