Free-electron lasers (FELs) are among the world's most intense artificial artificial sources of coherent light and are tunable to various wavelengths, including the X-ray spectrum. X-ray FELs (XFELs) are extremely useful for diffraction experiments to study molecules, materials, and quantum systems. A FEL consists of an electron accelerator and a structure of magnets called an undulator. The undulator has a periodic magnetic field, and when an electron beam passes through the undulator, the Lorentz force forces the electrons to oscillate and emit what is known asspontaneous undulator radiation. Initially, the undulator radiation is spontaneously emitted and incoherent. However, aAs the electrons interact with this initial spontaneous undulator radiation, they change their relative positions and form micro-bunches of electrons. These microbunches are shorter than the undulator radiation wavelength. Hence, the waves emitted by the electrons from the same microbunch arethey become in phase, meaning the radiation is now coherent with the radiation field, and the state of coherence develops. This process is known as self-amplified spontaneous emission (SASE). Due to the coherence, tThe radiation intensity grows exponentially along the undulator, forming several peaks in the radiation pulse known as SASE spikes. One technique for obtaining ultra-short laser pulses is to isolate single SASE spikes by controlling where, along the electron beam, the SASE spikes can grow. This growth limitation is archieved by modulating the electron energies, thus only allowing electrons at specific positions along the electron beam to radiate. In addition, to keep positive interference between undulator radiation from electrons with different energies, the energy modulation must be compensated with a gradient of the magnetic field amplitude of the undulator, so-called tapering. There are plans to implement this technique at one of the beamlines at the European X-ray FEL (EuXFEL) to generate attosecond X-ray pulses and study quantum systems. One goal of the design process is to choose design parameters for the electron beam's modulation amplitude and the undulator's tapering coefficient. These design parameters shall be chosen so that the XFEL will have as short pulse duration as possible while at the same time not getting too low peak power. This thesis aims to study the effect of electron energy modulation and undulator tapering on the SASE and how the modulation amplitude and the tapering coefficient affect the XFEL's peak power and pulse duration. A model was developed to simulate SASE with a modulated electron beam in a tapered undulator. With this model, a parameter scan gave the average peak power and pulse duration as functions of the modulation amplitude and the tapering coefficient. The parameter scan showed that the peak power and the pulse duration decrease as the modulation amplitude and the tapering coefficient increase. Therefore, a trade-off exists between high peak power and short pulse duration. It was possible to exclude sets of the parameters that gave too low peak power or long pulse duration. This study also found an optimum range for the tapering coefficient where the peak power had a local maximum without a significant increase in pulse duration. The physics behind this optimal tapering coefficient is also discussed in connection to the electrons' energy modulation.

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Approved for public release, distribution is unlimited / The Free Electron Laser has the potential to become a revolutionary weapon system. Deep magazines, low cost-per-shot, pinpoint accuracy, and speed of light delivery give this developing weapon system significant advantages over conventional systems. One limiting factor in high energy laser implementation is thermal blooming, a lensing effect which is caused by the quick heating of the atmosphere, so that the laser beam does not focus on the desired spot, thereby degrading the effectiveness of the laser on target. The use of multiple beam directors focusing on a target from a single platform may mitigate thermal blooming by allowing half of the laser's energy to travel through a given volume of air, so that they only overlap very near the target. Less energy traveling through a given volume of space means less heating, and therefore lessens the effects of thermal blooming. Also, simulations of FEL's were conducted modifying parameters such as the number of undulator periods, electron beam focus, the normalized Rayleigh length, and mirror output coupling, in order to determine optimum design parameters. New parameters for the next proposed FEL were simulated to examine the effect of mirror tilt on laser power and extraction as well. / Lieutenant, United States Navy

Information about protein structures is important in many areas of life sciences, including structure-based drug design. Gas phase methods, like electrospray ionization and mass spectrometry are powerful tools for the analysis of molecular interactions and conformational changes which complement existing solution phase methods. Novel techniques such as single particle imaging with X-ray free electron lasers are emerging as well. A requirement for using gas phase methods is that we understand what happens to proteins when injected into vacuum, and what is the relationship between the vacuum structure and the solution structure. Molecular dynamics simulations in combination with experiments show that protein structures in the gas phase can be similar to solution structures, and that hydrogen bonding networks and secondary structure elements can be retained. Structural changes near the surface of the protein happen quickly (ns-µs) during transition from solution into vacuum. The native solution structure results in a reasonably well defined gas phase structure, which has high structural similarity to the solution structure. Native charge locations are in some cases also preserved, and structural changes, due to point mutations in solution, can also be observed in vacuo. Proteins do not refold in vacuo: when a denatured protein is injected into vacuum, the resulting gas phase structure is different from the native structure. Native structures can be protected in the gas phase by adjusting electrospray conditions to avoid complete evaporation of water. A water layer with a thickness of less than two water molecules seems enough to preserve native conditions. The results presented in this thesis give confidence in the continued use of gas phase methods for analysis of charge locations, conformational changes and non-covalent interactions, and provide a means to relate gas phase structures and solution structures.

