A chromogen separation method for an image of a biological sample stained with four dyes obtained with a three channel imaging device, the method comprising: defining, from a priori knowledge, three dye combinations of three out of the four dyes spatially collocated in the biological sample; obtaining an image of a sample stained with four dyes with an imaging device having a red, green, and blue channel, the image thereby including a plurality of pixels each having a corresponding RGB triplet; projecting extinction coefficients of each dye, in each of the red, green and blue channels of the imaging device, onto an extinction coefficient plane included in a Maxwell equivalent plane, the extinction coefficient plane being defined by extinction coefficient components Ec R, Ec G and Ec B, where Ec R +Ec G +Ec B =1, such that the a priori known three dye combinations are represented by a triangle whose vertices correspond to locations on the extinction coefficient plane of the respective three out of the four dyes; projecting each RGB triplet onto the extinction coefficient plane to determine a corresponding location on the extinction coefficient plane and the corresponding three dye combination of the three out of the four dyes associated therewith by determining which triangle associated with a dye combination surrounds the location of the RGB triplet or which triangle associated with a dye combination is closest to the location of the RGB triple; and separating the image of the sample by determining the pixels in the image corresponding to each previously defined three dye combination in the extinction coefficient plane and by performing corresponding three dye chromogen separation for those pixels. defining, from a priori knowledge, three dye combinations of three out of the four dyes spatially collocated in the biological sample; obtaining an image of a sample stained with four dyes with an imaging device having a red, green, and blue channel, the image thereby including a plurality of pixels each having a corresponding RGB triplet; projecting extinction coefficients of each dye, in each of the red, green and blue channels of the imaging device, onto an extinction coefficient plane included in a Maxwell equivalent plane, the extinction coefficient plane being defined by extinction coefficient components Ec R, Ec G and Ec B, where Ec R +Ec G +Ec B =1, such that the a priori known three dye combinations are represented by a triangle whose vertices correspond to locations on the extinction coefficient plane of the respective three out of the four dyes; projecting each RGB triplet onto the extinction coefficient plane to determine a corresponding location on the extinction coefficient plane and the corresponding three dye combination of the three out of the four dyes associated therewith by determining which triangle associated with a dye combination surrounds the location of the RGB triplet or which triangle associated with a dye combination is closest to the location of the RGB triple; and separating the image of the sample by determining the pixels in the image corresponding to each previously defined three dye combination in the extinction coefficient plane and by performing corresponding three dye chromogen separation for those pixels.