Imaging of objects within tissue is enhanced by applying a contrast agent to a sample to be imaged to augment the emissions from an object, thereby forming a luminous object. The tissue is then illuminated and two image signals are recorded. The contrast agent is selected to bind to the object and provide spectral characteristics significantly different from that of the tissue for the two recorded image signals. The two image signals are subtracted to substantially minimize an image component resulting from the tissue and enhance an image component from the luminous object. The imaging methods and apparatus are particularly well suited for medical imaging where the object is diseased tissue such as tumors.