In this example, one of the controls was stained with BV711. BV711 appears brightest in detector V13. This is considered the target detector.

For a single-stained control that is brighter, the relative intensity in the target detector (V13) is significantly higher than in the non-target detectors. For a single-stained control that is dimmer, the relative intensity in the target detector (V13) is about the same as in the non-target detectors.

Relative brightness of the single-stained control	Gate used for unmixing	Spectrum Plot
Brighter
Dim
Very dim

This disparity is numerically reflected in the resulting unmixing matrix. While the target detector is assigned a normalized intensity value of 1 in all three cases, the values assigned to non-target detectors are higher when using a dimmer control. Since those values are closer to 1, the unmixing matrix expects more of the BV711 fluorophore to appear in the non-target detectors.

This difference is visualized on the plot below (the visualized mixing matrix). When a dimmer control is used, a higher unmixing matrix value (normalized intensity) is assigned to non-target detectors.

In this example, the other single-stained controls used to calculate unmixing were bright. Brightness of the BV711 control was varied. Unmixing was applied and a representative plot (BV711 vs PE-Dazzle594) is shown below. You can see that the accuracy of unmixing decreased as the brightness of the  BV711 control decreased.

Brighter Control	Dim Control	Very Dim Control

Plots displaying all parameters against BV711 for the three levels of brightness are shown below to further demonstrate the change in unmixing accuracy.

A brighter BV711 control yields accurate unmixing.

With a dimmer BV711 control, the unmixing is less accurate. For the events with high BV711 intensity, a smaller portion of the overall detected fluorescence is attributed to the other fluorophores.

With a very dim control, the unmixing is very inaccurate.