Tryptophan-to-heme electron transfer in ferrous myoglobins.

It was recently demonstrated that in ferric myoglobins (Mb) the fluorescence quenching of the photoexcited tryptophan 14 (*Trp(14)) residue is in part due to an electron transfer to the heme porphyrin (porph), turning it to the ferrous state. However, the invariance of *Trp decay times in ferric and ferrous Mbs raises the question as to whether electron transfer may also be operative in the latter. Using UV pump/visible probe transient absorption, we show that this is indeed the case for deoxy-Mb. We observe that the reduction generates (with a yield of about 30%) a low-valence Fe-porphyrin π [Fe(II)(porph(●-))] -anion radical, which we observe for the first time to our knowledge under physiological conditions. We suggest that the pathway for the electron transfer proceeds via the leucine 69 (Leu(69)) and valine 68 (Val(68)) residues. The results on ferric Mbs and the present ones highlight the generality of Trp-porphyrin electron transfer in heme proteins.

Harmonium: An Ultrafast Vacuum Ultraviolet Facility.

Harmonium is a vacuum ultraviolet (VUV) photon source built within the Lausanne Centre for Ultrafast Science (LACUS). Utilising high harmonic generation, photons from 20-110 eV are available to conduct steady-state or ultrafast photoelectron and photoion spectroscopies (PES and PIS). A pulse preserving monochromator provides either high energy resolution (70 meV) or high temporal resolution (40 fs). Three endstations have been commissioned for: a) PES of liquids; b) angular resolved PES (ARPES) of solids and; c) coincidence PES and PIS of gas phase molecules or clusters. The source has several key advantages: high repetition rate (up to 15 kHz) and high photon flux (1011 photons per second at 38 eV). The capabilities of the facility complement the Swiss ultrafast and X-ray community (SwissFEL, SLS, NCCR MUST, etc.) helping to maintain Switzerland's leading role in ultrafast science in the world.

Time-resolved ARPES at LACUS: Band Structure and Ultrafast Electron Dynamics of Solids.

The manipulation of the electronic properties of solids by light is an exciting goal, which requires knowledge of the electronic structure with energy, momentum and temporal resolution. Time- and angle-resolved photoemission spectroscopy (tr-ARPES) is the most direct probe of the effects of an optical excitation on the band structure of a material. In particular, tr-ARPES in the extreme ultraviolet (VUV) range gives access to the ultrafast dynamics over the entire Brillouin zone. VUV tr-ARPES experiments can now be performed at the ASTRA (ARPES Spectrometer for Time-Resolved Applications) end station of Harmonium, at LACUS. Its capabilities are illustrated by measurements of the ultrafast electronic response of ZrSiTe, a novel topological semimetal characterized by linearly dispersing states located at the Brillouin zone boundary.

The LOUVRE Laboratory: State-of-the-Art Ultrafast Ultraviolet Spectroscopies for Molecular and Materials Science.

We describe the facilities for ultraviolet studies in the femtosecond to nanosecond time domain. These facilities consist of: i) a set-up for deep-ultraviolet spectroscopy in the 260-380 nm range in both pump and probe pulses for transient absorption/reflectivity or two-dimensional spectroscopy studies; ii) a set-up for ultrafast fluorescence measurements with detection down to 300 nm. The capabilities of these set-ups are demonstrated by examples on molecular systems, biosystems, nanoparticles and solid materials.

Photo-induced oxidation of the uniquely liganded heme f in the cytochrome b6f complex of oxygenic photosynthesis.

