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.

Gas-phase Förster resonance energy transfer in mass-selected and trapped ions.

Förster Resonance Energy transfer (FRET) is a nonradiative process that may occur from an electronically excited donor to an acceptor when the emission spectrum of the donor overlaps with the absorption spectrum of the acceptor. FRET experiments have been done in the gas phase based on specially designed mass-spectroscopy setups with the goal to obtain structural information on biomolecular ions labeled with a FRET pair (i.e., donor and acceptor dyes) and to shed light on the energy-transfer process itself. Ions are accumulated in a radio-frequency ion trap or a Penning trap where mass selection of those of interest takes place, followed by photoexcitation. Gas-phase FRET is identified from detection of emitted light either from the donor, the acceptor, or both, or from a fragmentation channel that is specific to the acceptor when electronically excited. The challenge associated with the first approach is the collection and detection of photons emitted from a thin ion cloud that is not easily accessible while the second approach relies both on the photophysical and chemical behavior of the acceptor. In this review, we present the different instrumentation used for gas-phase FRET, including a discussion of advantages and disadvantages, and examples on how the technique has provided important structural information that is not easily obtainable otherwise. Furthermore, we describe how the spectroscopic properties of the dyes are affected by nearby electric fields, which is readily discernable from experiments on simple model systems with alkyl or π-conjugated bridges. Such spectral changes can have a significant effect on the FRET efficiency. Ideas for new directions are presented at the end with special focus on cold-ion spectroscopy.

Cryogenic Ion Fluorescence Spectroscopy: FRET in Rhodamine Homodimers and Heterodimers.

The internal electronic communication between two or more light-absorbers is fundamental for energy-transport processes, a field of large current interest. Here the intrinsic photophysics of homo- and heterodimers of rhodamine cations were studied where just two methylene units bridge the dyes. Gas-phase experiments were done on frozen molecular ions at cryogenic temperatures using the newly built LUNA2 mass spectroscopy setup in Aarhus. Both absorption (from fluorescence excitation) and dispersed-fluorescence spectra were measured. In the gas phase, there is no dielectric screening from solvent molecules, and the effect of charges on transition energies is maximum. Indeed, bands are redshifted compared to those of monomer dyes due to the electric field that each dye senses from the other in a dimer. Importantly, also, as two chemically identical dyes in a homodimer do not experience the same field along the long axis, each dye has separate absorption. At low temperatures, it is therefore possible to selectively excite one dye. Fluorescence is dominantly from the dye with the lowest transition energy no matter which dye is photoexcited. Hence this work unequivocally demonstrates Förster Resonance Energy Transfer even in homodimers where one dye acts as donor and the other as acceptor.

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.

Intrinsic fluorescence from firefly oxyluciferin monoanions isolated in vacuo.

Fireflies, click beetles, and railroad worms glow in the dark. The color varies from green to red among the insects and is associated with an electronically excited oxyluciferin formed catalytically by the luciferase enzyme. The actual color tuning mechanism has been, and still is, up for much debate. One complication is that oxyluciferin can occur in different charge states and isomeric forms. We present here emission spectra of oxyluciferin monoanions in vacuo at both room temperature and at 100 K recorded with a newly developed and unique mass-spectroscopy setup specially designed for gas-phase ion fluorescence spectroscopy. Ions are limited to the phenolate-keto and phenolate-enol forms that account for natural bioluminescence. At 100 K, fluorescence band maxima are at 599 ± 2 nm and 563 ± 2 nm for the keto and enol forms, respectively, and at 300 K about 5 nm further to the red. The bare-ion spectra, free from solvent effects, serve as important references as they reveal whether a protein microenvironment redshifts or blueshifts the emission, and they serve as important benchmarks for nontrivial excited-state calculations.

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.

Controlling Light-Induced Proton Transfer from the GFP Chromophore.

The front cover artwork is provided by the groups of Assoc. Prof. Anastasia V. Bochenkova (Lomonosov Moscow State University) and Prof. Lars H. Andersen (Aarhus University). The image shows the quantum nature of wavelength-dependent excited-state proton transfer in gas-phase H-bonded complexes of the GFP chromophore with an anionic proton acceptor. Read the full text of the Article at 10.1002/cphc.202100068.

Controlling Light-Induced Proton Transfer from the GFP Chromophore.

