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Soft x-ray microscopy is a powerful technique for natural-contrast, high-resolution imaging of organic materials. This Thesis describes new instrumentational and new image-processing methods to improve the image quality of the compact x-ray microscope at the Biomedical & X-Ray Physics division at KTH. The microscope is based on a laser-plasma source combined with different condenser optics, either multilayer mirrors or zone plates. Imaging is performed by micro zone plates. The microscope works in the water window (\lambda = 2.3-4.4 nm), where the attenuation lengths of oxygen and carbon differ strongly, providing high natural contrast for carbon-containing specimens in an aqueous environment. By optimizing the properties of the laser-plasma source and fabricating multilayer mirrors with high, uniform reflectivity, the performance of the microscope's illumination system could be improved and exposure times decreased significantly to about 2 min for imaging dry samples and 5 min for imaging wet samples. For imaging of wet samples, a wet-specimen chamber was developed, which is vacuum-compatible. Since it is horizontally mounted in the microscope, it offers advantages for investigations in polymer and soil science. To improve the quality of images taken by the compact x-ray microscope an image-restoration algorithm was developed. Denoising is performed by a filtering algorithm based on the discrete wavelet transform. This algorithm shows advantages compared to Fourier-based algorithms, since the filtering of spatial frequencies is done locally. An improvement in exposure time by a factor of about 2 could be realized without loss of image information. To stimulate experiments on functional imaging in x-ray microscopy an image-analysis algorithm for identifying colloidal-gold particles was developed. This algorithm is based on a combination of a threshold with respect to the local absorption and a shape discrimination, realized by fitting a Gaussian profile to the potential particles. The algorithm was evaluated and optimized on images taken by the transmission x-ray microscope at BESSY II. The size-selective identification and localization of single gold particles down to a diameter of 50 nm was demonstrated. / QC 20100920

The ultimate objective of this project was to develop a small, transportable X-ray microscope which would be able to view a wide range of biological specimens without the need for any type of sample preparation at a resolution greater than that obtainable by conventional light microscopy (ie. about 250nm). Of the various possible implementations of X-ray microscopy currently being investigated, contact microscopy was chosen as being the most suitable for the development of such a small-scale instrument, while at the same time minimizing the effects on image quality of radiation damage to the biological specimen. The requirement for a high brightness pulsed X-ray source of less than 50ns duration for illumination of the specimen was met by the production of laser generated plasmas. These were formed by focusing a 2.2J KrF laser beam, of wavelength 248nm and duration 20ns, onto the surface of one of a number of different target materials. In order to obtain the large intensities required for the production of a sufficiently high temperature plasma, a doubly pre-ionized, discharge-pumped amplifier KrF laser was developed. This was seeded by a smaller oscillator laser by means of a coupled unstable resonator configuration. A number of different cavity arrangements were investigated and an output beam divergence of 2.5 times the diffraction limit was achieved. The plasmas generated by focusing the laser beam to an intensity of 10¹⁴W/cm² onto carbon, titanium, molybdenum and tungsten targets were characterized as fully as was necessary for their use in the X-ray microscope. Preliminary investigations on the use of a grazing incidence ellipsoidal mirror to focus the emitted X-rays onto the specimen of the microscope were made and such an optical component was manufactured and tested. Finally, numerous images of a number of different biological specimens were made and resolutions of better than 100nm were achieved. Images were read out using a Park Scientific Instruments atomic force microscope, which enabled the entire microscopy process to be carried out in a single working day. The system is now in routine use and can produce more than ten images per session.

