Dynamic Imaging with X-ray Holography

X-ray holography is a type of coherent diffractive imaging where the phase information is physically encoded in the diffraction pattern by means of interference with a reference beam. The image of the diffracting specimen is obtained by a single Fourier transform of the interference pattern. X-ray holography is particularly well-suited for high resolution dynamic imaging because, intrinsically, the reconstructed image does not drift and the images show high contrast. Therefore, the motion of features between two images can be determined with a precision of better than 3 nm, as demonstrated recently. In this chapter, the technical aspects of X-ray holography are discussed from an end user perspective, focusing on what is required to obtain a high quality image in a short time. Specifically, the chapter discusses the key challenges of the technique, such as sample design and fabrication, beam requirements, suitable end stations, and how to implement pump-probe dynamic imaging. Good imaging parameters were found using simulations and experiments, and it is demonstrated how a deviation from the optimum value affects the image quality

Full phase diagram of isolated skyrmions in a ferromagnet

Magnetic skyrmions are topological quasi particles of great interest for data storage applications because of their small size, high stability, and ease of manipulation via electric current. Theoretically, however, skyrmions are poorly understood since existing theories are not applicable to small skyrmion sizes and finite material thicknesses. Here, we present a complete theoretical framework to determine the energy of any skyrmion in any material, assuming only a circular symmetric 360$^\circ$ domain wall profile and a homogeneous magnetization profile in the out-of-plane direction. Our model precisely agrees with existing experimental data and micromagnetic simulations. Surprisingly, we can prove that there is no topological protection of skyrmions. We discover and confirm new phases, such as bi-stability, a phenomenon unknown in magnetism so far. The outstanding computational performance and precision of our model allow us to obtain the complete phase diagram of static skyrmions and to tackle the inverse problem of finding materials corresponding to given skyrmion properties, a milestone of skyrmion engineering

Accurate calculation of the transverse anisotropy in perpendicularly magnetized multilayers

The transverse anisotropy constant and the related D\"oring mass density are key parameters of the one-dimensional model to describe the motion of magnetic domain walls. So far, no general framework is available to determine these quantities from static characterizations such as magnetometry measurements. Here, we derive a universal analytical expression to calculate the transverse anisotropy constant for the important class of perpendicular magnetic multilayers. All the required input parameters of the model, such as the number of repeats, the thickness of a single magnetic layer, and the layer periodicity, as well as the effective perpendicular anisotropy, the saturation magnetization, and the static domain wall width are accessible by static sample characterizations. We apply our model to a widely used multilayer system and find that the effective transverse anisotropy constant is a factor 7 different from the when using the conventional approximations, showing the importance of using our analysis scheme