The ultrafast behavior of the ferrous heme f from the cytochrome b6f complex of oxygenic photosynthesis is revealed by means of transient absorption spectroscopy. Benefiting from the use of microfluidic technologies for handling the sample as well as from a complementary frame-by-frame analysis of the heme dynamics, the different relaxation mechanisms from vibrationally excited states are disentangled and monitored via the shifts of the heme α-absorption band. Under 520 nm laser excitation, about 85% of the heme f undergoes pulse-limited photo-oxidation (<100 fs), with the electron acceptor being most probably one of the adjacent aromatic amino acid residues. After charge recombination in 5.3 ps, the residual excess energy is dissipated in 3.6 ps. In a parallel pathway, the remaining 15% of the hemes directly relax from their excited state in 2.5 ps. In contrast to a vast variety of heme-proteins, including the homologous heme c1 from the cytochrome bc1 complex, there is no evidence that heme f photo-dissociates from its axial ligands. Due to its unique binding, with histidine and an unusual tyrosine as axial ligands, the heme f exemplifies a dependence of ultrafast dynamics on the structural environment.

Photo-induced dynamics of the heme centers in cytochrome bc1.

The ultrafast response of cytochrome bc1 is investigated for the first time, via transient absorption spectroscopy. The distinct redox potentials of both c1- and b-hemes allow for a clear differentiation of their respective signals. We find that while the c1-heme photo-product exhibits the characteristics of a 5-coordinated species, the b-hemes presumably undergo photo-oxidation at a remarkably high quantum yield. The c1-heme iron-ligand recombination time is 5.4 ps, in agreement with previous reports on homologous cytochromes. The suggested photo-oxidized state of the b-hemes has a life-time of 6.8 ps. From this short life-time we infer that the electron acceptor must be within van der Walls contact with the heme, which points to the fact that the axial histidine residue is the electron acceptor. The different heme-responses illustrate the flexibility of the c1-heme ligation in contrast to the more rigid b-heme binding, as well as the higher electronic reactivity of the b-hemes within the bc1 complex. This study also demonstrates the remarkable connection between the heme local environment and its dynamics and, therefore, biological function.

A cascade through spin states in the ultrafast haem relaxation of met-myoglobin.

We report on a study of the early relaxation processes of met-Myoglobin in aqueous solution, using a combination of ultrafast broadband fluorescence detection and transient absorption with a broad UV-visible continuum probe at different pump energies. Reconstruction of the spectra of the transient species unravels the details of the haem photocycle in the absence of photolysis. Besides identifying a branching in the ultrafast relaxation of the haem, we show clear evidence for an electronic character of the intermediates, contrary to the commonly accepted idea that the early time relaxation of the haem is only due to cooling. The decay back to the ground state proceeds partially as a cascade through iron spin states, which seems to be a general characteristic of haem systems.

Tenfold sensitivity increase in streak camera detection by propagation synchronous integration without compromising time resolution.

For many applications that involve measuring ultrafast optical phenomena, the streak camera is the device of choice because of its excellent time resolution, its high sensitivity, the possibility to simultaneously measure lifetimes and spectra, and because it can capture the temporal dynamics in a single shot. Nevertheless, to obtain a good time resolution, often narrow slits have to be employed that restrict the image source area and, therefore, limit the light collection efficiency in the experiment. For some applications, it is therefore challenging to find an acceptable balance between the time resolution and signal-to-noise ratio. To overcome this limitation, we have devised the propagation synchronous integration principle for the streak camera, in which an effective spatio-dependent time-shift in the excitation of a sample is introduced and counteracted by the streak sweep, thereby effectively allowing for an increased image source area while maintaining the optimal time resolution. Using the Optronis streak camera with tunable streak sweep and large (1 mm) photocathode width, we could achieve a sevenfold increase in light collection efficiency without affecting the time resolution. Furthermore, we were also able to achieve an 11-fold increase in light collection at the cost of a 26% decrease in the time resolution.

Ultrabroadband femtosecond two-dimensional ultraviolet transient absorption.

We present a broadband two-dimensional transient absorption setup for the UV around 300 nm with a time resolution of 150 fs. A narrowband, frequency tunable pump pulse and a broadband probe pulse are generated from the output of a noncollinear optical parametric amplifier operated at 20 kHz repetition rate and combined in a spectrally resolved transient absorption experiment. The high repetition rate and low noise of the setup allow us to acquire high quality two-dimensional data as a function of time delay with an unsurpassed frequency window of 10,000 and 8000 cm(-1) along the probe and pump axis, respectively. The performance of the setup is demonstrated on 2,5-Diphenyloxazol dissolved in cyclohexane.