Tomography in soft X-ray microscopy is an emerging technique for obtaining quantitative 3D structural information about cells. One of its strengths, compared with other techniques, is that it can image intact cells in their near-native state at a few 10 nm’s resolution, without staining. However, the methods for reconstructing 3D-data rely on algorithms that assume projection data, which the images are generally not due to the imaging systems’ limited depth of focus. To bring out the full potential of tomography in soft X-ray microscopy an improved understanding of the image formation is desired. This Thesis reviews zone plate-based X-ray microscopy for biological imaging and the theory necessary for a numerical implementation of a 3D image formation model. Furthermore, a novel reconstruction approach is proposed that improves the overall resolution in a reconstruction of a tomographically imaged object. This is demonstrated by simulations and experiments. Finally, this Thesis covers work on the Stockholm X-ray microscope, including an upgrade of the X-ray source yielding unprecedented brightness for a compact system. With this upgrade it was possible to do high-quality imaging of cells in their near-native state with only 10 second exposures. / Tomografi i mjukröntgenmikroskopi är en ny teknik för att få ut kvantitativ strukturell 3D information om celler. Dess styrka jämfört med andra tekniker är att den kan avbilda intakta celler i deras nära naturliga tillstånd med ett par 10 nm upplösning, utan omfattande preparering. Dock är metoderna för att rekonstruera 3D-data beroende av algoritmer som antar projektionsdata, vilket bilderna i allmänhet inte är på grund av avbildningsystemens begränsade skärpedjup. För att få ut den fulla potentialen av tomografi i röntgenmikroskopi behövs en ökad förståelse för avbildningsprocessen. Denna avhandling behandlar zonplatte-baserad röntgenmikroskopi för biologisk avbildning och den nödvändiga teorin för en numerisk implementering av en avbildningsmodell i 3D. En ny rekonstruktionsmetod föreslås som förbättrar upplösningen i rekonstruktionen för ett tomografiskt avbildat objekt. Detta visas i simuleringar och experiment. Slutligen omfattar denna avhandling arbete på Stockholms mjukröntgenmikroskop, inklusive en uppgradering av röntgenkällan som ger oöverträffad ljusstyrka för ett kompakt system. Denna uppgradering möjliggör högkvalitativ avbildning av celler i deras nästan naturliga tillstånd med endast 10 sekunders exponering. / <p>QC 20160324</p>

This thesis describes the development of soft x-rayreflective optics, instrumentation and applications for compactsoft x-ray microscopy. The microscope is based on a table-topliquid-jet-target laser-plasma source in combination with aspherical normal-incidence multilayer condenser mirror andnanofabricated diffractive optics for imaging. High-resolutionimaging is performed at the wavelength 3.374 nm in thewater-window (2.3 - 4.4 nm), where natural contrast betweencarbon and oxygen allows imaging of unstained biologicalmaterial in their natural aqueous environment. The design and implementation of a compact soft x-rayreflectometer based on a laser-plasma source is described. Thereflectometer allows rapid and accurate characterization ofnormal-incidence multilayer coatings used at water-windowwavelengths. This instrument, which measures absolutereflectivity and multilayer period, is now used in thefabrication process, aiming to improve the soft x-raynormal-incidence multilayer condenser system of the compactsoft x-ray microscope. Latest results from the developmentprocess are presented. A new design of the compact soft x-ray microscope, withimprovements in mechanical and thermal stability, providesuser-friendly and daily operation. This includes also a newnozzle design for the liquid-jet-target laser-plasma source,which enables higher source stability and operation withcryogenic liquids. In addition, a new experimental arrangementunder construction is briefly described. It will utilize acondenser zone plate and operate at the wavelength 2.478nm. Finally, performance test of the compact soft x-raymicroscope is presented and discussed. In addition, a projectto explore the use of soft x-ray microscopy for imaging sensorycells is described. The high-resolution imaging of these cellswas performed at the synchrotron-based soft x-ray microscope atLawrence Berkeley National Laboratory (LBNL).

