Fig. 2.

Spectral and lifetime analysis of laurdan emission of control and cholesterol-depleted RBC (A) Spectral analysis of laurdan’s fluorescence. The normalized emission spectrum of the probe in healthy RBC is reported for control (green) and MβCD-treated (red) RBC, respectively. The red-shift in the emission wavelength as a consequence of the increased membrane micropolarity, results in a clockwise rotation of the center of mass of the clouds on the phasor plot. The spectral phasor analysis allows a fine polarity driven segmentation for RBC membranes. Pixels selected in the blue ROI corresponds to the regions with the highest membrane micropolarity, while the pixels in the red ROI corresponds to the lowest membrane micropolarity. Regions characterized by an intermediate micropolarity value are shown in a pseudo-colored scale from red to blue. Figure lookup table is reported along with the phasor plot. (B) decay curves in the green channel (emission 540/50 nm) of control and cholesterol depleted RBC. Decay curves are analyzed through lifetime phasor transformation to quantify and visualize changes in viscosity. The phasor distributions integrated for N = 100 cells (in each category) show the center of mass of the phasor that lies outside of the universal circle for the control cells, indicating a non-exponential decay. Upon depletion of cholesterol, the point rotates towards the universal circle. The lifetime phasor analysis allows a viscosity driven segmentation, from blue (low viscosity) to red (high viscosity). Figure lookup table is reported along with the phasor plot. The color-coded regions of interest are chosen as rectangles, with the shortest sides perpendicular to the line connecting the center of mass of the clouds in the phasor plot of untreated and treated cells. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)