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In preclinical cancer research, bioluminescence imaging with firefly luciferase and  D -luciferin has become a standard to monitor biological processes both  in vitro  and  in vivo . However, the emission maximum ( λ  max ) of bioluminescence produced by  D -luciferin is 562 nm where light is not highly penetrable in biological tissues. This emphasizes a need for developing a red-shifted bioluminescence imaging system to improve detection sensitivity of targets in deep tissue. Here we characterize the bioluminescent properties of the newly synthesized luciferin analogue, AkaLumine-HCl. The bioluminescence produced by AkaLumine-HCl in reactions with native firefly luciferase is in the near-infrared wavelength ranges (λ max =677 nm), and yields significantly increased target-detection sensitivity from deep tissues with maximal signals attained at very low concentrations, as compared with  D -luciferin and emerging synthetic luciferin CycLuc1. These characteristics offer a more sensitive and accurate method for non-invasive bioluminescence imaging with native firefly luciferase in various animal models.

A luciferin analogue generating near-infrared bioluminescence achieves highly sensitive deep-tissue imaging