Compensation – teasing apart the physical overlap among the emission spectra when an emission is detected in a detector designed to measure signal from another fluorochrome – is a critical, complex and important component of flow cytometry. Fluorochrome emission spectra will often spill over (known as “spillover”) into other detector channels and create a noise in the electronic signals; see the following example.

In this image, the only marker the cells are labeled with is CD45 FITC. You can see a false signal in the PE channel (top, right); it is there because of overlapping emission spectra. Once proper compensation is in place, the histograms should look similar to the following charts.