Emission spectra of bioluminescent reporters and interaction with mammalian tissue determine the sensitivity of detection in vivo

Key findings

All four codon-optimized luciferases showed increased activity from 25°C to 37°C, but CBLucs exhibited the greatest thermal activation (6.4-fold for CBGr68, 7.8-fold for CBRed) compared to FLuc (1.9-fold) and hRLuc (2.2-fold). FLuc uniquely displayed a 34-nm spectral red shift from 578 to 612 nm at 37°C, raising its emission fraction above 600 nm from 41% to 64%, approaching CBRed (75–80% above 600 nm).

Spectral imaging in liver, lung, and skin mouse models using 20-nm bandpass filters showed that blue/green-emitting luciferases (hRLuc, CBGr68) experienced the greatest tissue attenuation. Beetle luciferases transmitted 15–25% of photons through skin but only 1–2.5% through deep lung tissue. FLuc and CBRed, with peak emissions above 600 nm, showed the least attenuation and produced 307- and 315-fold greater liver bioluminescence signal than hRLuc (i.p. coelenterazine), respectively.

Gene fusions of CBGr68 and CBRed in both orientations (CB-GR and CB-RG) produced luciferases with broader emission spectra (480–750 nm) featuring dual peaks corresponding to the λmax of each component. The CB-RG fusion produced the highest liver bioluminescence signal (497.8-fold relative to hRLuc i.p.), exceeding all single luciferases. Differential green-light attenuation in deep tissues indicated that these dual-wavelength fusions may enable signal depth estimation in spectral imaging strategies.