Using X-ray absorption spectra to monitor specific radiation damage to anomalously scattering atoms in macromolecular crystallography

Radiation damage in macromolecular crystals is not suppressed even at 90 K. This is particularly true for covalent bonds involving an anomalous scatterer (such as bromine) at the peak wavelength'. It is shown that a series of absorption spectra recorded on a brominated RNA faithfully monitor the extent of cleavage. The continuous spectral changes during irradiation preserve an isosbestic point', each spectrum being a linear combination of zero' and infinite' dose spectra. This easily yields a good estimate of the partial occupancy of bromine at any intermediate dose. The considerable effect on the near-edge features in the spectra of the crystal orientation versus the beam polarization has also been examined and found to be in good agreement with a previous study. Any significant influence of the (C-Br bond/beam polarization) angle on the cleavage kinetics of bromine was also searched for, but was not detected. These results will be useful for standard SAD/MAD experiments and for the emerging radiation-damage-induced phasing' method exploiting both the anomalous signal of an anomalous scatterer and the isomorphous' signal resulting from its cleavage.

Time-resolved X-ray and Optical Studies of Metalloproteins and Iridium Complexes

Mahsa Silatani

A wide variety of physicochemical processes at a molecular level, in particular charge or energy transfer, electronic and vibrational relaxation, are at the origin of biological functionality of proteins and organometallic compounds. The work reported in this thesis is devoted to the study of these molecular dynamics in selected organometallic systems. In particular, it focuses on the role of these dynamics in the ultrafast photophysics and photochemistry of Myoglobin and iridium complexes having a transition metal active center of d6 configuration. The nature of ligand recombination to the active centre has a strong impact on the reactivity and activation of heme proteins. Using picosecond (ps) Fe K-edge X-ray absorption spectroscopy (XAS) we probed the NO-heme recombination with direct sensitivity to the Fe-NO binding in physiological solution. The transient XAS at 70 ps and 300 ps are identical, but they deviate from the difference between the static spectra of deoxyMb and nitrosyl myoglobin demonstrating the formation of an intermediate species, supposedly the 6-coordinated domed form that is populated on a time scale of ~200 ps and relaxes in ~30 ps. A broadband femtosecond (fs) luminescence study of Ir(ppy)3 (Ir1), Ir(ppy)2(pic) (Ir2), [Ir(ppy)2(bpy)]+ (Ir3) and Ir(ppz)3 (Ir4) provides the first extensive picture of their ligand dependence. After excitation to a ligand-centered state (266 nm), we directly clock the relaxation cascade of Ir1 leading to the lowest metal-to-ligand charge transfer (3MLCT) state. The cascade proceeds within â€ 10 fs, being faster than some of the high-frequency modes of the system. In Ir4 the 3MLCT state decays (530 fs) by non-radiative channels. For Ir1-3, the early emission (100s ps the emission evolves to the steady-state showing evidence of dual emission for Ir1,2 due to a double-well minimum of the lowest 3MLCT state. For Ir3, the final emission stems from a ppy to bpy inter-ligand CT state. The studies of the electronic and geometric structure of Ir1-4 by Ir L3 XAS in solution reveals overall identical above-edge multiple scattering resonances and EXAFS indicating similar bond lengths and angles. Simulation of the XAS using TDDFT and FEFF9 shows good overall agreement with the experiment. The near-edge region of the spectra shows distinct differences among the studied complexes. Ir4 has stronger WL intensity than Ir1, indicating weaker Ï-back donation. The XAS spectra of Ir2,3 indicate larger 5d-electron density on the iridium. The XAS spectra of Ir1 and Ir4 show weak pre-edge resonance due to metal (2p3/2)-to-ligand charge transfer excitations. The absence of the pre-edge in Ir2,3 is due to the reduced covalency of the bonds. However, the existence of such transitions remains open for debate since Ir+3 has only occupied t2g orbitals in the low spin configuration. Using ps XAS, we have successfully captured the transient spectrum at the Ir L3-edge of both Ir1 and Ir4 probed 150 ps after laser excitation in solution. Upon excitation of a MLCT band (355 nm), we observed oxidation state change of Ir from +3 to +4. In DCM, the transient spectra are almost identical for both Ir1,4. The only significant changes in the transient spectra are those observed around the WL. The calculations reproduce the blue shift of the absorption spectrum due to the reduced electron density on the Ir, consistent with the MLCT excitation.