This thesis describes the development of a sub-60-nm full-period resolution compact soft x-ray microscope operating in the water-window region (2.3-4.4 nm). Soft x-ray water-window microscopy is a powerful technique for high-resolution imaging of organic materials exploiting the natural contrast mechanism between carbon and oxygen, cf. Sect. 4.1. The thesis discusses the components of, as well as the integration of the microscope, including liquid-jet laser-plasma x-ray sources, optics, simulations, and image-processing tools. Liquid-jet-target laser-plasma sources for generation of soft x-rays and extreme-ultraviolet radiation are compact sources with high brightness. The work focused on improved target stability, decreased debris, and accurate source characterization. For x ray microscopy applications a liquid-jet target delivery system allowing cryogenic liquids was developed. Source characterization was performed for two different liquid-jet targets: Methanol and liquid nitrogen. For extreme-ultraviolet lithography applications, the potential use of a liquid-tin-jet laser-plasma source was explored including conversion efficiency and debris measurements. High quality optics are essential in the development of compact x-ray microscopes. For soft x-ray wavelengths, zone plates and multilayer mirrors are used to focus or redirect radiation. This thesis describes the development and characterization of a condenser zone plate suitable for use in a compact soft x-ray microscope operating at λ = 2.478 nm. It also investigates the possibility to perform differential interference contrast microscopy in the water window using a single diffractive optical element. An arrangement for rapid and accurate determination of absolute and local diffraction efficiency of soft x-ray zone plates using a compact laser-plasma source has been developed. The instrument is used to characterize the zone plates fabricated at the Biomedical &amp; X-Ray Physics division at KTH. Through a collaboration with the Fraunhofer-Institut in Jena, Germany, a large diameter spherical Cr/Sc multilayer mirror, suitable as condenser in the compact x-ray microscope, was developed and characterized. The mirror is designed for λ = 3.374 nm and shows a high, and uniform reflectivity of 3%. This increases the photon flux by an order of magnitude compared to the W/B4C mirrors previously used. The thesis describes the development of a compact soft x-ray microscope with sub-60-nm full-period resolution. It can operate at two different wavelengths in the water window using the soft x-ray laser-plasma sources combined with the condenser optics described above. Imaging is performed by zone plate objectives. The microscope is capable of imaging hydrated biological samples with thicknesses up to ~10 μm. Improvements made to the mechanical design has turned it into a user friendly instrument allowing daily operation. A numerical method was developed to study the effects of partially coherent illumination on 2D imaging. To stimulate experiments on functional imaging in x-ray microscopy an image-analysis algorithm for identifying colloidal-gold particles was developed. Size selective identification and localization of single gold particles down to a diameter of 50 nm was demonstrated. / QC 20100819

X-ray microscopy is a well-established technique for nanoscale imaging. Zone plates are used as microscope objectives and provide high resolution, approaching 10 nm, currently limited by fabrication issues. This Thesis presents zone plate optics that achieve either high resolution or phase contrast in x-ray microscopy. The high-resolution optics use high orders of the zone plate, which alleviates the demands on fabrication, and the phase-contrast optics are single-element diffractive optical elements that produce contrast by Zernike or differential-interference contrast methods. The advantage of phase contrast in x-ray microscopy is shorter exposure times, and is crucial in the hard x-ray regime. Microscopy in the absorption‑contrast region of the water-window (2.34 - 4.37 nm) also benefits from these optics. The development of the optics for a laboratory soft x-ray microscope spans from theoretical and numerical analysis of coherence and stray light to experimental implementation and testing. The laboratory microscope uses laser-produced plasma-sources in the water-window and is unique in its design and performance. It will be shown that the laboratory microscope in its current form is a user-oriented and stable instrument, and has been used in a number of applications. The implementation of a cryogenic sample stage for tomographic imaging of biological samples in their natural environment has enabled applications in biology, and 3D x-ray microscopy of cells was performed for the first time with a laboratory instrument. / QC 20101130

