Reporter Expression and Tissue Depth Quantification Using Bright and Broad-Range Spectrum Bioluminescence Probes

Bioluminescence imaging is widely used to study biological processes in small animal models. Such technique suffers from absorptive and scattering properties of tissues preventing to assess depth and expression of a given bioluminescent reporter. We developed a broad-band highly bioluminescent probe and studied its selective absorption according to tissue depth using tissue-mimicking phantom. Our probe, named YFP-Nluc consisting of the Yellow Fluorescent Protein (YFP) fused with the Nluc luciferase (Nluc), emits two distinctive peaks at 444 nm (Nluc) and 530 nm (YFP) upon bioluminescence resonance energy transfer (BRET). Upon expression in HEK cells, we show that the YFP-Nluc emission spectra is selectively and gradually modified upon increase of the hemoglobin concentration simulating multiple tissue depths. Furthermore, we found a linear correlation between the hemoglobin concentration and the bioluminescence ratio of YFP-Nluc corresponding to the spectra band-pass of 530-550 and 570-590 nm. Such probe opens possibility to determine, in the context of dual bioluminescence small animal imaging, the depth and signal strength without requiring the use of complex and time-consuming tomography based methods.