In this book, the author describes the development of the experimental diffraction setup and structural analysis of non-crystalline particles from material science and biology. Recent advances in X-ray free electron laser (XFEL)-coherent X-ray diffraction imaging (CXDI) experiments allow for the structural analysis of non-crystalline particles to a resolution of 7 nm, and to a resolution of 20 nm for biological materials. Now XFEL-CXDI marks the dawn of a new era in structural analys of non-crystalline particles with dimensions larger than 100 nm, which was quite impossible in the 20th century. To conduct CXDI experiments in both synchrotron and XFEL facilities, the author has developed apparatuses, named KOTOBUKI-1 and TAKASAGO-6 for cryogenic diffraction experiments on frozen-hydrated non-crystalline particles at around 66 K. At the synchrotron facility, cryogenic diffraction experiments dramatically reduce radiation damage of specimen particles and allow tomography CXDI experiments. In addition, in XFEL experiments, non-crystalline particles scattered on thin support membranes and flash-cooled can be used to efficiently increase the rate of XFEL pulses. The rate, which depends on the number density of scattered particles and the size of X-ray beams, is currently 20-90%, probably the world record in XFEL-CXDI experiments. The experiment setups and results are introduced in this book. The author has also developed software suitable for efficiently processing of diffraction patterns and retrieving electron density maps of specimen particles based on the diffraction theory used in CXDI.

Understanding the intricate details of muscle contraction has a long-standing tradition in biophysical research. X-ray diffraction has been one of the key techniques to resolve the nanometer-sized molecular machinery involved in force generation. Modern, powerful X-ray sources now provide billions of X-ray photons in time intervals as short as microseconds, enabling fast time-resolved experiments that shed further light on the complex relationship between muscle structure and function. Another approach harnesses this power by repeatedly performing such an experiment at different locations in a sample. With millions of repeated exposures in a single experiment, X-ray diffraction can seamlessly be turned into a raster imaging method, neatly combining real- and reciprocal space information. This thesis has focused on the advancement of this scanning scheme and its application to soft biological tissue, in particular muscle tissue. Special emphasis was placed on the extraction of meaningful, quantitative structural parameters such as the interfilament distance of the actomyosin lattice in cardiac muscle. The method was further adapted to image biological samples on a range of scales, from isolated cells to millimeter-sized tissue sections. Due to the ‘photon-hungry’ nature of the technique, its full potential is often exploited in combination with full-field imaging techniques. From the vast set of microscopic tools available, coherent full-field X-ray imaging has proven to be particularly useful. This multimodal approach allows to correlate two- and three-dimensional images of cells and tissue with diffraction maps of structure parameters. With the set of tools developed in this thesis, scanning X-ray diffraction can now be efficiently used for the structural analysis of soft biological tissues with overarching future applications in biophysical and biomedical research.

The past 70 years of muscle research have profoundly shaped our current understanding of the structure and function of muscle. X-ray diffraction became a key method in its structural analysis and yielded valuable insights into the molecular arrangement of the contraction apparatus. This work employs an extension of the X-ray diffraction methodology, scanning X-ray diffraction, for structural imaging of biological cells and tissue. With this technique periodicites in a structure on the order of several nanometers can be detected and, by raster scanning of the X-ray beam over the sample, imag...

The use of coherent x rays for microscopic imaging has seen a rapid and ongoing development within the past decade, driven by an increasing availability of highly brilliant and coherent sources worldwide. Accordingly, novel methods have been developed, which replace the microscope‘s objective lens by a numerical reconstruction scheme. The aim of the present work is to study how very recent experimental and algorithmic developments in the field can be implemented towards a highly sensitive and fully quantitative microscopy method for imaging of biological cells. To this end, different experimental approaches are studied, based on coherent far-field as well as near-field diffraction. At first, an application of the novel ptychographic imaging method to single biological cells is presented. In particular, it is demonstrated how weakly scattering biological specimens can be imaged with fully quantitative density contrast. Alongside, a sueccessful extension of the method towards soft x-ray energies is described.In the second part of the work it is shown how x-ray waveguides can be used as a point source for propagation-based microscopy of single cells in the hard x-ray regime. The specifically devised iterative reconstruction scheme allows for full quantitativity and high sensitivity and thus enables an application to single biological cells. The work contains a thorough introduction into the x-ray optical methods applied and aims at a useful and self-contained overview on aspects of signal and Fourier theory relevant for the used numerical propagation schemes.