Green Fluorescent Protein (GFP) is known to undergo excited-state proton transfer (ESPT). Formation of a short H-bond favors ultrafast ESPT in GFP-like proteins, such as the GFP S65T/H148D mutant, but the detailed mechanism and its quantum nature remain to be resolved. Here we study in vacuo, light-induced proton transfer from the GFP chromophore in hydrogen-bonded complexes with two anionic proton acceptors, I- and deprotonated trichloroacetic acid (TCA- ). We address the role of the strong H-bond and the quantum mechanical proton-density distribution in the excited state, which determines the proton-transfer probability. Our study shows that chemical modifications to the molecular network drastically change the proton-transfer probability and it can become strongly wavelength dependent. The proton-transfer branching ratio is found to be 60 % for the TCA complex and 10 % for the iodide complex, being highly dependent on the photon energy in the latter case. Using high-level ab initio calculations, we show that light-induced proton transfer takes place in S1 , revealing intrinsic photoacid properties of the isolated GFP chromophore in strongly bound H-bonded complexes. ESPT is found to be very sensitive to the topography of the highly anharmonic potential in S1 , depending on the quantum-density distribution upon vibrational excitation. We also show that the S1 potential-energy surface, and hence excited-state proton transfer, can be controlled by altering the chromophore microenvironment.

The Effect of an Electric Field on the Spectroscopic Properties of the Isolated Green Fluorescent Protein Chromophore Anion.

Here we uncover the direct effect of a high electric field on the absorption by the Green Fluorescent Protein chromophore anion isolated in vacuo based on gas-phase action spectroscopy. Betaine is a strong molecular dipole that creates an electric field of ∼70 MV/cm when attached to the ion at the phenolate oxygen, more than half the actual field from the protein matrix and pointing in the same direction. Nevertheless, the shift in absorption is limited (0.08 eV), supporting earlier conclusions, but subject to much debate, that the protein is rather innocent in perturbing the transition energy. The betaine complexes are readily made by electrospray ionization and in contrast to the bare ions, they dissociate after one-photon absorption. Also, electron detachment is not an open channel complicating the bare ion case. As steric constraints are absent in vacuo, the possibility of turning on fluorescence by an electric field can be tested from experiments on complexes with betaine.

Elucidation of the intrinsic optical properties of hydrogen-bonded and protonated flavin chromophores by photodissociation action spectroscopy.

A model system of the flavin chromophore was synthesized and investigated for its intrinsic optical properties by gas phase action spectroscopy using an ion storage ring. An ammonium group was anchored to this flavin chromophore to allow its transfer to the gas phase by electrospray ionization and for studying the influence of hydrogen bonding and a nearby positive charge. According to calculations one of the hydrogen atoms of the ammonium group favorably forms an intramolecular ionic hydrogen bond to one of the oxygen atoms of the flavin chromophore, and this interaction was found to cause a blueshift of the S0 → S1 transition and a redshift of the S0 → S2 transition. For comparison, the S0 → S1 transition shows little solvent dependence (only in regard to the degree of fine structure). In addition, the influence of protonation of the flavin chromophore was elucidated by experimental and theoretical studies of a simple flavin system. While the position of the S0 → S1 absorption was at identical positions in the gas phase for the intramolecularly hydrogen-bonded and protonated flavin systems, the S0 → S2 absorption was further redshifted for the protonated species. This redshift resulting from protonation was also observed in solution.

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.

Intrinsic photoisomerization dynamics of protonated Schiff-base retinal.

The retinal protonated Schiff-base (RPSB) in its all-trans form is found in bacterial rhodopsins, whereas visual rhodopsin proteins host 11-cis RPSB. In both cases, photoexcitation initiates fast isomerization of the retinal chromophore, leading to proton transport, storage of chemical energy or signaling. It is an unsolved problem, to which degree this is due to protein interactions or intrinsic RPSB quantum properties. Here, we report on time-resolved action-spectroscopy studies, which show, that upon photoexcitation, cis isomers of RPSB have an almost barrierless fast 400 fs decay, whereas all-trans isomers exhibit a barrier-controlled slow 3 ps decay. Moreover, formation of the 11-cis isomer is greatly favored for all-trans RPSB when isolated. The very fast photoresponse of visual photoreceptors is thus directly related to intrinsic retinal properties, whereas bacterial rhodopsins tune the excited state potential-energy surface to lower the barrier for particular double-bond isomerization, thus changing both the timescale and specificity of the photoisomerization.