Data normalization and scaling based on the hygromycin controls. Measured dye intensities at different temperatures and under different PMT gains are shown. Lines connect the median intensities under the three most common pairs of Cy5/Cy3 PMT settings at each temperature. (a) Before normalization, the Cy5 (red) values are more variable. (b) After normalization, the Cy5 lines are aligned to Cy3 (green) lines, and most red dots are inside green circles. (c) Quadratic curve fit of the difference in log intensities against the difference in Cy3 PMT settings. (d) Intensity values can then be scaled toward the middle Cy3 750 line.