A system and method that images biological samples and uses chromophores to analyze the imaged samples. The chromophore analysis can be done by itself or in conjunction with fluorophore analysis in High Content Imaging systems. To perform chromophore analysis the biological samples can be labeled with different chromophores and imaged using transmitted light that is at least partially absorbed by the chromophores. To also perform fluorophore analysis the samples can also be labeled with fluorophores that are excited by excitation light. The chromophore analysis and fluorophore analysis can be performed separately or concurrently using a High Content Imaging system. The system provides the expanded capability by illuminating the chromophore-labeled samples with transmitted light of different wavelengths and automatically detecting the images which represent the differential absorption of the colored lights by the sample.