towards time-resolved imaging of molecular structure

We demonstrate an experimental method to record snapshot diffraction images of polyatomic gas-phase molecules, which can, in a next step, be used to probe time-dependent changes in the molecular geometry during photochemical reactions with femtosecond temporal and angstrom spatial resolution. Adiabatically laser-aligned 1-ethynyl-4-fluorobenzene (C8H5F) molecules were imaged by diffraction of photoelectrons with kinetic energies between 31 and 62 eV, created from core ionization of the fluorine (1s) level by ≈80 fs x-ray free-electron-laser pulses. Comparison of the experimental photoelectron angular distributions with density functional theory calculations allows relating the diffraction images to the molecular structure

Imaging single cells in a beam of live cyanobacteria with an X-ray laser

There exists a conspicuous gap of knowledge about the organization of life at mesoscopic levels. Ultra-fast coherent diffractive imaging with X-ray free-electron lasers can probe structures at the relevant length scales and may reach sub-nanometer resolution on micron-sized living cells. Here we show that we can introduce a beam of aerosolised cyanobacteria into the focus of the Linac Coherent Light Source and record diffraction patterns from individual living cells at very low noise levels and at high hit ratios. We obtain two-dimensional projection images directly from the diffraction patterns, and present the results as synthetic X-ray Nomarski images calculated from the complex-valued reconstructions. We further demonstrate that it is possible to record diffraction data to nanometer resolution on live cells with X-ray lasers. Extension to sub-nanometer resolution is within reach, although improvements in pulse parameters and X-ray area detectors will be necessary to unlock this potential

Imaging Molecular Structure through Femtosecond Photoelectron Diffraction on Aligned and Oriented Gas-Phase Molecules

This paper gives an account of our progress towards performing femtosecond time-resolved photoelectron diffraction on gas-phase molecules in a pump-probe setup combining optical lasers and an X-ray Free-Electron Laser. We present results of two experiments aimed at measuring photoelectron angular distributions of laser-aligned 1-ethynyl-4-fluorobenzene (C8H5F) and dissociating, laseraligned 1,4-dibromobenzene (C6H4Br2) molecules and discuss them in the larger context of photoelectron diffraction on gas-phase molecules. We also show how the strong nanosecond laser pulse used for adiabatically laser-aligning the molecules influences the measured electron and ion spectra and angular distributions, and discuss how this may affect the outcome of future time-resolved photoelectron diffraction experiments.Comment: 24 pages, 10 figures, Faraday Discussions 17

Imaging single cells in a beam of live cyanobacteria with an X-ray laser

Citation: van der Schot, G., Svenda, M., Maia, F., Hantke, M., DePonte, D. P., Seibert, M. M., . . . Ekeberg, T. (2015). Imaging single cells in a beam of live cyanobacteria with an X-ray laser. Nature Communications, 6, 9. doi:10.1038/ncomms6704There exists a conspicuous gap of knowledge about the organization of life at mesoscopic levels. Ultra-fast coherent diffractive imaging with X-ray free-electron lasers can probe structures at the relevant length scales and may reach sub-nanometer resolution on micron-sized living cells. Here we show that we can introduce a beam of aerosolised cyanobacteria into the focus of the Linac Coherent Light Source and record diffraction patterns from individual living cells at very low noise levels and at high hit ratios. We obtain two-dimensional projection images directly from the diffraction patterns, and present the results as synthetic X-ray Nomarski images calculated from the complex-valued reconstructions. We further demonstrate that it is possible to record diffraction data to nanometer resolution on live cells with X-ray lasers. Extension to sub-nanometer resolution is within reach, although improvements in pulse parameters and X-ray area detectors will be necessary to unlock this potential.Additional Authors: Almeida, N. F.;Odic, D.;Hasse, D.;Carlsson, G. H.;Larsson, D. S. D.;Barty, A.;Martin, A. V.;Schorb, S.;Bostedt, C.;Bozek, J. D.;Rolles, D.;Rudenko, A.;Epp, S.;Foucar, L.;Rudek, B.;Hartmann, R.;Kimmel, N.;Holl, P.;Englert, L.;Loh, N. T. D.;Chapman, H. N.;Andersson, I.;Hajdu, J.;Ekeberg, T

