Absorption and fluorescence spectroscopy of cold proflavine ions isolated in the gas phase.

Proflavine, a fluorescent cationic dye with strong absorption in the visible, has been proposed as a potential contributor to diffuse interstellar bands (DIBs). To investigate this hypothesis, it is essential to examine the spectra of cold and isolated ions for comparison. Here, we report absorption spectra of proflavine ions, trapped in a liquid-nitrogen-cooled ion trap filled with helium-buffer gas, as well as fluorescence spectra to provide further information on the intrinsic photophysics. We find absorption- and fluorescence-band maxima at 434.2 ± 0.1 and 434.7 ± 0.3 nm, corresponding to a Stokes shift of maximum 48 cm-1, which indicates minor differences between ground-state and excited-state geometries. Based on time-dependent density functional theory, we assign the emitting state to S2 as its geometry closely resembles that of S0, whereas the S1 geometry differs from that of S0. As a result, simulated spectra involving S1 exhibit long Franck-Condon progressions, absent in the experimental spectra. The latter displays well-resolved vibrational features, assigned to transitions involving in-plane vibrational modes where the vibrational quantum number changes by one. Dominant transitions are associated with vibrations localized on the NH2 moieties. Experiments repeated at room temperature yield broader spectra with maxima at 435.5 ± 1 nm (absorption) and 438.0 ± 1 nm (fluorescence). We again conclude that prevalent fluorescence arises from S2, i.e., anti-Kasha behavior, in agreement with previous work. Excited-state lifetimes are 5 ± 1 ns, independent of temperature. Importantly, we exclude the possibility that a narrow DIB at 436.4 nm originates from cold proflavine cations as the band is redshifted compared to our absorption spectra.

Cryogenic fluorescence spectroscopy of oxazine ions isolated in vacuo.

Recent developments in fluorescence spectroscopy have made it possible to measure both absorption and dispersed fluorescence spectra of isolated molecular ions at liquid-nitrogen temperatures. Absorption is here obtained from fluorescence-excitation experiments and does not rely on ion dissociation. One large advantage of reduced temperature compared to room-temperature spectroscopy is that spectra are narrow, and they provide information on vibronic features that can better be assigned from theoretical simulations. We report on the intrinsic spectroscopic properties of oxazine dyes cooled to about 100 K. They include six cations (crystal violet, darrow red, oxazine-1, oxazine-4, oxazine-170 and nile blue) and one anion (resorufin). Experiments were done with a home-built setup (LUNA2) where ions are stored, mass-selected, cooled, and photoexcited in a cylindrical ion trap. We find that the Stokes shifts are small (14-50 cm-1), which is ascribed to rigid geometries, that is, there are only small geometrical changes between the electronic ground and excited states. However, both the absorption and the emission spectra of darrow-red cations are broader than those of the other ionic dyes, which is likely associated with a less symmetric electronic structure and more non-zero Franck-Condon factors for the vibrational progressions. In the case of resorufin, the smallest ion under study, vibrational features are assigned based on calculated spectra.

Cryogenic Fluorescence Spectroscopy of Ionic Fluorones in Gaseous and Condensed Phases: New Light on Their Intrinsic Photophysics.

Fluorescence spectroscopy of gas-phase ions generated through electrospray ionization is an emerging technique able to probe intrinsic molecular photophysics directly without perturbations from solvent interactions. While there is ample scope for the ongoing development of gas-phase fluorescence techniques, the recent expansion into low-temperature operating conditions accesses a wealth of data on intrinsic fluorophore photophysics, offering enhanced spectral resolution compared with room-temperature measurements, without matrix effects hindering the excited-state dynamics. This perspective reviews current progress on understanding the photophysics of anionic fluorone dyes, which exhibit an unusually large Stokes shift in the gas phase, and discusses how comparison of gas- and condensed-phase fluorescence spectra can fingerprint structural dynamics. The capacity for temperature-dependent measurements of both fluorescence emission and excitation spectra helps establish the foundation for the use of fluorone dyes as fluorescent tags in macromolecular structure determination. We suggest ideas for technique development.

Triangular Rhodamine Triads and Their Intrinsic Photophysics Revealed from Gas-Phase Ion Fluorescence Experiments.