Modulation of the bacterial cell wall by N‐acetylmuramoyl‐L‐alanine amidases

The bacterial cell wall is a highly dynamic structure that undergoes constant change in order to fulfill its various tasks, which range from physical protection against exterior stress and maintaining homoeostasis to immune evasion. A major component of the bacterial cell wall is the peptidoglycan network (PGN). Built up by a carbohydrate backbone of repeating units of N‐acetylglucosamine and N‐acetylmuramic acid linked to a peptide stem containing nonproteinogenic amino acids, PGN is a net‐like structure that harbors various proteins and anchors further components of the cell wall. Depending on the composition of the peptide stems and the type of cross‐linkage between the peptide stems, PGN can be a very dense network or a rather loose mesh. N‐acetylmuramoyl‐L‐alanine amidases cleave the amide bond between the carbohydrate backbone and the peptide stem and represent a class of PGN‐modulating enzymes that ensure its plasticity. My work focused on this class of enzymes in order to better understand the mechanisms that underlie PGN cleavage, and thus its plasticity, by using biochemical and cell biological tools in combination with X‐ray crystallography. The bifunctional major autolysin AtlA of Staphylococcus aureus contains a glucosaminidase and an amidase, which are post‐translationally processed and separated. Deletion of AtlA leads to cell clusters with irregular division patterns, indication a crucial role in cell division. I solved the atomic structure of the catalytic domain of the amidase, AmiA‐cat, in complex with its substrate component muramoyltetrapeptide. Close investigation of the molecular interactions between enzyme and substrate, along with the analyses of the apo‐structure and enzymatic activity assays, elucidated the likely reaction mechanism as well as substrate specificity. Since the intact substrate, including the scissile bond, is present in the complex structure, it moreover serves as a starting point for therapeutics against methicillin‐resistant Staphylococcus aureus. Further studies with AmiA‐cat in this regard involve a fragment‐based screening approach using X‐ray crystallography and the production and evaluation of therapeutic antibodies against AmiA‐cat as possible active agents. AmiC2 of the filamentous cyanobacterium Nostoc punctiforme fulfills a unique task in order to enable communication of neighboring cells within a filament. In contrast to cell‐splitting amidases, AmiC2 drills holes into the septal disk that separates neighboring cells, thus generating a nanopore array used for nutrient exchange and communication. My cooperation partner located AmiC2 in the maturating septum and I solved the structure of the catalytic domain of this enzyme, AmiC2‐cat. In comparison with the homologous enzyme AmiC E. coli, a regulatory α‐helix is missing, and AmiC2‐cat exhibits high activity, which can be abolished by mutation of a catalytic glutamate. Ongoing research is focused on the mechanism that governs activity and specificity of this unusual amidase. In particular, I study the separate and / or cooperative influence of the additional domains, AMIN‐A, AMIN‐B, and the proline‐rich linker of the AmiC2 holo‐enzyme on catalysis and specificity. Furthermore, in cooperation, I am working on elucidating the exact chemical composition of Nostoc PGN, perhaps even differences between nascent, septal, and mature PGN. The results will be essential to generate complex structures, and elucidating potential PGN differences will provide insights into specificity.Die Bakterienzellwand ist eine hochdynamische Struktur, die einem ständigen Wandel unterliegt, um ihre verschiedenen Aufgaben zu erfüllen. Diese reichen von physischem Schutz gegen äußere Belastungen über die Aufrechterhaltung der Zellhomöostase bis zur Immunevasion. Ein Hauptbestandteil der bakteriellen Zellwand ist das Peptidoglycan (PGN). Es ist aus einem Kohlenhydratgerüst mit abwechselnden Einheiten von N‐Acetylglucosamin und N‐Acetylmuraminsäure sowie einem Peptidstamm, der auch nicht‐proteinogene Aminosäuren beinhaltet, aufgebaut. PGN ist eine netzartige Struktur, die außerdem verschiedene Proteine und weitere Komponenten der Zellwand verankert. Je nach Zusammensetzung des Peptidstammes selbst und der Art der Vernetzung zwischen den Peptidstämmen kann das PGN ein sehr dichtes Netz oder ein eher lockeres Geflecht sein. N‐Acetylmuramoyl‐L‐Alanin‐Amidasen spalten die Amidbindung zwischen dem Kohlenhydratgerüst und dem Peptidstamm und stellen eine Klasse von PGN‐modulierenden Enzymen dar, die seine Plastizität sicherstellen. Die vorliegende Arbeit konzentriert sich auf diese Enzymklasse und soll zum Verständnis der zugrunde liegenden Mechanismen jener enzymatischen Spaltung beitragen, die für die Plastizität von PGN verantwortlich ist. Dieser Fragestellung wurde mit Hilfe biochemischer und zellbiologischer Methoden sowie der Röntgenstrukturanalyse nachgegangen. Das bi‐funktionelle Major Autolysin AtlA von Staphylococcus aureus besteht aus einer Glucosaminidase und einer Amidase, welche posttranslational voneinander getrennt werden. Das gezielte Abschalten (Knockout, Nullmutante) von AtlA führt zu Zellclustern mit unregelmäßigem Teilungsmuster, was eine entscheidende Rolle bei der Zellteilung nahelegt. Ich habe die atomare Struktur der katalytischen Domäne der Amidase, AmiA‐cat, im Komplex mit ihrem Substratbestandteil Muramoyltetrapeptid gelöst. Sowohl die genaue Untersuchung der molekularen Wechselwirkungen zwischen Enzym und Substrat sowie die Analyse der apo‐ Struktur als auch enzymatische Aktivitätstests haben Anhaltspunkte für den wahrscheinlichen Reaktionsmechanismus sowie die Substratspezifität des Enzyms geliefert. Da das intakte Substrat einschließlich der zu spaltenden Bindung in der Komplexstruktur vorhanden ist, dient sie ferner als ein guter Startpunkt für Therapeutika gegen den Methicillin‐resistenten Staphylococcus aureus. Weitere Studien mit AmiA‐cat in diese Richtung beinhalten neben einem fragmentbasierten Screeningansatz unter Zuhilfenahme von Röntgenkristallographie auch die Produktion und Tests von therapeutischen Antikörpern gegen AmiA‐cat als mögliche Wirkstoffe. AmiC2 des filamentösen Cyanobakteriums Nostoc punctiforme führt eine einzigartige Reaktion aus, um die Kommunikation von benachbarten Zellen innerhalb eines Filaments zu ermöglichen. Im Gegensatz zu zellspaltenden Amidasen bohrt AmiC2 Löcher in das Septum, welches Nachbarzellen voneinander trennt. Dadurch entsteht ein Nanopore Array, das für Nährstoffaustausch und Kommunikation verwendet wird. Meine Kooperationspartner haben AmiC2 im ausreifenden Septum lokalisiert, und ich habe die Struktur der katalytischen Domäne dieses Enzyms gelöst (AmiC2‐cat). Interessanterweise fehlt eine regulatorische α‐Helix, wie man sie in dem homologen Enzym AmiC E. coli findet. AmiC2‐cat ist katalytisch sehr aktiv, was durch die Mutation eines katalytischen Glutamats aber aufgehoben werden kann. Weitergehende Forschung zielt auf die Aufklärung des Mechanismus ab, der die Aktivität und Spezifität dieser ungewöhnlichen Amidase regelt. Insbesondere wird momentan der getrennte und / oder kooperative Einfluss der zusätzlichen Domänen des AmiC2‐Holoenzyms ‐ AMIN‐A, AMIN‐B sowie Prolin‐reicher Linker ‐ auf die Katalyse und Spezifität von AmiC2‐cat erforscht. Außerdem wird die genaue chemische Zusammensetzung des PGN von Nostoc untersucht, um den physiologischen Liganden von AmiC2 für eine Komplexstruktur zu identifizieren. Weiterhin könnten eventuelle Unterschiede zwischen jungem, septalem und reifem PGN eine Rolle bei der enzymatischen Spezifität spielen

