Figure 1.

Using color to indicate undisplayed measurement values. (A) The three different color mapping functions are shown for the IFNγ measurement. In the first graphic, the histogram of IFNγ fluorescence for stimulated, CD4⁺ T cells is shown in black (see Supplemental Figure 1 for gating). Each of the colored lines shows the mapping of IFNγ intensity (abscissa) to a shade of blue (heat map shown on the right side of the graphic). The purple line represents “uniform” mapping, where the color varies smoothly from black to full blue intensity linearly (with respect to the displayed fluorescence scale), bounded by the 1^st and 99^th percentiles of fluorescence. The orange line represents “percentile” mapping, where the color changes according to the fraction of events below any given intensity. For this, the cumulative display function (CDF) is used; here, the median IFNγ fluorescence is assigned the mid-point color between black and blue. The green line illustrates the “clustered” mapping (see text for derivation). The grey lines identify the relative IFNγ fluorescence intensity that maps to the midpoint of blue color intensity for each of the three different functions. The three heatmaps above the histogram show the complete output color mapping as a function of IFNγ intensity. The three bivariate dot plots illustrate the result using each of the three different color mapping functions. (B) The same illustration as in (A), but using the CD45RA measurement to determine the intensity of the red color assigned to each event. Note how the “clustered” function tends to give events in the same cluster the same color; the red color intensity changes most rapidly in the CD45RA distribution where there are relatively fewer events. (C) The color mapping schemes illustrated in (A) and (B) are simultaneously applied. The first box illustrates the mapping of color within the bivariate distribution of IFNγ and CD45RA; cells expressing both markers will be drawn with a mixture of red and blue, resulting in shades of violet; cells expressing neither will be close to black. The two biviariate graphs show the results of this color mapping. (D) Adding a third parameter, CCR5, to control the green color, results in the full range of colors being used. The triangle illustrates roughly the mixture of colors that are achieved by different amounts of each parameter: for example, events expressing only CD45RA will be red; those with CD45RA and CCR5 will be yellow (i.e., red + green); cells expressing all three markers will be white. Note that it is impossible in two dimensions to display a legend for all possible colors combinations; the triangle is only a rough guide. The two bivariate displays show the full color mapping result; subsets of cells can be readily identified by the color scheme.