Charge transfer in dissociating iodomethane and fluoromethane molecules ionized by intense femtosecond X-ray pulses

Citation: Boll, R., Erk, B., Coffee, R., Trippel, S., Kierspel, T., Bomme, C., . . . Rudenko, A. (2016). Charge transfer in dissociating iodomethane and fluoromethane molecules ionized by intense femtosecond X-ray pulses. Structural Dynamics, 3(4). doi:10.1063/1.4944344Additional Authors: Marchenko, T.;Miron, C.;Patanen, M.;Osipov, T.;Schorb, S.;Simon, M.;Swiggers, M.;Techert, S.;Ueda, K.;Bostedt, C.;Rolles, D.;Rudenko, A.Ultrafast electron transfer in dissociating iodomethane and fluoromethane molecules was studied at the Linac Coherent Light Source free-electron laser using an ultraviolet-pump, X-ray-probe scheme. The results for both molecules are discussed with respect to the nature of their UV excitation and different chemical properties. Signatures of long-distance intramolecular charge transfer are observed for both species, and a quantitative analysis of its distance dependence in iodomethane is carried out for charge states up to I21+. The reconstructed critical distances for electron transfer are in good agreement with a classical over-the-barrier model and with an earlier experiment employing a near-infrared pump pulse. © 2016 Author(s)

Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser

Citation: Ekeberg, T., Svenda, M., Seibert, M. M., Abergel, C., Maia, F. R. N. C., Seltzer, V., . . . Hajdu, J. (2016). Single-shot diffraction data from the Mimivirus particle using an X-ray free-electron laser. Scientific Data, 3. doi:10.1038/sdata.2016.60Free-electron lasers (FEL) hold the potential to revolutionize structural biology by producing X-ray pules short enough to outrun radiation damage, thus allowing imaging of biological samples without the limitation from radiation damage. Thus, a major part of the scientific case for the first FELs was three-dimensional (3D) reconstruction of non-crystalline biological objects. In a recent publication we demonstrated the first 3D reconstruction of a biological object from an X-ray FEL using this technique. The sample was the giant Mimivirus, which is one of the largest known viruses with a diameter of 450 nm. Here we present the dataset used for this successful reconstruction. Data-analysis methods for single-particle imaging at FELs are undergoing heavy development but data collection relies on very limited time available through a highly competitive proposal process. This dataset provides experimental data to the entire community and could boost algorithm development and provide a benchmark dataset for new algorithms

Diffraction data of core-shell nanoparticles from an X-ray free electron laser

abstract: X-ray free-electron lasers provide novel opportunities to conduct single particle analysis on nanoscale particles. Coherent diffractive imaging experiments were performed at the Linac Coherent Light Source (LCLS), SLAC National Laboratory, exposing single inorganic core-shell nanoparticles to femtosecond hard-X-ray pulses. Each facetted nanoparticle consisted of a crystalline gold core and a differently shaped palladium shell. Scattered intensities were observed up to about 7 nm resolution. Analysis of the scattering patterns revealed the size distribution of the samples, which is consistent with that obtained from direct real-space imaging by electron microscopy. Scattering patterns resulting from single particles were selected and compiled into a dataset which can be valuable for algorithm developments in single particle scattering research.The final version of this article, as published in Scientific Data, can be viewed online at: https://www.nature.com/articles/sdata20174

Three-dimensional view of ultrafast dynamics in photoexcited bacteriorhodopsin

Bacteriorhodopsin (bR) is a light-driven proton pump. The primary photochemical event upon light absorption is isomerization of the retinal chromophore. Here we used time-resolved crystallography at an X-ray free-electron laser to follow the structural changes in multiphoton-excited bR from 250 femtoseconds to 10 picoseconds. Quantum chemistry and ultrafast spectroscopy were used to identify a sequential two-photon absorption process, leading to excitation of a tryptophan residue flanking the retinal chromophore, as a first manifestation of multiphoton effects. We resolve distinct stages in the structural dynamics of the all-trans retinal in photoexcited bR to a highly twisted 13-cis conformation. Other active site sub-picosecond rearrangements include correlated vibrational motions of the electronically excited retinal chromophore, the surrounding amino acids and water molecules as well as their hydrogen bonding network. These results show that this extended photo-active network forms an electronically and vibrationally coupled system in bR, and most likely in all retinal proteins

