Significance: Bioluminescence imaging and tomography (BLT) are used to study biologically relevant activity, typically within a mouse model. A major limitation is that the underlying optical properties of the volume are unknown, leading to the use of a “best” estimate approach often compromising quantitative accuracy.

Aim: An optimization algorithm is presented that localizes the spatial distribution of bioluminescence by simultaneously recovering the optical properties and location of bioluminescence source from the same set of surface measurements.

Approach: Measured data, using implanted self-illuminating sources as well as an orthotopic glioblastoma mouse model, are employed to recover three-dimensional spatial distribution of the bioluminescence source using a multi-parameter optimization algorithm.

Results: The proposed algorithm is able to recover the size and location of the bioluminescence source while accounting for tissue attenuation. Localization accuracies of <1  mm are obtained in all cases, which is similar if not better than current “gold standard” methods that predict optical properties using a different imaging modality.

Conclusions: Application of this approach, using in-vivo experimental data has shown that quantitative BLT is possible without the need for any prior knowledge about optical parameters, paving the way toward quantitative molecular imaging of exogenous and indigenous biological tumor functionality.