PrismaLuc luciferase enables tunable near-infrared bioluminescence for in vivo imaging.

Bioluminescence imaging (BLI) enables non-invasive monitoring of biological processes in living animals, yet most bioluminescent reporters emit at fixed wavelengths with limited spectral flexibility, constraining their use in multiplexed and deep-tissue applications. Bioluminescence resonance energy transfer (BRET) offers a solution by coupling a luciferase to a proximal fluorescent protein or synthetic fluorophore that absorbs the emitted light and re-emits it at a longer wavelength. However, existing BRET systems built from luciferase-fluorescent protein fusions are limited by suboptimal energy transfer efficiency and the fixed photophysical properties of their genetically encoded chromophores. We recently introduced PrismaLuc, a high-efficiency BRET reporter, in which a circularly permuted NanoLuc is inserted into HaloTag, enabling efficient energy transfer to a covalently bound synthetic fluorophore whose emission wavelength is selected by choice of ligand. In our initial report, PrismaLuc was paired with JF549 HaloTag ligand for two-colour cell-based assays. Here, we extend PrismaLuc to in vivo bioluminescence imaging using far-red and near-infrared HaloTag ligands. In cultured cells, labelling with JF608 and JF669 HaloTag ligands produced emission peaks at 640 and 690 nm, respectively. Direct comparison with established BRET reporters revealed greater photon output at wavelengths above 665 nm, which are less attenuated in tissue. In tumour-bearing mice, PrismaLuc:JF669 produced an emission peak of approximately 700 nm, and in vivo fluorescence imaging confirmed sufficient JF669 HaloTag ligand bioavailability to label PrismaLuc expressed in xenograft tumours. Finally, dual-colour imaging in mice co-implanted with NanoLuc- and PrismaLuc-expressing cells demonstrated clear spectral separation, enabling multiplexed detection within the same animal. Together, these results establish PrismaLuc as a tunable platform for near-infrared and dual-colour bioluminescence imaging in vivo.