Quantitative in vivo dual-color bioluminescence imaging in the mouse brain

Bioluminescence imaging (BLI) is an optical imaging method that can be translated from the cell culture dish in vitro to cell tracking in small animal models in vivo. In contrast to the more widely used fluorescence imaging, which requires light excitation, in BLI the light is exclusively generated by the enzyme luciferase. The luciferase gene can be engineered to target and monitor almost every cell and biological process quantitatively in vitro and even from deep tissue in vivo. While initially used for tumor imaging, bioluminescence was recently optimized for mouse brain imaging of neural cells and monitoring of viability or differentiation of grafted stem cells. Here, we describe the use of bright color-shifted firefly luciferases (Flucs) based on the thermostable x5 Fluc that emit red and green for effective and quantitative unmixing of two human cell populations in vitro and after transplantation into the mouse brain in vivo. Spectral unmixing predicts the ratio of luciferases in vitro and a mixture of cells precisely for cortical grafts, however, with less accuracy for striatal grafts. This dual-color approach enables the simultaneous visualization and quantification of two cell populations on the whole brain scale, with particular relevance for translational studies of neurological disorders providing information on stem cell survival and differentiation in one imaging session in vivo.