The recent availability of X-ray Free Electron Lasers (XFELs) has opened a completely new and unexplored regime for the study of light-matter interactions. The extremely bright intensities delivered by XFELs can couple many photons into the target, turning well known interactions such as photoionization and scattering into new, non-linear, complex many-body phenomena. This thesis reports theoretical investigations aiming to improve the understanding of the fundamental processes and dynamics triggered by intense X-ray pulses, with a special focus in finite systems such as molecules and clusters. Sequential multiple photoionization in atomic clusters was investigated, where previous observations were extended for higher charge states where direct photoionization is frustrated. Through a rate equation study and subsequent molecular dynamics simulations, it was found that frustrated ionization is partially responsible for the low-energy peak observed in the electron energy spectrum. The influence of plasma evaporation over the formation of the sequential low-energy peak was also investigated, identifying the effects of the system size and photon energy. Multiple channel ionization was also investigated for the case of fullerenes. This is done through a series of studies, starting from a simplified rate equation scheme, and culminating with full molecular dynamics simulations. From these results, a good insight was obtained over the origin, physical meaning, and relevant parameters that give rise to the complicated features observed in the electronic spectra. The mechanisms responsible of all these features are expected to be present in other systems, making these results quite general. Diffractive imaging of biomolecules was studied in a final step, with a particular focus on the influence of intramolecular charge transfer mechanisms. To this end a conformer of T4 Lysozyme was used, a representative enzyme with well known structure. Charge migration is found to allow for additional processes such as proton ejection, a mechanism which enables an efficient release of energy from the system. This mechanism considerably suppresses structural damage for heavy ions, improving the quality of the measured diffraction patterns.

Les centres de rayonnement synchrotron et les Lasers à Électrons Libres (LEL) sont des sources de rayonnement utilisant des Électrons relativistes. Les onduleurs en constituent un élément important : ces instruments qui génèrent un champ magnétique périodique le long de la trajectoire des électrons, guident ces derniers de façon à les faire rayonner ou à rendre l’onde lumineuse émise plus intense. Ce travail porte sur l’étude d’onduleurs de différents types appliqués au Synchrotron SOLEIL, et sur leur possible application sur un LEL. Une première partie du travail porte sur la réalisation pour SOLEIL de deux onduleurs de type APPLE-II. Ce type d’onduleurs permet d’émettre du rayonnement de polarisation variable et les deux onduleurs construits présentent certaines spécificités par rapport à la plupart des onduleurs de ce type. Le premier (HU36) génère un champ magnétique de courte période tout en conservant une valeur crête relativement importante, ce qui lui permet de rayonner sur une large gamme spectrale. Ces caractéristiques entrainent cependant une hystérésis expliquée par la déformation des supports. Les moyens mis en œuvre pour atteindre ces caractéristiques magnétiques et pour réduire l’hystérésis sont présentés. Le deuxième onduleur APPLE-II construit (HU64) présente la double particularité d’être quasipériodique (ou apériodique) afin de réduire la pollution rayonnée aux harmoniques de la longueur d’onde fondamentale, et de permettre l’accès à de la polarisation linéaire inclinée sur une plage de 180° grâce à son châssis étendu. L’optimisation magnétique de sa structure apériodique est détaillée et les difficultés découlant des configurations étendues sont expliquées. Dans un deuxième temps, la réalisation pour SOLEIL d’un troisième onduleur d’un tout