When ionic dyes are close together, the internal Coulomb interaction may affect their photophysics and the energy-transfer efficiency. To explore this, we have prepared triangular architectures of three rhodamines connected to a central triethynylbenzene unit (1,3,5-tris(buta-1,3-diyn-1-yl)benzene) based on acetylenic coupling reactions and measured fluorescence spectra of the isolated, triply charged ions in vacuo. We find from comparisons with previously reported monomer and dimer spectra that while polarization of the π-system causes redshifted emission, the separation between the rhodamines is too large for a Stark shift. This picture is supported by electrostatic calculations on model systems composed of three linear and polarizable ionic dyes in D3h configuration: The electric field that each dye experiences from the other two is too small to induce a dipole moment, both in the ground and the excited state. In the case of heterotrimers that contain either two rhodamine 575 (R575) and one R640 or one R575 and two R640, emission is almost purely from R640 although the polarization of the π-system expectedly diminishes the dipole-dipole interaction.

Empirical Calibration of a Cylindrical Ion Trap for Mass-Selected Gas-Phase Fluorescence Spectroscopy.

The ion motion in a quadrupole ion trap of hyperbolic geometry is well described by the Mathieu equations. A simpler cylindrical ion trap has also gained significance and has been used by us for fluorescence-spectroscopy experiments. This design allows for the easy replacement of the end-cap with a mesh, enhancing the photon collection. It is crucial to obtain a firm understanding of the ion motion in cylindrical ion traps and their capability as mass spectrometers. We present here an empirical method of calibrating a cylindrical ion trap based on fluorescence detection. This can be done nearly background-free in a pulsed experiment. The ions are located at the center of the trap, where the field is primarily quadrupolar, and here an effective Mathieu description is found through an effective geometry parameter. In spectroscopy experiments, high buffer-gas pressures are needed to efficiently cool the ions, which complicates the ions' motion and hence their stability. Still, simulations show that the stability diagram closely aligns with the Mathieu diagram, albeit shifted due to collisions. We map the stability diagram for six molecular ions by fluorescence collection from four cations and two anions spanning m/z from 212 to 647. The stability diagram is parametrized through the Mathieu functions with an m/z-dependent effective geometry parameter and a q-dependent shrinkage of the diagram. Based on the calibration, we estimate the mass resolution to be +7/-3 Da for ions with masses in the hundreds of Da.

Cryogenic Absorption and Emission Spectroscopy of the Oxyluciferin Anion In Vacuo.

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.

Effect of Freezing out Vibrational Modes on Gas-Phase Fluorescence Spectra of Small Ionic Dyes.

While action spectroscopy of cold molecular ions is a well-established technique to provide vibrationally resolved absorption features, fluorescence experiments are still challenging. Here we report the fluorescence spectra of pyronin-Y and resorufin ions at 100 K using a newly constructed setup. Spectra narrow upon cooling, and the emission maxima blueshift. Temperature effects are attributed to the population of vibrational excited levels in S1, and that frequencies are lower in S1 than in S0. This picture is supported by calculated spectra based on a Franck-Condon model that not only predicts the observed change in maximum, but also assigns Franck-Condon active vibrations. In-plane vibrational modes that preserve the mirror plane present in both S0 and S1 of resorufin and pyronin Y account for most of the observed vibrational bands. Finally, at low temperatures, it is important to pick an excitation wavelength as far to the red as possible to not reheat the ions.

A new setup for low-temperature gas-phase ion fluorescence spectroscopy.

Here, we present a new instrument named LUNA2 (LUminescence iNstrument in Aarhus 2), which is purpose-built to measure dispersed fluorescence spectra of gaseous ions produced by electrospray ionization and cooled to low temperatures (<100 K). LUNA2 is, as an earlier room-temperature setup (LUNA), optimized for a high collection efficiency of photons and includes improvements based on our operational experience with LUNA. The fluorescence cell is a cylindrical Paul trap made of copper with a hole in the ring electrode to permit laser light to interact with the trapped ions, and one end-cap electrode is a mesh grid combined with an aspheric condenser lens. The entrance and exit electrodes are both in physical contact with the liquid-nitrogen cooling unit to reduce cooling times. Mass selection is done in a two-step scheme where, first, high-mass ions are ejected followed by low-mass ions according to the Mathieu stability region. This scheme may provide a higher mass resolution than when only one DC voltage is used. Ions are irradiated by visible light delivered from a nanosecond 20-Hz pulsed laser, and dispersed fluorescence is measured with a spectrometer combined with an iCCD camera that allows intensification of the signal for a short time interval. LUNA2 contains an additional Paul trap that can be used for mass selection before ions enter the fluorescence cell, which potentially is relevant to diminishing RF heating in the cold trap. Successful operation of the setup is demonstrated from experiments with rhodamine dyes and oxazine-4, and spectral changes with temperature are identified.