Described herein are systems and methods for obtaining a three-dimensional (3D) representation of the distribution of fluorescent probes inside a sample, such as a mammal. Using a) fluorescent light emission data from one or more images, b) a surface representation of the mammal, and c) computer-implemented photon propagation models, the systems and methods produce a 3D representation of the fluorescent probe distribution in the mammal. The distribution may indicate--in 3D--the location, size, and/or brightness or concentration of one or more fluorescent probes in the mammal.