Fragmentation Dynamics of Small Molecules upon Multiple Ionization by X-Ray Free-Electron Laser Pulses

The ionization and fragmentation dynamics of small molecules (CH3SeH, C2H5SeH, CH3I, and ICl) triggered by intense ultrashort soft X-ray pulses delivered by the Linac Coherent Light Source are investigated employing coincident three-dimensional ion momentum spectroscopy. This work aims at investigating the role of the molecular environment in multiple inner-shell photoionization and the accompanying electronic relaxation processes by studying molecular systems containing a single constituent of high nuclear charge Z, i.e. selenium or iodine, such that photoabsorption is almost exclusively localized at the core-shells of these heavy atoms. By comparing the level of ionization for the molecules containing selenium or iodine with results on isolated krypton and xenon atoms, signatures of efficient charge redistribution within the molecular environment are observed. Measured kinetic energies and angular distributions of the ionic fragments in comparison to the outcome of a simple Coulomb explosion model allow tracking down the evolution of the molecular geometry, revealing considerable displacement of the nuclei on the time scale of sequential multiple ionization. The results obtained have considerable implications for coherent diffractive imaging, providing a direct measure of radiation damage (displacement of nuclei and electronic rearrangement) on the time scale of the X-ray pulse and the length scale of the individual atoms

Studying protein dynamics with X-ray free-electron lasers: Opportunities & Limitations