autre type est présentée. Il s’agit d’un onduleur hybride sous-vide appelé U20. Des pôles intercalés entre les aimants guident les lignes de champ de façon à augmenter la densité de flux magnétique sur axe, et l’assemblage magnétique ainsi obtenu est installé dans la chambre à vide, ce qui permet de réduire l’entrefer minimum à 5.5 mm et donc, d’accroitre considérablement la valeur crête du champ magnétique généré. Compte-tenu des connaissances acquises pendant la construction des deux onduleurs APPLE-II et de l’onduleur sous-vide, l’étude d’un onduleur combinant ces deux aspects est ensuite proposée. Cet onduleur de tyoe « APPLE-II sous-vide » serait extrêmement polyvalent puisqu’il permettrait l’émission de rayonnement de différentes polarisations sur une large gamme spectrale. L’étude de principe menée ici met en évidence les nombreuses difficultés à surmonter et propose différentes solutions. La dernière partie du travail porte sur l’étude de l’interaction qui s’opère dans le cas d’un LEL au sein de l’onduleur entre les électrons et l’onde lumineuse, conduisant à l’amplification de cette dernière. Des expériences menées en configuration SASE et en mode injecté sur le SPARC (Italie, Frascati) sont rapportées, puis des calculs sont réalisés dans le cadre du projet LUNEX5, pour évaluer l’efficacité de cette interaction dans un onduleur apériodique, et dans l’onduleur APPLE-II sous-vide envisagé précédemment. / Storage rings and Free Electron Lasers (FEL) are relativistic electron based radiation sources, in which an important element is a device called undulator. This apparatus generates a periodic magnetic field along the electron trajectory, guiding the particles in such a way that they lose energy to the benefit of the radiation. This work deals with the study of several undulators of different types, and also their application on FEL sources. A first part of the work is dedicated to the construction of two APPLE-II type undulators for SOLEIL. Undulators of this type are able to emit radiation of variable polarization states, and the two studied devices present some special characteristics with respect to most of APPLE-II undulators. The first one (HU36) generates a short period high value magnetic field, leading to radiation emitted on a large spectral range. These parameters brought about a hysteresis explained by the deformation of the magnet supports, which was reduced. The second APPLE-II undulator (HU64) is characterized by two particularities: its field is quasi-periodic (or aperiodic) in order to reduce the pollution emitted at harmonics of the fundamental wavelength, and it is based on a special frame enabling radiation of tilted linear polarization on the full range of 180°. The optimization of the aperiodic structure is detailed and the difficulties brought about by the extended frame are explained. The third undulator built for SOLEIL is a hybrid in-vacuum one. Ferromagnetic poles guide the magnetic flux lines in order to increase the on-axis flux density, and the magnetic part of the undulator is included inside the vacuum chamber, which leads to a higher value of the generated field. Starting from the knowledge of both APPLE-II and in-vacuum undulators, the study of a device combining both characteristics is then proposed. Such an in-vacuum APPLE-II undulator would consist in a very performant and flexible apparatus because it could emit radiation of variable polarization on a large spectral range. The study points out the technical difficulties and suggests some solutions. The last part of the work deals with the study of the interaction realized in a FEL between electrons and radiation, resulting in the amplification of the light. Experiments performed in both SASE and injected configurations at the FEL SPARC (Italy, Frascati) are reported. Then calculations are made in the case of the FEL French project LUNEX5 to estimate the interaction efficiency when realized inside a quasi-periodic undulator and also through the previously studied in-vacuum APPLE-II one.

