Quantum yield of near-infrared bioluminescence with firefly luciferin analog: AkaLumine

• The bioluminescence quantum yield for d -AkaLumine with Photinus pyralis is 4.0±0.5%. • This bioluminescence quantum yield is independent of the pH value. • The bioluminescence spectrum for d -AkaLumine is fit well by two Gaussian components. • The decay rate of bioluminescence with d -AkaLumine at pH 8.0 is 7.0 × 10 -2 sec -1 . • This decay rate is very sensitive to the pH value. We investigated bioluminescence with d - AkaLumine, which is a firefly luciferin analog producing 675-nm light through chemical reaction with O 2 , ATP, and Mg 2+ in native firefly luciferase, and is useful for in vivo imaging. Using a total-photon flux spectrometer system calibrated for 450–950 nm, we evaluated the bioluminescence quantum yield for d - AkaLumine, characterizing the concentration of d - AkaLumine solutions using qNMR spectroscopy and HPLC measurements. We found that the bioluminescence quantum yield for d - AkaLumine with Photinus pyralis is insensitive to pH and that its value is 4.0±0.5%. The bioluminescence spectrum for d - AkaLumine is well fit by two simple Gaussian components. The decay rate for d - AkaLumine is very sensitive to the pH value, and it increases to 7.0 × 10 -2 sec -1 at pH 8.0. For stable absorption spectra for 4–20-μM AkaLumine aqueous solutions, we determined the molar extinction coefficient at 490 nm to be 3.33 × 10 4 L/mol cm.