A method of unmixing an image of a biological sample stained with at least two stains comprising: deriving a total optical density for each channel in R, G, B color space from pixel intensity values in an image or a portion of the image having a first stain and a second stain, wherein at least the first stain is a concentration-dependent stain, wherein: the total optical density corresponding to a respective channel in the R, G, B color space includes a set of optical density values associated with the respective channel; the concentration-dependent stain includes a precipitate forming stain that causes a light-scattering effect on the image or on the portion of the image; obtaining a plurality of prospective first stain color reference vectors in the optical density converted RGB color space, each of the plurality of prospective first stain color reference vectors characterizing the first stain at a different concentration; obtaining at least one second stain color reference vector; deriving a series of candidate color systems from the first stain and second stain color reference vectors, each candidate color system comprising one of the prospective first stain color reference vectors from the plurality of prospective first stain color reference vectors and one of the at least one second stain color reference vector; selecting an optimal color system from the series of candidate color system, the optimal color system selected by (i) computing a reconstruction error for each candidate color system, wherein the reconstruction error of a candidate color system is computed based at least in part on a difference between a first one of the derived total optical density and a reconstructed total optical density, and wherein the reconstructed total optical density is generated based on the prospective first stain color reference vector corresponding to the candidate color system and one of the at least one second stain color reference vector corresponding to the candidate color system and the second and third one of the derived total optical density for each channel, and (ii) determining, from the series of candidate color systems, the candidate color system having a minimal reconstruction error; and unmixing the image using the selected optimal color system. deriving a total optical density for each channel in R, G, B color space from pixel intensity values in an image or a portion of the image having a first stain and a second stain, wherein at least the first stain is a concentration-dependent stain, wherein: the total optical density corresponding to a respective channel in the R, G, B color space includes a set of optical density values associated with the respective channel; the concentration-dependent stain includes a precipitate forming stain that causes a light-scattering effect on the image or on the portion of the image; the total optical density corresponding to a respective channel in the R, G, B color space includes a set of optical density values associated with the respective channel; the concentration-dependent stain includes a precipitate forming stain that causes a light-scattering effect on the image or on the portion of the image; obtaining a plurality of prospective first stain color reference vectors in the optical density converted RGB color space, each of the plurality of prospective first stain color reference vectors characterizing the first stain at a different concentration; obtaining at least one second stain color reference vector; deriving a series of candidate color systems from the first stain and second stain color reference vectors, each candidate color system comprising one of the prospective first stain color reference vectors from the plurality of prospective first stain color reference vectors and one of the at least one second stain color reference vector; selecting an optimal color system from the series of candidate color system, the optimal color system selected by (i) computing a reconstruction error for each candidate color system, wherein the reconstruction error of a candidate color system is computed based at least in part on a difference between a first one of the derived total optical density and a reconstructed total optical density, and wherein the reconstructed total optical density is generated based on the prospective first stain color reference vector corresponding to the candidate color system and one of the at least one second stain color reference vector corresponding to the candidate color system and the second and third one of the derived total optical density for each channel, and (ii) determining, from the series of candidate color systems, the candidate color system having a minimal reconstruction error; and (i) computing a reconstruction error for each candidate color system, wherein the reconstruction error of a candidate color system is computed based at least in part on a difference between a first one of the derived total optical density and a reconstructed total optical density, and wherein the reconstructed total optical density is generated based on the prospective first stain color reference vector corresponding to the candidate color system and one of the at least one second stain color reference vector corresponding to the candidate color system and the second and third one of the derived total optical density for each channel, and (ii) determining, from the series of candidate color systems, the candidate color system having a minimal reconstruction error; and unmixing the image using the selected optimal color system.