Les protéines fluorescentes photocommutables (RSFPs) ont la propriété de passer d’un état fluorescent à un état non-fluorescent en réponse à la lumière. Cette propriété en fait des outils de marquage pour la microscopie de super-résolution (ou nanoscopie). Le mécanisme de photocommutation implique l’isomérisation du chromophore ainsi qu’un changement d’état de protonation de ce dernier. Le mécanisme a été très étudié par différentes approches de spectroscopie et de simulation mais reste encore mal compris, l’ordre séquentiel des évènements est notamment encore débattu. Certains de ces évènements de la photocommutation se déroulent à des échelles de temps très courtes, ce qui rend difficile l’étude structurale par cristallographie des rayons X à l’aide des sources synchrotron actuelles dont la résolution temporelle est encore limitée. Les lasers à électrons libres (XFELs) sont une nouvelle source de rayons X produisant des impulsions suffisamment courtes pour permettre l’étude structurale des intermédiaires précoces ou à courte durée de vie qui se forment ou cours de la photocommutation, et suffisamment brillantes pour permettre la collecte de données cristallographiques sur des cristaux de tailles nano- et micrométrique. L’utilisation de ce nouveau genre d’instrument a permis l’émergence de la cristallographie sérielle, une nouvelle approche de la cristallographie des rayons X. Cette approche a depuis été adaptée aux lignes synchrotrons.Le travail présenté ici se focalise sur l’étude de rsEGFP2, une protéine fluorescente photocommutable de la famille de la GFP. Il y est décrit la mise au point d’un protocole de microcristallisation permettant l’obtention d’échantillons en vue d’une expérience de cristallographie résolue en temps au XFEL. Un mécanisme de photocommutation y est proposé à travers le résultat de deux expériences sur les deux XFELs actuellement opérationnels, à des échelles de temps différentes, dévoilant un chromophore « twisté » à l’état excité ainsi qu’un état cis protoné de ce dernier. La caractérisation structurale des variants de rsEGFP2 par cristallographie d’oscillation « classique » combinée à la découverte fortuite d’une conformation alternée du chromophore dans l’état non-fluorescent, issue d’expérience de cristallographie sérielle, apporte un complément d’explication des propriétés photophysiques de la protéine. / Reversibly switchable fluorescent proteins (RSFPs) are able to reversibly toggle between a fluorescent on-state and a non-fluorescent off-state under visible light irradiation. This property makes them a suitable marker used in super-resolution microscopy (or nanoscopy). The photo-switching mechanism involves isomerisation of the chromophore and a change of its protonation state. This mechanism has been well studied but remains poorly understood. The structural nature and the sequential order of atomistic events are still under debate. Some of them take place on the ultra-fast time scale and make structural investigation by X-ray crystallography impossible using current synchrotron radiation sources whose temporal resolution they offer is limited. X-ray free electron lasers (XFELs) are a new kind of X-ray source producing femtosecond pulses that allow structural investigation of ultra-fast intermediates during photoswitching. They are also so bright that crystallographic data collection from micro- and nanometer-sized crystals became possible. The bright and short XFEL pulses required a new methodology to be developed, the so-called serial crystallography methodology. This method is now being adapted to synchrotron radiation facilities.Here is presented a time-resolved crystallography study of the reversibly switchable green fluorescent protein 2 (rsEGFP2). A microcrystallization protocol is described allowing the preparation of suitable samples in large amounts for time-resolved serial crystallography experiments. A photoswitching mechanism of rsEGFP2 is proposed based on crystallographic results obtained from data collected at the two XFEL facilities currently fully operational, i.e. the LCLS in the USA and SACLA in Japan. In particular, the structure of two photoswitching intermediates have been determined, one featuring a twisted chromophore in the excited state and the other displaying a protonated cis isomer of the chromophore in the ground state. The structural characterization of rsEGFP2 variants by traditional oscillation crystallography combined with the serendipitous discovery of an alternate chromophore conformation in the off-state during an XFEL experiment provided unique insight into the photophysical behavior of the protein.