Functional characterization of VirB/VirD4 and Icm/Dot type IV secretion systems from the plant-pathogenic bacterium Xanthomonas euvesicatoria

IntroductionMany Gram-negative plant- and animal-pathogenic bacteria employ type IV secretion (T4S) systems to transport proteins or DNA/protein complexes into eukaryotic or bacterial target cells. T4S systems have been divided into minimized and expanded T4S systems and resemble the VirB/VirD4 T4S system from the plant pathogen Agrobacterium tumefaciens and the Icm/Dot T4S system from the human pathogen Legionella pneumophila, respectively. The only known plant pathogen with both types of T4S systems is Xanthomonas euvesicatoria which is the causal agent of bacterial spot disease on pepper and tomato plants.Results and discussionIn the present study, we show that virB/virD4 and icm/dot T4S genes are expressed and encode components of oligomeric complexes corresponding to known assemblies of VirB/VirD4 and Icm/Dot proteins. Both T4S systems are dispensable for the interaction of X. euvesicatoria with its host plants and do not seem to confer contact-dependent lysis of other bacteria, which was previously shown for the chromosomally encoded VirB/VirD4 T4S system from Xanthomonas axonopodis pv. citri. The corresponding chromosomal T4S gene cluster from X. euvesicatoria is incomplete, however, the second plasmid-localized vir gene cluster encodes a functional VirB/VirD4 T4S system which contributes to plasmid transfer. In agreement with this finding, we identified the predicted relaxase TraI as substrate of the T4S systems from X. euvesicatoria. TraI and additional candidate T4S substrates with homology to T4S effectors from X. axonopodis pv. citri interact with the T4S coupling protein VirD4. Interestingly, however, the predicted C-terminal VirD4-binding sites are not sufficient for T4S, suggesting the contribution of additional yet unknown mechanisms to the targeting of T4S substrates from X. euvesicatoria to both VirB/VirD4 and Icm/Dot T4S systems

Photon correlation spectroscopy with heterodyne mixing based on soft-x-ray magnetic circular dichroism

Many magnetic equilibrium states and phase transitions are characterized by fluctuations. Such magnetic fluctuation can in principle be detected with scattering-based x-ray photon correlation spectroscopy (XPCS). However, in the established approach of XPCS, the magnetic scattering signal is quadratic in the magnetic scattering cross section, which results not only in often prohibitively small signals but also in a fundamental inability to detect negative correlations (anticorrelations). Here, we propose to exploit the possibility of heterodyne mixing of the magnetic signal with static charge scattering to reconstruct the first-order (linear) magnetic correlation function. We show that the first-order magnetic scattering signal reconstructed from heterodyne scattering now directly represents the underlying magnetization texture. Moreover, we suggest a practical implementation based on an absorption mask rigidly connected to the sample, which not only produces a static charge scattering signal but also eliminates the problem of drift-induced artificial decay of the correlation functions. Our method thereby significantly broadens the range of scientific questions accessible by magnetic x-ray photon correlation spectroscopy

Reference shape effects on Fourier transform holography

Soft-x-ray holography which utilizes an optics mask fabricated in direct contact with the sample, is a widely applied x-ray microscopy method, in particular, for investigating magnetic samples. The optics mask splits the x-ray beam into a reference wave and a wave to illuminate the sample. The reconstruction quality in such a Fourier-transform holography experiment depends primarily on the characteristics of the reference wave, typically emerging from a small, high-aspect-ratio pinhole in the mask. In this paper, we study two commonly used reference geometries and investigate how their 3D structure affects the reconstruction within an x-ray Fourier holography experiment. Insight into these effects is obtained by imaging the exit waves from reference pinholes via high-resolution coherent diffraction imaging combined with three-dimensional multislice simulations of the x-ray propagation through the reference pinhole. The results were used to simulate Fourier-transform holography experiments to determine the spatial resolution and precise location of the reconstruction plane for different reference geometries. Based on our findings, we discuss the properties of the reference pinholes with view on application in soft-x-ray holography experiments