EPFL2015

Supercontinuum generation in silicon nitride (Si_3N_4) waveguides for middle infrared spectroscopy

Eirini Tagkoudi

The middle-infrared (mid-IR) spectral range hosts the most intense roto-vibrational absorption lines of many important molecules. Particularly, mid-IR spectroscopy constitutes a unique tool for identifying and quantifying molecular species through their mid-IR spectral fingerprints. Exploiting efficient ways to access the mid-IR is of supreme importance for spectroscopic applications and environmental monitoring. Supercontinuum (SC) sources have been a common way to access this spectral region, using femtosecond fiber lasers and optical fibers as the broadening medium. However, emphasis has recently shifted towards compact SC sources based on integrated platforms. Among the different materials studied for the integration of SC, stoichiometric silicon nitride,

\mathrm{Si_3N_4}

, is one of the most promising. With a transparency window extending from the visible to mid-IR, CMOS compatibility, and a mature fabrication technology combined with flexibility in design,

\mathrm{Si_3N_4}

waveguides represent an attractive solution for on-chip SC generation. The thesis's main aim is to develop a tunable mid-IR SC source based on

\mathrm{Si_3N_4}

waveguides that can efficiently reach the spectral region between 3 - 4

\mathrm{\mu m}

, enabling mid-IR spectroscopy experiments. Towards this goal, nonlinear SC dynamics based on anomalous and all-normal-dispersion (ANDi) pumping regimes are explored in different waveguide designs. In the first part of the thesis, the development of a tunable mid-IR source is presented. Starting from a tunable turn-key femtosecond 2

\mathrm{\mu m}

fiber laser located in the anomalous dispersion regime of cladded waveguides, mid-IR soliton-induced dispersive waves (DWs) are generated. A synergy of proper waveguide geometry and pump positioning enables favorable dispersion profiles towards the mid-IR while suppressing the generation into the visible. Moreover, by switching to a waveguide with different dimensions, the DW can be tuned between the entire 3 - 4

\mathrm{\mu m}

spectral region, reaching record efficiencies up to 35%. The milliwatt output powers of the mid-IR DWs guarantee their utilization in gas spectroscopy. In the second part, the application of the on-chip generated mid-IR source in direct absorption spectroscopy is demonstrated for the first time. The most efficient DW generated at 3.0

\mathrm{\mu m}

is used to detect acetylene in a proof-of-principle spectroscopic experiment. However, due to DWs' limited bandwidth, parallel gas detection is not possible. By leveraging a waveguide that generates a DW centered at 3.5

\mathrm{\mu m}

and fine-tuning the pump wavelength, a larger bandwidth of the DW source is achieved, enabling simultaneous detection of multiple gas-phase species. Using the broad mid-IR DW from a single

\mathrm{Si_3N_4}

waveguide, detection of acetylene, methane, and ethane is successfully demonstrated, with hundreds of ppm detection limit. Finally, a polarization-sensitive SC source based on an un-cladded

\mathrm{Si_3N_4}

waveguide is studied. The waveguide is engineered to yield ANDi at 2

\mathrm{\mu m}

for the TM mode and anomalous dispersion regime for TE mode excitation. A flat and highly coherent ANDi SC or an octave-spanning soliton fission-driven SC source can be selectively generated by simply switching between the pump's polarization states. As a result, the SC has the potential for versatile applications, from broadband on-chip sensing to pulse compression.

EPFL2021