Coherent imaging is a promising method in the field of x-ray microscopy allowing for the nondestructive determination of the interior structure of radiation-hard samples with a spatial resolution that is only limited by the fluence on the sample and the scattering strength of the sample. Ultimately, the achievable spatial resolution is limited by the wavelength of the incoming x-ray radiation. Combining coherent imaging with scanning microscopy to a method called ptychography enables one to also probe extended objects. In this method, a sample is scanned through a defined coherent x-ray beam and at each scan point a diffraction pattern is recorded with a diffraction camera located in the far field of the sample. Neighboring illuminated areas must have a certain overlap to guarantee the collection of sufficient information about the object for a subsequent successful and unique computational reconstruction of the object. Modern ptychographic reconstruction algorithms are even able to reconstruct the complex-valued transmission function of the sample and the complex illumination wave field at the same time. Once the 2D transmission function of a sample is known, it is an obvious step forward to combine ptychography with tomographic techniques yielding the 3D internal structure of an object with unprecedented spatial resolution. Here, projections at varying angular positions of the sample are generated via ptychographic scans and are subsequently used for the tomographic reconstruction. In this thesis the development of 3D ptychography is described. It includes the description of the required experimental environment, the numerical implementation of ptychographic phase retrieval and tomographic reconstruction routines, and a detailed analysis of the performance of 3D ptychography using an example of an experiment carried out at beamline P06 of PETRA III at DESY in Hamburg. In that experiment the investigated object was a Mo/UO2 thin film, which is a simplified model for spent nuclear fuel from nuclear power plant reactors. Such models find application in systematic scientific investigations related to the safe disposal of nuclear waste. We determined the three-dimensional interior structure of this sample with an unprecedented spatial resolution of at least 18 nm. The measurement of the fluorescence signal at each scan point of the ptychograms delivers the two- and three-dimensional elemental distribution of the sample with a spatial resolution of 80 nm. Using the fluorescence data, we assigned the chemical element to the area of the corresponding phase shift in the ptychographic reconstruction of the object phase and to the corresponding refractive index decrement in the tomographic reconstruction. The successful demonstration of the feasibility of the 3D ptychography motivates further applications, for instance, in the field of medicine, of material science, and of basic physical research. / Kohärente Bildgebung ist eine vielversprechende Methode der Röntgenmikroskopie. Sie ermöglicht die zerstörungsfreie Bestimmung der inneren Struktur von strahlenharten Untersuchungsobjekten mit einer räumlichen Auflösung, die im Prinzip nur von der integralen Anzahl der Photonen auf der Probe sowie deren Streukraft abhängt. Letztendlich stellt die Wellenlänge der verwendeten Röntgenstrahlung eine Grenze für die erreichbare räumliche Auflösung dar. Die Kombination der kohärenten Bildgebung mit der Rastermikroskopie zur sogenannten Ptychographie eröffnet die Möglichkeit, auch ausgedehnte Objekte mit hoher Auflösung zu untersuchen. Dabei wird die Probe mit einem räumlich begrenzten, kohärenten Röntgenstrahl abgerastert und an jedem Rasterpunkt ein Beugungsbild von einer im Fernfeld platzierten Beugungskamera registriert. Die Beleuchtungen benachbarter Rasterpunkte müssen dabei zu einem bestimmten Prozentsatz überlappen, um genügend Informationen für eine anschließende computergestützte und eindeutige Rekonstruktion des Objektes sicherzustellen. Moderne Rekonstruktionsalgorithmen ermöglichen sogar die gleichzeitige Rekonstruktion der Transmissionsfunktion des Objektes und der Beleuchtungsfunktion des eintreffenden Röntgenstrahls. Die Verknüpfung der Ptychographie mit der Tomographie zur 3D-Ptychographie ist der nahe liegende Schritt, um nun auch die dreidimensionale innere Struktur von Objekten mit hoher räumlicher Auflösung zu bestimmen. Die Projektionen an den verschiedenen Winkelpositionen der Probe werden dabei mittels ptychographischer Abrasterung der Probe erzeugt und anschließend der tomographischen Rekonstruktion zugrunde gelegt. In dieser Arbeit wird die Entwicklung der 3D-Ptychographie beschrieben. Das beinhaltet die Beschreibung der experimentellen Umgebung, der numerischen Implementierung des ptychographischen und des tomographischen Rekonstruktionsalgorithmus als auch eine detaillierte Darstellung der Durchführung der 3D-Ptychographie am Beispiel eines Experiments, welches unter Verwendung des modernen Nanoprobe-Aufbaus des Strahlrohres P06 am PETRA III Synchrotronring des DESY in Hamburg durchgeführt wurde. Als Untersuchungsobjekt diente dabei ein dünner Mo/UO2-Film, der ein vereinfachtes Modell für die in Reaktoren von Atomkraftwerken verbrauchten Brennstäbe darstellt und deshalb im Bereich des Umweltschutzes Anwendung findet. Die dreidimensionale Struktur der Probe wurde mit einer - für diese Methode bisher einmaligen - räumlichen Auflösung von 18 nm bestimmt. Die Messung des von der Probe kommenden Fluoreszenz-Signals an jedem Rasterpunkt der Ptychogramme ermöglichte zusätzlich die Bestimmung der zwei- und dreidimensionalen Elementverteilung innerhalb der Probe mit einer räumlichen Auflösung von 80 nm. Anhand der Fluoreszenzdaten konnte sowohl den Bereichen verschiedener Phasenschübe in den ptychographischen Rekonstruktionen der Objektphase als auch den verschiedenen Werten des Dekrementes des Brechungsindex in der tomographischen Rekonstruktion, das entsprechende chemische Element zugeordnet werden. Die erfolgreiche Demonstration der Durchführbarkeit der 3D-Ptychographie motiviert weitere zukünftige Anwendungen, z. B. auf dem Gebiet der Medizin, der Materialforschung und der physikalischen Grundlagenforschung.

