Red-shifted bioluminescent emitters allow improved in vivo tissue penetration and signal quantification, and have led to the development of beetle luciferin analogues that elicit red-shifted bioluminescence with firefly luciferase. However, unlike natural luciferin, none have been shown to emit different colors with different luciferases. We have synthesized and tested the first dual color, far-red to near infrared (nIR) emitting analogue of beetle luciferin, which akin to natural luciferin exhibits pH dependent fluorescence spectra and emits bioluminescence of different colors with different engineered Fluc enzymes. Our analogue produces different far-red to nIR emission maxima with different Fluc mutants up to .max 706 nm. This is the furthest red-shifted form of bioluminescence reported without the requirement of a resonance energy transfer acceptor and such improvement should allow tissues to be more effectively probed using multiparametric deep-tissue bioluminescence imaging
