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Bioluminescence tomography (BLT) is an inverse source problem that localizes and quantifies bioluminescent probe distribution in 3-D. The generic BLT model is ill-posed, leading to nonunique solutions and aberrant reconstruction in the presence of measurement noise and optical parameter mismatches. In this paper, we introduce the knowledge of the number of bioluminescence sources to stabilize the BLT problem. Based on this regularized BLT model, we develop a differential evolution-based reconstruction algorithm to determine the source locations and strengths accurately and reliably. Then, we evaluate this novel approach in numerical, phantom, and mouse studies.