Functional electric field changes in photoactivated proteins revealed by ultrafast Stark spectroscopy of the Trp residues.

Ultrafast transient absorption spectroscopy of wild-type bacteriorhodopsin (WT bR) and 2 tryptophan mutants (W86F and W182F) is performed with visible light excitation (pump) and UV probe. The aim is to investigate the photoinduced change in the charge distribution with 50-fs time resolution by probing the effects on the tryptophan absorption bands. A systematic, quantitative comparison of the transient absorption of the 3 samples is carried out. The main result is the absence in the W86F mutant of a transient induced absorption band observed at approximately 300-310 nm in WT bR and W182F. A simple model describing the dipolar interaction of the retinal moiety with the 2 tryptophan residues of interest allows us to reproduce the dominant features of the transient signals observed in the 3 samples at ultrashort pump-probe delays. In particular, we show that Trp(86) undergoes a significant Stark shift induced by the transient retinal dipole moment. The corresponding transient signal can be isolated by direct subtraction of experimental data obtained for WT bR and W86F. It shows an instantaneous rise, followed by a decay over approximately 500 fs corresponding to the isomerization time. Interestingly, it does not decay back to zero, thus revealing a change in the local electrostatic environment that remains long after isomerization, in the K intermediate state of the protein cycle. The comparison of WT bR and W86F also leads to a revised interpretation of the overall transient UV absorption of bR.

Vibrational relaxation and intersystem crossing of binuclear metal complexes in solution.

The ultrafast vibrational-electronic relaxation upon excitation into the singlet (1)A(2u) (dσ*→pσ) excited state of the d(8)-d(8) binuclear complex [Pt(2)(P(2)O(5)H(2))(4)](4-) has been investigated in different solvents by femtosecond polychromatic fluorescence up-conversion and femtosecond broadband transient absorption (TA) spectroscopy. Both sets of data exhibit clear signatures of vibrational relaxation and wave packet oscillations of the Pt-Pt stretch vibration in the (1)A(2u) state with a period of 224 fs, that decay on a 1-2 ps time scale, and of intersystem crossing (ISC) into the (3)A(2u) state. The vibrational relaxation and ISC times exhibit a pronounced solvent dependence. We also extract from the TA measurements the spectral distribution of the wave packet at a given delay time, which reflects the distribution of Pt-Pt bond distances as a function of time, i.e., the structural dynamics of the system. We clearly establish the vibrational relaxation and coherence decay processes, and we demonstrate that PtPOP represents a clear example of a harmonic oscillator that does not comply with the optical Bloch description due to very efficient coherence transfer between vibronic levels. We conclude that a direct Pt-solvent energy dissipation channel accounts for the vibrational cooling in the singlet state. ISC from the (1)A(2u) to the (3)A(2u) state is induced by spin-vibronic coupling with a higher-lying triplet state and/or (transient) symmetry breaking in the (1)A(2u) excited state. The particular structure, energetics, and symmetry of the molecule play a decisive role in determining the relatively slow rate of ISC, despite the large spin-orbit coupling strength of the Pt atoms.

Ultrafast X-ray absorption studies of the structural dynamics of molecular and biological systems in solution.

We review our recent studies of excited state structures and dynamics of chemical and biological systems with pico- and femtosecond X-ray absorption spectroscopy in the liquid phase.

Ultrafast (bio)physical and (bio)chemical dynamics.

We give an overview of our recent work on ultrafast dynamics of chemical (organic dyes, metal-complexes, colloidal quantum dots), and biological (retinal and haem proteins) systems in the liquid phase, studied with a variety of ultrafast optical techniques from the infrared to the ultraviolet.

Ultrafast excited-state dynamics of [Re(L)(CO)3(bpy)]n complexes: involvement of the solvent.