Since its first experimental demonstration in 1999, Coherent X-Ray Diffractive Imaging has become one of the most promising high resolution X-Ray imaging techniques using coherent radiation produced by brilliant synchrotron storage rings. The ability to directly invert diffraction data with the help of advanced algorithms has paved the way for microscopic investigations and wave-field analyses on the spatial scale of nanometres without the need for inefficient imaging lenses. X-Ray phase contrast which is a measure of the electron density is an important contrast mode of soft biological specimens. For the case of many dominant elements of soft biological matter, the electron density can be converted into an effective mass density offering a unique quantitative information channel which may shed light on important questions such as DNA compaction in the bacterial nucleoid through ‚weighing with light‘. In this work X-Ray phase contrast maps have been obtained from different biological samples by exploring different methods. In particular, the techniques Ptychography and Waveguide-Holographic-Imaging have been used to obtain twodimensional and three-dimensional mass density maps on the single-cell-level of freeze-dried cells of the bacteria Deinococcus radiodurans, Bacillus subtilis and Bacillus thuringiensis allowing, for instance, to estimate the dry weight of the bacterial genome in a near native state. On top of this, reciprocal space information from coherent small angle X-Ray scattering (cellular Nano-Diffraction) of the fine structure of the bacterial cells has been recorded in a synergistic manner and has been analysed down to a resolution of about 2.3/nm exceeding current limits of direct imaging approaches. Furthermore, the dynamic range of present detector technology being one of the major limiting factors of ptychographic phasing of farfield diffraction data has been significantly increased. Overcoming this problem for the case of the very intense X-Ray beam produced by Kirkpatrick-Baez mirrors has been explored by using semi-transparent central stops.

The advances and technical improvements of X-ray imaging techniques, taking advantage of X-ray focussing optics and high intensity synchrotron sources, nowadays allow for the use of X-rays to probe the cellular nanoscale. Importantly, X-rays permit thick samples to be imaged without sectioning or slicing. In this work, two macromolecules, namely keratin intermediate filament (IF) proteins and DNA, both essential components of cells, were studied by X-ray techniques. Keratin IF proteins make up an integral part of the cytoskeleton of epithelial cells and form a dense intracellular network of bundles. This network is built from monomers in a hierarchical fashion. Thus, the keratin structure formation spans a large range of length scales from a few nanometres (monomers) to micrometres (networks). Here, keratin was studied at three different scales: i) filaments, ii) bundles and iii) networks. Solution small-angle X-ray scattering revealed distinct structural and organisational characteristics of these highly charged polyelectrolyte filaments, such as increasing radius with increasing salt concentration and spatial accumulation of ions depending on the salt concentration. The results are quantified by employing advanced modelling of keratin IFs by a core cylinder fl anked with Gaussian chains. Scanning micro- diffraction was used to study keratin at the bundle scale. Very different morphologies of keratin bundles were observed at different salt conditions. At the network scale, new imaging approaches and analyses were applied to the study of whole cells. Ptychography and scanning X-ray nano-diffraction imaging were performed on the same cells, allowing for high resolution in real and reciprocal space, thereby revealing the internal structure of these networks. By using a fitting routine based on simulations of IFs packed on a hexagonal lattice, the radius of each fi lament and distance between fi laments were retrieved. In mammalian cells, each nucleus contains 2 nm-thick DNA double helices with a total length of about 2 m. The DNA strands are packed in a highly hierarchical manner into individual chromosomes. DNA was studied in intact cells by visible light microscopy and scanning X-ray nano-diffraction, unveiling the compaction und decompaction of DNA during the cell cycle. Thus, we obtained information on the aggregation state of the nuclear DNA at a real space resolution on the order of few hundreds nm. To exploit to the reciprocal space information, individual diffraction patterns were analysed according to a generalised Porod’s law at a resolution down to 10 nm. We were able to distinguish nucleoli, heterochromatin and euchromatin in the nuclei and follow the compaction and decompaction during the cell division cycle.

In 1979, a conference on x-ray microscopy was organized by the New York Academy of Sciences, and in 1983, the Second Interna tional Symposium on X-ray Imaging was organized by the Akademie der Wissenschaften in Gottingen, Federal Republic of Germany. This volume contains the contributions to the symposium "X-ray Microscopy '86", held in Taipei, Taiwan, the Republic of China in August 1986. This is the first volume which intends to provide up-to date information on x-ray imaging to biologists, therefore, emphasis was given to specimen preparation techniques and image interpreta tion. Specimen preparation represents a major part of every microscopy work, therefore, it should be strongly emphasized in this emerging field of x-ray microscopy. Theoretically, x-ray microscopy offers the potential for the study of unfixed, hydrated biological ma terials. Since very few biological system can be directly observed without specimen preparation, we would like to emphasize that new information on biological specimens can only be obtained if the speci men is properly prepared. In the past decade, many of the published x-ray images were obtained from poorly prepared biological speci mens, mainly air-dried materials. Therefore, one of the goals of this conference is to bring the importance of specimen preparation to the attention of x-ray microscopy community. X-ray microscopy can be subdivided into several major areas. They are the classic x-ray projection microscope, x-ray contact imag ing (microradiography) and the more recent x-ray scanning micro scope, x-ray photoelectron microscope and x-ray imaging microscope.