The invention provides for surface mapping of in-vivo imaging subjects using a single camera and a moveable stage on which a subject animal for in-vivo imaging is placed. Images are taken and the stage is moved by known amounts along the optical axis, and the heights of individual features on the subject are determined through analysis of focus, given the known stage displacement. Alternatively, height of sub-regions of the subject are determined through analysis of focus. A mesh or other surface can be constructed from individual features, to provide a surface map of the subject. Accuracy of 0.5 mm or better can be attained for mice and similarly sized subjects.