<p>This thesis describes the development of soft x-rayreflective optics, instrumentation and applications for compactsoft x-ray microscopy. The microscope is based on a table-topliquid-jet-target laser-plasma source in combination with aspherical normal-incidence multilayer condenser mirror andnanofabricated diffractive optics for imaging. High-resolutionimaging is performed at the wavelength 3.374 nm in thewater-window (2.3 - 4.4 nm), where natural contrast betweencarbon and oxygen allows imaging of unstained biologicalmaterial in their natural aqueous environment.</p><p>The design and implementation of a compact soft x-rayreflectometer based on a laser-plasma source is described. Thereflectometer allows rapid and accurate characterization ofnormal-incidence multilayer coatings used at water-windowwavelengths. This instrument, which measures absolutereflectivity and multilayer period, is now used in thefabrication process, aiming to improve the soft x-raynormal-incidence multilayer condenser system of the compactsoft x-ray microscope. Latest results from the developmentprocess are presented.</p><p>A new design of the compact soft x-ray microscope, withimprovements in mechanical and thermal stability, providesuser-friendly and daily operation. This includes also a newnozzle design for the liquid-jet-target laser-plasma source,which enables higher source stability and operation withcryogenic liquids. In addition, a new experimental arrangementunder construction is briefly described. It will utilize acondenser zone plate and operate at the wavelength 2.478nm.</p><p>Finally, performance test of the compact soft x-raymicroscope is presented and discussed. In addition, a projectto explore the use of soft x-ray microscopy for imaging sensorycells is described. The high-resolution imaging of these cellswas performed at the synchrotron-based soft x-ray microscope atLawrence Berkeley National Laboratory (LBNL).</p>

This thesis summarizes the present status of the nanofabrication of diffractive optics, i.e. zone plates, and test objects for soft x-ray microscopy at KTH. The emphasis is on new and improved fabrication processes for nickel and germanium zone plates. A new concept in which nickel and germanium are combined in a zone plate is also presented. The main techniques used in the fabrication are electron beam lithography for the patterning, followed by plasma etching and electroplating for the structuring of the optical materials. The process for fabricating nickel zone plates has been significantly improved. The reproducibility of the electroplating step has been increased by the implementation of an in-situ rate measurement and an end-point detection method. We have also shown that pulse plating can be used to obtain zone plates with a uniform height profile. New plating mold materials have been introduced and electron-beam curing of the molds has been investigated and implemented to increase their mechanical stability so that pattern collapse in the electroplating step can be avoided. The introduction of cold development has improved the achievable resolution of the process. This has enabled the fabrication of zone plates with outermost zone widths down to 16 nm. The nickel process has also recently been adapted to fabrication of gold structures intended for test objects and hard x-ray zone plates. For the fabrication of germanium zone plates we developed a highly anisotropic plasma-etch process using Cl2 feed and sidewall passivation. Germanium zone plates have been fabricated with zone widths down to 30 nm. The diffraction efficiency is comparable to that of nickel zone plates, but the process does not involve electroplating and thus has for potential for highyield fabrication. The combination of nickel and germanium is a new fabrication concept that provides a means to achieve high diffraction efficiency even for thin nickel. The idea is to fabricate a nickel zone plate on a germanium film. The nickel zone plate itself is then used as etch mask for a highly selective CHF3- plasma etch into the germanium layer. Proof of principle experiments showed an efficiency increase of about a factor of two for nickel zone plates with a 50- nm nickel thickness. / QC 20100728