In-vivo optical molecular imaging methods for producing an image of an animal are described. A time series of image data sets of an optical contrast substance in the animal is acquired using an optical detector Each image data set is obtained at a selected time and has the same plurality of pixels, with each pixel having an associated value. The image data sets are analyzed to identify a plurality of distinctive time courses, and respective pixel sets are determined from the plurality of pixels which correspond to each of the time courses. In one embodiment, each pixel set is associated with an identified anatomical or other structure, and an anatomical image map of the animal can be generated which includes one or more of the anatomical structures.