Protein structure and function are intimately connected. To deduce the mechanisms underlying specific functions, it is therefore of high interest to investigate structural changes during a reaction. Recently, the development of serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) has attracted a great deal of attention by enabling time-resolved (TR) experiments at atomic spatial and femtosecond temporal resolution, thereby allowing unprecedented insight into protein dynamics. The high intensity of the XFEL pulse destroys any sample that has been exposed to the focused beam. A new protein crystal thus needs to be supplied for each pulse. This is typically achieved using a continuously flowing jet. For light-triggered reactions, an optical pulse starts the reaction in crystals of photosensitive proteins and the X-ray pulse then interrogates the system after a given time interval. For such experiments there are two main issues: First, appropriate conditions have to be found for triggering the reaction of interest. Second, the measurement of weak signals is severely limited by the low data collection rate (≤ 120 Hz) at first-generation XFELs. Moreover high sample consumption is an issue at these X-ray sources. The goals of this thesis were therefore twofold: In the first part, techniques were developed to enable studying the ultrafast isomerization following photon absorption by bacteriorhodopsin in a TR-SFX experiment. Extending these results, light-matter interactions changing the incident excitation intensity were quantified based on experiments and calculations. This allowed establishing guidelines how to generally determine appropriate excitation conditions in SFX employing light triggering. These findings are fundamental to avoid multiphoton artefacts arising from excessive excitation and are thus essential for studying biological reactions which take place almost exclusively in the single photon regime. In the second part of this thesis, opportunities and challenges of SFX experiments at next-generation XFELs were explored. These new machines generate X-ray pulses at MHz peak repetition rate and promise significantly higher throughput and more efficient sample usage. However, the short spacing between pulses introduces new challenges: it needs to be ensured that fresh sample is supplied sufficiently fast for each X-ray pulse. Moreover, it has been shown that the XFEL pulse launches shock waves in the sample carrying jet. These may damage sample probed by subsequent pulses. Here, first experiments at MHz peak repetition rate were conducted to investigate both issues. It was demonstrated that data collection of undamaged sample is indeed possible at 1.1 MHz repetition rate. At shorter pulse intervals (corresponding to 4.5 and 9.2 MHz), shock wave induced damage may lead to a significant loss in diffraction resolution of the crystal and even to structural changes in the protein. Together, the results of this thesis delineate the limitations of (TR-) SFX due to XFEL induced shock damage and pave the way towards exploiting the promising capabilities of MHz XFELs, in particular for studying biologically relevant light-triggered reactions in proteins.Proteinstruktur und –funktion sind eng miteinander verbunden. Um die zugrundeliegenden Mechanismen aufzuklären, ist es daher von hohem Interesse, strukturelle Änderungen während einer Reaktion zu verfolgen. Die Entwicklung serieller Femtosekunden-Kristallographie (SFX) an Freie-Elektronen-Lasern im Röntgenbereich (XFEL) hat folglich durch die einmalige Kombination von atomarer räumlicher und Femtosekunden zeitlicher Auflösung viel Aufmerksamkeit erregt, da sie beispiellose Einblicke in die Struktur und Dynamik von Proteinen erlaubt. XFEL Pulse besitzen eine solch hohe Intensität, dass die Probe letztendlich zerstört und für jeden Puls ein neuer Proteinkristall benötigt wird. Ein Flüssigkeitsstrahl (Jet) liefert daher kontinuierlich frisches Material. Mit diesem Ansatz lassen sich auch lichtgesteuerte Reaktionen beobachten, indem ein optischer Puls die Reaktion in einem Kristall aus photosensitiven Proteinen startet, und der Röntgenpuls nach einer festgelegten Zeit das System abfragt. Bei dieser Herangehensweise gibt es zwei grundlegende Probleme: Erstens müssen geeignete Bedingungen zum Starten der Reaktion gefunden werden. Zweitens ist an XFELs der ersten Generation die Messung schwacher Signale durch die geringe Repetitionsrate (≤ 120 Hz) limitiert, die zudem zu einem hohen Probenverbrauch führt. Diese Arbeit hat daher zwei Ziele: Im ersten Teil wurden Methoden entwickelt, die die Grundlage für das Verfolgen der ultraschnellen lichtinduzierten Isomerisierung in Bacteriorhodopsin mittels SFX bildet. Anknüpfend daran wurden die Anregungsintensität ändernde Licht-Materie-Wechselwirkungen mithilfe von Experimenten und Berechnungen quantifiziert, sodass ein allgemeiner Leitfaden für die Bestimmung passender Anregungsbedingungen aufgestellt werden konnte. Dies ist ein entscheidender Schritt für das Vermeiden biologisch irrelevanter Multiphotonen-Effekte. Im zweiten Teil der Arbeit wurden die Chancen und Herausforderungen von SFX an neuen XFELs untersucht, die Röntgenpulse mit bis zu MHz Wiederholrate produzieren können und dadurch versprechen, Durchsatz und Probeneffizienz zu erhöhen. Durch die kurzen Pulsabstände entstehen jedoch neue Probleme: einerseits muss die Zufuhr neuer Kristalle in den Strahl schnell genug geschehen. Andererseits wurde gezeigt, dass der XFEL Puls im Jet Schockwellen auslöst, die die Probe schädigen und so die Messung mit schnell aufeinanderfolgenden Pulsen beeinträchtigen könnte. In dieser Arbeit wurden erste Experimente bei MHz Wiederholrate durchgeführt und beide Problematiken untersucht. Messungen bei 1.1 MHz konnten erfolgreich ohne Beeinträchtigung durchgeführt werden. Es wurde aber auch gezeigt, dass bei kürzeren Pulsintervallen (entsprechend 4.5 und 9.2 MHz) die Schockwelle die Probe schädigen kann und dadurch zu einer reduzierten Auflösung der Kristalle, sowie zu Strukturänderungen im Protein führen können. Die Ergebnisse dieser Arbeit sind wegweisend für das Ausschöpfen der vielversprechenden Möglichkeiten von MHz XFELs, insbesondere für das Beobachten biologisch relevanter, ultraschneller, lichtinduzierter Reaktionen in Proteinen