<p>Information about protein structures is important in many areas of life sciences, including structure-based drug design. Gas phase methods, like electrospray ionization and mass spectrometry are powerful tools for the analysis of molecular interactions and conformational changes which complement existing solution phase methods. Novel techniques such as single particle imaging with X-ray free electron lasers are emerging as well. A requirement for using gas phase methods is that we understand what happens to proteins when injected into vacuum, and what is the relationship between the vacuum structure and the solution structure.</p><p>Molecular dynamics simulations in combination with experiments show that protein structures in the gas phase can be similar to solution structures, and that hydrogen bonding networks and secondary structure elements can be retained. Structural changes near the surface of the protein happen quickly (ns-µs) during transition from solution into vacuum. The native solution structure results in a reasonably well defined gas phase structure, which has high structural similarity to the solution structure. </p><p>Native charge locations are in some cases also preserved, and structural changes, due to point mutations in solution, can also be observed in vacuo. Proteins do not refold in vacuo: when a denatured protein is injected into vacuum, the resulting gas phase structure is different from the native structure.</p><p>Native structures can be protected in the gas phase by adjusting electrospray conditions to avoid complete evaporation of water. A water layer with a thickness of less than two water molecules seems enough to preserve native conditions.</p><p>The results presented in this thesis give confidence in the continued use of gas phase methods for analysis of charge locations, conformational changes and non-covalent interactions, and provide a means to relate gas phase structures and solution structures.</p>

Infrared multiple photon dissociation (IRMPD) spectroscopy has been coupled with electronic structure calculations in order to elucidate the structures of several small biological molecules including: uracil, 5-fluorouracil, 5-fluorocytosine, ferulic acid, and a number of their related analogs. IRMPD is a powerful technique, that when combined with electronic structure calculations can provide convincing evidence for the structural characterization of ions in the gas phase. Isomers of uracil and 5-fluorouracil (5-FU) have been characterized by calculations performed at the MP2(full)/aug-cc-pVTZ level of theory; however, infrared multiple photon dissociation spectroscopy experiments proved to be unsuccessful for these species. Geometry optimization and frequency calculations have isolated the dominant isomer(s) for neutral and deprotonated uracil and 5-fluorouracil, along with several cluster interactions involving water, methanol, ammonia, and methylamine. For both uracil and 5-FU, a single relevant neutral isomer was determined, with each isomer existing in the diketo, as opposed to the enol form. Following the deprotonation of this neutral isomer, both uracil and 5-FU were permitted to form anionic cluster ions with water, methanol, ammonia, or methylamine, and based on the relative Gibbs free energies (298 K) of the calculated isomers, relevant cluster interactions were determined. For each cluster, several sites of intramolecular interaction were found to exist; however, interaction at the site of deprotonation was the most favourable in every instance. Ionic hydrogen bond interactions have been found in several clusters formed by 5-fluorocytosine (5-FC). The chloride and trimethylammonium cluster ions, in addition to the cationic and anionic dimers have been characterized by infrared multiple photon dissociation (IRMPD) spectroscopy and electronic structure calculations performed at the B2PLYP/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. IRMPD spectra in combination with calculated spectra and relative energetics have indicated, quite conclusively, that a single isomer for each 5-FC cluster that is likely being observed experimentally except in the case of the anionic dimer, in which a combination of isomers is probable. For the 5-FC-trimethylammonium cluster specifically, the calculated spectrum of the lowest energy isomer matches the experimental spectrum remarkably well. Interestingly, the cationic dimer of 5-FC was found to have a single energetically relevant isomer (Cationic-IV) in which a unique tridentate ionic hydrogen bond interaction is formed. The three sites of intramolecular ionic hydrogen bonds in this isomer interact very efficiently, leading to a significantly large calculated enthalpy of binding of 180 kJ/mol. The magnitude of the calculated binding energy for this species, in combination with the strong correlation between the simulated and IRMPD spectra, indicates that the tridentate-bound dimer is observed predominantly in experiment. Comparison of the calculated relative Gibbs free energies (298 K) for this species with several of the other isomers considered also supports the likelihood of the dominant protonated dimer existing as Cationic-IV. Protonated ferulic acid has been characterized using infrared multiple photon dissociation spectroscopy and electronic structure calculations at the B3LYP/6-311+G(d,p) level of theory. Neutral ferulic acid has been determined to undergo protonation on the carbonyl oxygen of the acid group, forming an ion of m/z 195. Due to its extensively conjugated structure, protonated ferulic acid (m/z 195) is observed to yield three stable fragment ions in IRMPD experiments. It is proposed that two parallel fragmentation pathways of protonated ferulic acid are being observed. First, proton transfer occurs from the carbonyl oxygen to the hydroxyl oxygen within the acid group, resulting in the loss of water and subsequently carbon monoxide, forming ions of m/z 177 and 149, respectively. The second proposed fragmentation pathway undergoes proton transfer from the phenolic group to the methoxy group resulting in loss of methanol and rearrangement to a five-membered ring of m/z 163. IRMPD spectra have been obtained for the ions m/z 195 and m/z 177, and anharmonic calculations have been performed on these species at the B3LYP/6-311+G(d,p) level of theory. The calculated anharmonic spectra for these ions match the experimental spectrum exceptionally well and strongly support the proposed fragmentation mechanisms.

