Annette Svendsen

Experimental studies of the photophysics of gas-phase fluorescent protein chromophores

To better understand the photophysics of photoactive proteins, the absorption bands of several gas-phase biomolecules have been studied recently at the electrostatic heavy-ion storage ring ELISA by a photo-fragmentation technique. In the present paper we discuss the involved photophysics and photochemistry for protonated and deprotonated model chromophores of the Green Fluorescent Protein (GFP) and the Red Fluorescent Protein (RFP). We show specifically that the delayed dissociation after photoabsorption can be understood in terms of a thermally activated process of the Arrhenius type. The rate of dissociation as a function of time after laser excitation is modeled in a calculation which is based on calculated heat capacities of the chromophores. Absorption of only one photon is enough to dissociate the deprotonated GFP chromophore on a time scale of milliseconds whereas absorption of two to three photons occurs for other chromophore ions. The difference is attributed to different activation energies, pre-exponential factors and locations of the absorption bands. We obtain activation energies for the dissociation that are of the order of 1–3 eV. Collision-induced dissociation experiments were performed to help identifying the fragmentation channels. Loss of methyl is found to be the dominant fragmentation channel for the deprotonated GFP chromophore and is also likely to be important for the protonated GFP chromophore at high temperatures. Other channels are open for the RFP chromophores. For the deprotonated RFP chromophore there is evidence that dissociation occurs through a non-trivial dissociation with substantial rearrangement.

Dissociative recombination of rare gas hydride ions: II. ArH +

A storage ring measurement of the rate coefficient for the production of neutral Ar in e + ArH + collisions is described. It is found that the recombination rate is too small to measure at low centre-of-mass energies but the combined rate coefficient for dissociative recombination and dissociative excitation increases above 2.5 eV displaying peaks centred at 7.5 eV, 16 and 26 eV. Calculated potential energy curves for the ground and excited states of ArH + are presented and these aid in the elucidation of the recombination and excitation processes observed at higher energies. The implications for plasma modelling are discussed.

The dissociative recombination of fluorocarbon ions: II. CF+

The dissociative recombination and excitation of CF+ have been measured at the ASTRID and CRYRING storage rings. Though examination of the available potential energy curves would suggest that the recombination rate would be large for this ion, in fact a rate constant of 5.2 ± 1.0 × 10−8 (Te/300)−0.8 cm3 s−1 was found. The recombination cross section at low energies falls off to a minimum at 0.5 eV centre-of-mass collision energy but exhibits resonances at energies above this. The dissociative excitation cross section leading to C+ + F was also measured and this displays an onset beginning at about 7 eV.

A cylindrical quadrupole ion trap in combination with an electrospray ion source for gas-phase luminescence and absorption spectroscopy

A relatively simple setup for collection and detection of light emitted from isolated photo-excited molecular ions has been constructed. It benefits from a high collection efficiency of photons, which is accomplished by using a cylindrical ion trap where one end-cap electrode is a mesh grid combined with an aspheric condenser lens. The geometry permits nearly 10% of the emitted light to be collected and, after transmission losses, approximately 5% to be delivered to the entrance of a grating spectrometer equipped with a detector array. The high collection efficiency enables the use of pulsed tunable lasers with low repetition rates (e.g., 20 Hz) instead of continuous wave (cw) lasers or very high repetition rate (e.g., MHz) lasers that are typically used as light sources for gas-phase fluorescence experiments on molecular ions. A hole has been drilled in the cylinder electrode so that a light pulse can interact with the ion cloud in the center of the trap. Simulations indicate that these modifications to the trap do not significantly affect the storage capability and the overall shape of the ion cloud. The overlap between the ion cloud and the laser light is basically 100%, and experimentally >50% of negatively charged chromophore ions are routinely photodepleted. The performance of the setup is illustrated based on fluorescence spectra of several laser dyes, and the quality of these spectra is comparable to those reported by other groups. Finally, by replacing the optical system with a channeltron detector, we demonstrate that the setup can also be used for gas-phase action spectroscopy where either depletion or fragmentation is monitored to provide an indirect measurement on the absorption spectrum of the ion.

