Firefly bioluminescence quantum yield and colour change by pH-sensitive green emission

Firefly bioluminescence(1-19) is the most well-known ideal photoemitter system in biophotonics, known in particular for its extremely high quantum yield, 88 +/- 25% (refs 2,3) or higher(4-6), and its magnificent pH-dependent emission-colour change(3,7) between yellow-green and red, modelled as the chemical equilibrium between two corresponding states(8-14). However, the need for re-examination has also been discussed(4-6). In this letter we quantify quantum yields and colour changes using our new total-photon-flux spectrometer(20,21). We determine the highest quantum yield to be 41.0 +/- 7.4% (1 standard deviation (s.d.) estimate, coverage factor k = 1), and find that bioluminescence spectra are systematically decomposed into one pH-sensitive and two pH-insensitive gaussian components. There is no intensity conversion between yellow-green and red emissions through pH equilibrium, but simple intensity variation of the pH-sensitive gaussian peak at 2.2 eV causes the changes in emission colours. This represents a paradigm shift in the concept of colour determination from long-standing interpretation based on pH equilibrium.