Cryogenic Absorption and Emission Spectroscopy of the Oxyluciferin Anion In Vacuo

Abstract

Bioluminescence from fireflies, click beetles, and railroad worms ranges in color from green-yellow to orange to red. The keto form of oxyluciferin is considered a key emitter species in the proposed mechanisms to account for color variation. To establish the intrinsic photophysics in the absence of a microenvironment, we present experimental and theoretical gas-phase absorption and emission spectra of the 5,5-dimethyloxyluciferin anion (keto form) at room and cryogenic temperatures as well as lifetime measurements based on fluorescence. The theoretical model includes all 75 vibrational modes. The spectral impact of the large number of excited states at elevated temperatures is captured by an effective state distribution. At low temperature, spectral congestion is greatly reduced, and the observed well-resolved vibrational features are assigned to multiple Franck−Condon progressions involving different vibrational modes. An in-plane ∼60 cm −1 scissoring mode is found to be involved in the dominant progressions.

Cryogenic fluorescence spectroscopy of oxazine ions isolated in vacuo

“…This gives rise to more than a doubling of the Stokes shift from 14 cm À1 to 37 cm À1 . The Stokes shifts measured for the oxazines are similar to those for rhodamine cations (17-25 cm À1 ) 26 but smaller than those for fluorone anions and that for the dimethyl-oxyluciferin anion; in the latter case the shift is an order of magnitude larger (360 cm À1 ) 14 Both absorption and fluorescence spectra of rhodamine cations are, however, much less well-resolved than those of oxazines, which is likely associated with differences in the importance of low frequency modes.…”

“…The fluorescence spectrum (blue) exhibits maximum intensity at 565.5 AE 0.5 nm, and the spectrum is significantly broader but still shows appreciable vibrational structure. In our previous work 9,14,25,26 on a range of similar fused three-ring systems (pyronin Y, rhodamines, fluorones) and dimethyl-oxyluciferin, the fluorescence spectra were also broader and more congested than the fluorescence-excitation spectra. We showed that it was caused by an increased effective temperature of the ions due to excess energy deposited by the absorbed laser photon.…”

“…The instrument (LUNA2) for measuring fluorescence from mass-selected ions isolated in vacuo has been described in detail previously. 14,37 Ions are accumulated in an octopole ion trap and guided to a cylindrical Paul trap every 50 ms (20 Hz). The end-cap electrodes of the trap are in direct thermal contact with a copper container filled with liquid nitrogen.…”

“…11 In the case of fluorescent species, another approach can, however, also be taken as the yield of emitted photons as a function of excitation wavelength indirectly represents the absorption (a fluorescence-excitation spectrum). 10,13,14 This technique also benefits from cryogenic temperatures where fluorescence quantum yields often are higher than at room temperature. Finally, but not least, the combination of absorption and emission spectra provides valuable information on excited-state dynamics and Stokes shifts, the latter defined as the energy difference between the absorption-and fluorescence-band maxima.…”

“…Finally, but not least, the combination of absorption and emission spectra provides valuable information on excited-state dynamics and Stokes shifts, the latter defined as the energy difference between the absorption-and fluorescence-band maxima. 13,14 In the case of isolated molecular ions, the Stokes shift is a proxy of the geometry change in the excited state.…”