Ultrafast excited-state dynamics of [Re(L)(CO)(3)(bpy)](n) (L = Cl, Br, n = 0; L = 4-ethyl-pyridine (Etpy), n = 1+; bpy = 2,2'-bipyridine) have been investigated in dimethylformamide (DMF) solution by fluorescence up-conversion (FlUC) and UV-vis transient absorption (TA) with approximately 100 fs time resolution. TA was also measured in the [1-ethyl-3-methyl-imidazolium]BF(4) ionic liquid. The complexes show a very broad fluorescence band at 540-550 nm at zero time delay, which decays with 100-140 fs (depending on L) by intersystem crossing (ISC) to a pipi* intraligand ((3)IL) and a Re(L)(CO)(3) --> bpy charge-transfer ((3)CT) excited states. A second emission decay component (1.1-1.7 ps), apparent in the red part of the spectrum, is attributed to (3)IL --> (3)CT conversion, leaving phosphorescence from the lowest (3)CT state as the only emission signal at longer time delays. The triplet conversion is slower in DMF than acetonitrile, commensurate with solvation times. Full assignment of the excited-state absorption at long delay times is obtained by TD-DFT calculations on the lowest triplet state, showing that the 373 nm band is the sole diagnostics of bpy reduction in the CT excited state. Bands in the visible are due to Ligand-to-Metal-Charge-Transfer (LMCT) transitions. Time-resolved UV-vis absorption spectra exhibit a units-of-ps rise of all absorption features attributed to (3)IL --> (3)CT conversion as well as electronic and vibrational relaxation, and a approximately 15 ps rise of only the 373 nm pipi*(bpy(*-)) band, which slows down to approximately 1 ns in the ionic liquid solvent. It is proposed that this slow relaxation originates mainly from restructuring of solvent molecules that are found very close to the metal center, inserted between the ligands. The solvent thus plays a key role in controlling the intramolecular charge separation, and this effect may well be operative in other classes of metal-based molecular complexes.

Compact portable multiphoton microscopy reveals histopathological hallmarks of unprocessed lung tumor tissue in real time.

During lung cancer operations a rapid and reliable assessment of tumor tissue can reduce operation time and potentially improve patient outcomes. We show that third harmonic generation (THG), second harmonic generation (SHG) and two-photon excited autofluorescence (2PEF) microscopy reveals relevant, histopathological information within seconds in fresh unprocessed human lung samples. We used a compact, portable microscope and recorded images within 1 to 3 seconds using a power of 5 mW. The generated THG/SHG/2PEF images of tumorous and nontumorous tissues are compared with the corresponding standard histology images, to identify alveolar structures and histopathological hallmarks. Cellular structures (tumor cells, macrophages and lymphocytes) (THG), collagen (SHG) and elastin (2PEF) are differentiated and allowed for rapid identification of carcinoid with solid growth pattern, minimally enlarged monomorphic cell nuclei with salt-and-pepper chromatin pattern, and adenocarcinoma with lipidic and micropapillary growth patterns. THG/SHG/2PEF imaging is thus a promising tool for clinical intraoperative assessment of lung tumor tissue.

Aqueous solvation dynamics at metal oxide surfaces.

Broadband transient absorption (TA) spectroscopy, three-pulse photon echo peak shift (3PEPS), and anisotropy decay measurements were used to study the solvation dynamics in bulk water and interfacial water at ZrO(2) surfaces, using Eosin Y as a probe. The 3PEPS results show a multiexponential behavior with two subpicosecond components that are similar in bulk and interfacial water, while a third component of several picoseconds is significantly lengthened at the interface. The bandwidth correlation function from TA spectra exhibits the same behavior, and the TA spectra are well reproduced using the doorway-window picture with the time constants from PEPS. Our results suggest that interfacial water is restricted to a thickness of less than 5 A. Also the high-frequency collective dynamics of water does not seem to be affected by the interface. On the other hand, the increase of the third component may point to a slowing down of diffusional motion at the interface, although other effects, may play a role, which are discussed.

