In ZEN the calculation of the ratio value R of an individual pixel xy in case of a dual wavelength dye is determined as followed:

where:
Ch1xy = 0 if (Ch1xy - background of Ch1) < Threshold of Ch1
Ch1xy = Ch1xy - background of Ch1 if (Ch1xy - background of Ch1) > Threshold of Ch1
The same is true for Ch2xy using the corresponding values for background and threshold.