Determination of the background threshold.

<p>(<b>A</b>) Two 8-bit greyscale images of wells showing high (<b>left</b>) and lower (<b>right</b>) intensities of cell staining with similar colony density. (<b>B</b>) For each case, the colony area percentage is plotted as a function of the applied intensity threshold. At this point, the colony area percentage corresponds to the percentage of the well area that is selected based on the criterion that each pixel in the area has an intensity value below a given intensity threshold. (<b>C</b>) First and (<b>D</b>) second derivatives of the colony area percentage function shown in (<b>B</b>), which allow identifying the correct intensity threshold. After the correct threshold has been identified, the colony area parameter gives the percentage of the well area that is occupied by cells. In all plots (<b>B–D</b>), the highlighted region represents the intensity range where only cells are selected. Above that intensity threshold the background starts to be included, which identifies this intensity value as the background threshold.