Bioluminescent imaging has proven to be a valuable tool for monitoring physiological and pathological activities at cellular and molecular levels in living small animals. Using biological techniques, target cells can be tagged with reporters which generate characteristic photons in a wide spectrum covering the infra-red range. Part of the diffused light can reach the body surface of a subject/specimen (e.g., a small animal), be separated into several spectral bands using optical means, and collected by a sensitive camera. Systems and methods are disclosed herein for multi-spectral bioluminescence tomography (MBLT), in which an image of an underlying 3D bioluminescent source distribution is synergistically reconstructed from spectrally resolved datasets externally measured. This MBLT process involves two or multiple imaging modalities that produce structural information of the object and optical properties of the object as well to enable and improve the quality of MBLT.