A simple and rapid method for determining the linearity of a flow cytometer amplification system

We describe a simple and rapid method for determining the linearity of a flow cytometer amplification system. The method is based on a fundamental characteristic of linear amplifiers: The difference between two amplified signals increases linearly with increasing amplifier gain. Two populations of beads or cells, differing slightly in fluorescence intensity, are analyzed by the flow cytometer at increasing photomultiplier tube high-voltage settings. The distribution of the populations' mean difference versus mean position is a straight line intersecting the origin for linear amplifiers. Although some types of nonlinearities cannot be detected with this technique, deviations from linearity indicate nonlinear components in the flow cytometer amplification system. The correlation coefficient is used to quantify degree of nonlinearity. We also describe a method for amplifier nonlinearity compensation.