Characterization of a new electrostatic storage ring for photofragmentation experiments

We describe the design of and the first commissioning experiments with a newly constructed electrostatic storage ring named SAPHIRA (Storage Ring in Aarhus for PHoton-Ion Reaction Analysis). With an intense beam of Cu(-) at 4 keV, the storage ring is characterized in terms of the stored ion beam decay rate, the longitudinal spreading of an injected ion bunch, as well as the direct measurements of the transverse spatial distributions under different conditions of storage. The ion storage stability in SAPHIRA was investigated systematically in a selected region of its electrical configuration space.

The dissociative recombination of fluorocarbon ions III: CF+2 and CF+3

Cross sections and branching ratios are presented for the dissociative recombination of the CF+2 and CF+3 ions with electrons. It is found that the channel producing CF + F is dominant for the reaction with CF+2 and the production of CF2 + F is dominant for the reaction with CF+3. The cross sections for these two ions are very similar.

Origin of the Intrinsic Fluorescence of the Green Fluorescent Protein

Green fluorescent protein, GFP, has revolutionized biology, due to its use in bioimaging. It is widely accepted that the protein environment makes its chromophore fluoresce, whereas the fluorescence is completely lost when the native chromophore is taken out of GFP. By the use of a new femtosecond pump-probe scheme, based on time-resolved action spectroscopy, we demonstrate that the isolated deprotonated GFP chromophore can be trapped in the first excited state when cooled to 100 K. The trapping is shown to last for 1.2 ns, which is long enough to establish conditions for fluorescence and consistent with calculated trapping barriers in the electronically excited state. Thus, GFP fluorescence is traced back to an intrinsic chromophore property, and by improving excited-state trapping, protein interactions enhance the molecular fluorescence.

Lifetime measurement of the metastable Be- ion using an electrostatic ion storage ring

The lifetime of the metastable Be-(2s2p2 4P3/2) ion has been measured to be 43.40±0.10 µs by means of an electrostatic heavy-ion storage ring. This value is in good agreement with the previously reported lifetime obtained using a magnetic storage ring, but more than 3% longer than the lifetime recently measured utilizing a newly constructed electrostatic ion trap.

Electron scattering on OH−(H2O)n clusters (n=0–4)

The cross sections for electron scattering on OH-(H2O)n for n = 0-4 were measured from threshold to approximately 50 eV. All detachment cross sections were found to follow the classical prediction given earlier [Phys. Rev. Lett. 74, 892 (1995)] with a threshold energy for electron-impact detachment that increased upon sequential hydration, yielding values in the range from 4.5 eV +/- 0.2 eV for OH- to 12.10 eV +/- 0.5 eV for OH-(H2O)4. For n > or = 1, we found that approximately 80% of the total reaction events lead to electron detachment plus total dissociation of the clusters into the constituent molecules of OH and H2O. Finally, we observed resonances in the cross sections for OH-(H2O)3 and for OH-(H2O)4. The resonances were located at approximately 15 eV and were ascribed to the formation of dianions in excited states.

Analysis of ionic photofragments stored in an electrostatic storage ring

A new method to analyze the properties of fragment ions created in storage ring experiments is presented. The technique relies on an acceleration of ionic fragments immediately after production whereby the fragments are stored in the storage ring. To obtain a fragment mass spectrum, the storage ring is exploited as an electrostatic analyzer (ESA) in which case the number of stored fragment ions is recorded as a function of the applied acceleration potential. However, the storage ring can additionally be employed as a time-of-flight (TOF) instrument by registering the temporal distribution of fragment ions. It is demonstrated that the combined ESA-TOF operation of the ring allows not only to determine fragment masses with much better resolution compared to the ESA mode alone but also enables the extraction of detailed information on the fragmentation dynamics. The method is described analytically and verified with photodissociation experiments on stored Cl2 (-) at an excitation wavelength of 530 nm.