A plastic mouse phantom (XPM2, Perkin Elmer, Fig. 4), with two internal light sources, was imaged using the BLDOT system. Images were taken at , 620, and 640 nm with 10-nm bandwidth filters (Thorlabs Inc.) and at , 623, and 643 nm, where each filter had a different bandwidth: 22, 32, and 34 nm, respectively (Semrock Inc.). The emission characteristics of the sources within the XPM2 phantom have been previously characterized and the spectral optical properties have been experimentally determined23 and are accounted for within the reconstruction. Surface measurements were calibrated using a free-space model,24 which uses the bioluminescence images taken during the experiment along with information about the dimensions of the imaging system, to model the transport of light through the space between the surface of the imaging subject and the CCD. This enables accurate surface measurements to be calculated from raw CCD data.