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The ability to track the biodistribution and fate of multiple cell populations administered to rodents has the potential to facilitate the understanding of biological processes in a range of fields including regenerative medicine, oncology, and host/pathogen interactions. Bioluminescence imaging is an important tool for achieving this goal, but current protocols rely on systems that have poor sensitivity or require spectral decomposition. Here, we show that a bioluminescence resonance energy transfer reporter (BRET) based on NanoLuc and LSSmOrange in combination with firefly luciferase enables the unambiguous discrimination of two cell populations  in vivo  with high sensitivity. We insert each of these reporter genes into cells using lentiviral vectors and demonstrate the ability to monitor the cells' biodistribution under a wide range of administration conditions, including the venous or arterial route, and in different tissues including the brain, liver, kidneys, and tumours. Our protocol allows for the imaging of two cell populations in the same imaging session, facilitating the overlay of the signals and the identification of anatomical positions where they colocalise. Finally, we provide a method for postmortem confirmation of the presence of each cell population in excised organs.

Multicolour In Vivo Bioluminescence Imaging Using a NanoLuc‐Based BRET Reporter in Combination with Firefly Luciferase