A versatile setup for ultrafast broadband optical spectroscopy of coherent collective modes in strongly correlated quantum systems.

A femtosecond pump-probe setup is described that is optimised for broadband transient reflectivity experiments on solid samples over a wide temperature range. By combining high temporal resolution and a broad detection window, this apparatus can investigate the interplay between coherent collective modes and high-energy electronic excitations, which is a distinctive characteristic of correlated electron systems. Using a single-shot readout array detector at frame rates of 10 kHz allows resolving coherent oscillations with amplitudes <10-4. We demonstrate its operation on the charge-transfer insulator La2CuO4, revealing coherent phonons with frequencies up to 13 THz and providing access into their Raman matrix elements.

Ultrafast electronic and vibrational relaxations in mixed-ligand dithione-dithiolato Ni, Pd, and Pt complexes.

Ultrafast excited-state dynamics of planar Pt, Pd, and Ni dithione-dithiolato complexes were investigated by transient absorption spectroscopy on the femtosecond-picosecond timescale. All studied complexes show a common photobehaviour, although individual kinetics parameters and quantum yields vary with the metal, the dithione ligand and, namely the solvent (DMF, MeCN). Laser pulse irradiation at 800 nm populates the lowest singlet excited state of a dithiolato → dithione charge transfer character, (1)LL'CT. The optically excited state undergoes a solvation-driven sub-picosecond electronic relaxation that enhances the dithione/dithiolato charge separation. The (1)LL'CT state decays with a 1.9-4.5 ps lifetime by two simultaneous pathways: intersystem crossing (ISC) to the lowest triplet state (3)LL'CT and non-radiative decay to the ground state. ISC occurs on a ∼6 ps timescale, virtually independent of the metal, whereas the rate of the non-radiative decay to the ground state decreases on going from Ni (2 ps) to Pd (3 ps) and Pt (∼10 ps). (3)LL'CT is initially formed as a vibrationally excited state. Its equilibration (cooling) takes place on a picosecond timescale and is accompanied by a competitive decay to the ground state. Equilibrated (3)LL'CT is populated with a quantum yield of less than 50%, depending on the metal: Pt > Pd > Ni. (3)LL'CT is long-lived for Pt and Pd (≫500 ps) and short-lived for Ni (∼15 ps). Some of the investigated complexes also exhibit spectral changes due to vibrational cooling of the singlet (2-3 ps, depending on the solvent). Rotational diffusion occurs with lifetimes in the 120-200 ps range. Changing the dithione (Bz2pipdt/(i)Pr2pipdt) as well as dithiolate/diselenolate (dmit/dsit) ligands has only small effects on the photobehavior. It is proposed that the investigated dithione-dithiolato complexes could act as photooxidants (*E(o) ≈ +1.2 V vs. NHE) utilizing their lowest excited singlet ((1)LL'CT), provided that the excited-state electron transfer is ultrafast, competitive with the picosecond decay. On the other hand, the efficiency of any triplet-based processes would be severely limited by the low quantum yield of the triplet population.

Ultrafast tryptophan-to-heme electron transfer in myoglobins revealed by UV 2D spectroscopy.

Tryptophan is commonly used to study protein structure and dynamics, such as protein folding, as a donor in fluorescence resonant energy transfer (FRET) studies. By using ultra-broadband ultrafast two-dimensional (2D) spectroscopy in the ultraviolet (UV) and transient absorption in the visible range, we have disentangled the excited state decay pathways of the tryptophan amino acid residues in ferric myoglobins (MbCN and metMb). Whereas the more distant tryptophan (Trp(7)) relaxes by energy transfer to the heme, Trp(14) excitation predominantly decays by electron transfer to the heme. The excited Trp(14)→heme electron transfer occurs in <40 picoseconds with a quantum yield of more than 60%, over an edge-to-edge distance below ~10 angstroms, outcompeting the FRET process. Our results raise the question of whether such electron transfer pathways occur in a larger class of proteins.