Figure 2. Fluorescein fluorescence intensities at multiple gain settings.

(A) Fluorescence intensities for samples of 1 nM to 2 μM 6-carboxy fluorescein were recorded at gains of 55% (black, solid), 59% (red circle), 65% (blue circle), 74% (green circle), 81% (purple circle), 96% (black square), 83% × 4 (red square), 100% × 4 (blue square) and 100% × 8 (green square). Data were collected at 5,000 RPM and 20°C. The background fluorescence of a buffer blank was subtracted for each gain setting and data sets were fit to a linear function with a zero offset. In all cases, the linear correlation coefficient R is greater than 0.999. Inset: the data at a gain setting of 55% plotted on a linear scale with the 1 and 2 μM points included. B) Overlay of data recorded at multiple gain settings. The data at different gains were normalized using their fitted slopes to give a signal amplitude of 1 at a concentration of 500 nM 6-carboxy fluorescein. The solid line is a linear fit to the combined data with a correlation coefficient of 0.99994. The data sets at each gain were truncated to remove points where the intensities are less than 5% of the maximum.