Infrared multiple photon dissociation (IRMPD) spectroscopy has been coupled with electronic structure calculations in order to elucidate the structures of several small biological molecules including: uracil, 5-fluorouracil, 5-fluorocytosine, ferulic acid, and a number of their related analogs. IRMPD is a powerful technique, that when combined with electronic structure calculations can provide convincing evidence for the structural characterization of ions in the gas phase. Isomers of uracil and 5-fluorouracil (5-FU) have been characterized by calculations performed at the MP2(full)/aug-cc-pVTZ level of theory; however, infrared multiple photon dissociation spectroscopy experiments proved to be unsuccessful for these species. Geometry optimization and frequency calculations have isolated the dominant isomer(s) for neutral and deprotonated uracil and 5-fluorouracil, along with several cluster interactions involving water, methanol, ammonia, and methylamine. For both uracil and 5-FU, a single relevant neutral isomer was determined, with each isomer existing in the diketo, as opposed to the enol form. Following the deprotonation of this neutral isomer, both uracil and 5-FU were permitted to form anionic cluster ions with water, methanol, ammonia, or methylamine, and based on the relative Gibbs free energies (298 K) of the calculated isomers, relevant cluster interactions were determined. For each cluster, several sites of intramolecular interaction were found to exist; however, interaction at the site of deprotonation was the most favourable in every instance. Ionic hydrogen bond interactions have been found in several clusters formed by 5-fluorocytosine (5-FC). The chloride and trimethylammonium cluster ions, in addition to the cationic and anionic dimers have been characterized by infrared multiple photon dissociation (IRMPD) spectroscopy and electronic structure calculations performed at the B2PLYP/aug-cc-pVTZ//B3LYP/6-311+G(d,p) level of theory. IRMPD spectra in combination with calculated spectra and relative energetics have indicated, quite conclusively, that a single isomer for each 5-FC cluster that is likely being observed experimentally except in the case of the anionic dimer, in which a combination of isomers is probable. For the 5-FC-trimethylammonium cluster specifically, the calculated spectrum of the lowest energy isomer matches the experimental spectrum remarkably well. Interestingly, the cationic dimer of 5-FC was found to have a single energetically relevant isomer (Cationic-IV) in which a unique tridentate ionic hydrogen bond interaction is formed. The three sites of intramolecular ionic hydrogen bonds in this isomer interact very efficiently, leading to a significantly large calculated enthalpy of binding of 180 kJ/mol. The magnitude of the calculated binding energy for this species, in combination with the strong correlation between the simulated and IRMPD spectra, indicates that the tridentate-bound dimer is observed predominantly in experiment. Comparison of the calculated relative Gibbs free energies (298 K) for this species with several of the other isomers considered also supports the likelihood of the dominant protonated dimer existing as Cationic-IV. Protonated ferulic acid has been characterized using infrared multiple photon dissociation spectroscopy and electronic structure calculations at the B3LYP/6-311+G(d,p) level of theory. Neutral ferulic acid has been determined to undergo protonation on the carbonyl oxygen of the acid group, forming an ion of m/z 195. Due to its extensively conjugated structure, protonated ferulic acid (m/z 195) is observed to yield three stable fragment ions in IRMPD experiments. It is proposed that two parallel fragmentation pathways of protonated ferulic acid are being observed. First, proton transfer occurs from the carbonyl oxygen to the hydroxyl oxygen within the acid group, resulting in the loss of water and subsequently carbon monoxide, forming ions of m/z 177 and 149, respectively. The second proposed fragmentation pathway undergoes proton transfer from the phenolic group to the methoxy group resulting in loss of methanol and rearrangement to a five-membered ring of m/z 163. IRMPD spectra have been obtained for the ions m/z 195 and m/z 177, and anharmonic calculations have been performed on these species at the B3LYP/6-311+G(d,p) level of theory. The calculated anharmonic spectra for these ions match the experimental spectrum exceptionally well and strongly support the proposed fragmentation mechanisms.