3D Multimodal X-ray microscopy of biological specimens

Studies of biological systems typically require the application of several complementary methods able to yield statistically-relevant results at a unique level of sensitivity. Long penetration depth of X-rays makes them particularly suitable for non-destructive volumetric investigations of whole cells and tissue sections, providing structural and elemental specificity at nanoscale spatial resolutions.X-ray ptychography is a promising imaging technique for sub-100-nm structural studies of weakly-scattering extended biological specimens. It provides quantitative phase-contrast maps revealing the morphology of an investigated object. In addition, its scanning nature allows for a simultaneous acquisition of nanoscale X-ray fluorescence, yielding the element distributions at an unmatched sensitivity.Thus, the goal of this PhD project has been to combine X-ray fluorescence and ptychography to enable a robust correlation of elemental distributions with respect to the cellular morphology. Exploiting a highly intense and coherent X-ray beam at beamline P11 of the low-emittance synchrotron light source PETRA III in Hamburg, Germany, a versatile multimodal scanning X-ray microscope was developed in the framework of this PhD project. This thesis describes the consecutive stages of the microscope’s development and highlights its application in structural determination of biological samples.In the first stage of this PhD project, a 2D long-range scanning unit was developed, enabling seamless, serial measurements of many targets at nanometer precision. With this setup, the PhD work allowed to optimise the correlative imaging method at higher incident photon energies for mapping of first-row transition metals up to iron, while using a nano-focussed beam. In this way, it addressed limitations of the so-far presented demonstrations, restrained to light-element mapping in the context of organelles of single cells. The correlative imaging method was then used to quantify the iron distributions in a population of macrophages treated with Mycobacterium-tuberculosis-targeting iron-oxide nanocontainers in the context of their sub-cellular structure obtained by ptychography. In the second application, the calcium content in a human bone matrix was mapped in close proximity to osteocyte lacunae (perilacunar matrix). A concurrently acquired ptychographic image was then used to remove the mass-thickness effect from the raw calcium map.In the second stage of this work, the concept of concurrent ptychography and X-ray fluorescence was extended to 3D correlative imaging in the framework of computed tomography. For this purpose, an upgraded 3D scanning X-ray microscope was built, commissioned, and used in volumetric imaging of two specimens, using ptychographic X-ray computed tomography. The final performance of the 3D microscope reached far beyond the throughputs of the available ptychographic tomography setups which, until now, have hindered application of the method by a broader biomedical community. A tomogram of the nano-porous glass demonstrated the fastest on-the-fly ptychographic tomography, to date, at an isotropic spatial resolution of 52 nm. In the second application, ptychographic tomography of a chiton’s radular tooth provided a quantitative insight into one of the nature’s hardest biominerals, at a Nyquist-sampling limited spatial resolution of 65 nm.The work has been concluded with a prospect for future operation and opportunities of the correlative method of simultaneous ptychographic and X-ray fluorescence imaging at upcoming diffraction-limited synchrotron light sources

Zernike x-ray ptychography

We present an imaging technique combining Zernike phase-contrast imaging and ptychography. The contrast formation is explained by following the theory of Zernike phase-contrast imaging. The method is demonstrated with x-rays at a photon energy of 6.2 keV, showing how ptychographic reconstruction of a phase sample leads to a Zernike phase-contrast image appearing in the amplitude reconstruction. In addition, the results presented in this Letter indicate an improvement of the resolution of the reconstructed object in the case of Zernike ptychography compared with the conventional one

Influence of finite spatial coherence on ptychographic reconstruction

X-ray ptychography is an ultrahigh-resolution scanning coherent diffractive imaging technique, allowing quantitative measurements of extended samples and a simultaneous reconstruction of the illuminating wavefront. Recent development of the mixed-state reconstruction algorithm has triggered a certain interest in utilizing partially coherent X-ray sources for ptychography. Here, we study how finite spatial coherence influences the reconstructed image of a test structure. Our work shows that use of a highly coherent illumination provides images with better spatial resolution and fewer artefacts than the approach with partial coherence

Multimodal imaging of cubic Cu2O@AuCu_2O@Au nanocage formation via galvanic replacement using X-ray ptychography and nano diffraction

Being able to observe the formation of multi-material nanostructures in situ, simultaneously from a morphological and crystallographic perspective, is a challenging task. Yet, this is essential for the fabrication of nanomaterials with well-controlled composition exposing the most active crystallographic surfaces, as required for highly active catalysts in energy applications. To demonstrate how X-ray ptychography can be combined with scanning nanoprobe diffraction to realize multimodal imaging, we study growing Cu$_2$O nanocubes and their transformation into Au nanocages. During the growth of nanocubes at a temperature of 138 °C, we measure the crystal structure of an individual nanoparticle and determine the presence of (100) crystallographic facets at its surface. We subsequently visualize the transformation of Cu$_2$O into Au nanocages by galvanic replacement. The nanocubes interior homogeneously dissolves while smaller Au particles grow on their surface and later coalesce to form porous nanocages. We finally determine the amount of radiation damage making use of the quantitative phase images. We find that both the total surface dose as well as the dose rate imparted by the X-ray beam trigger additional deposition of Au onto the nanocages. Our multimodal approach can benefit in-solution imaging of multi-material nanostructures in many related fields

Status of the crystallography beamlines at PETRA III

Since 2013, three beamlines for macromolecular crystallography are available to users at the third-generation synchrotron PETRA III in Hamburg: P11, P13 and P14, the latter two operated by EMBL. Beamline P11 is operated by DESY and is equipped with a Pilatus 6M detector. Together with the photon flux of $2×10^{13}$ ph/s provided by the very brilliant X-ray source of PETRA III, a full data set can be typically collected in less than 2min. P11 provides state-of-the-art microfocusing capabilities with beam sizes down to 1×1 $\mu$ $m^{2}$, which makes the beamline ideally suited for investigation of microcrystals and serial crystallography experiments. An automatic sample changer allows fast sample exchange in less than 20s, which enables high-throughput crystallography and fast crystal screening. For sample preparation, an S2 biosafety laboratory is available in close proximity to the beamline

Multimodal X-ray imaging of nanocontainer-treated macrophages and calcium distribution in the perilacunar bone matrix

Studies of biological systems typically require the application of several complementary methods able to yield statistically-relevant results at a unique level of sensitivity. Combined X-ray fluorescence and ptychography offer excellent elemental and structural imaging contrasts at the nanoscale. They enable a robust correlation of elemental distributions with respect to the cellular morphology. Here we extend the applicability of the two modalities to higher X-ray excitation energies, permitting iron mapping. Using a long-range scanning setup, we applied the method to two vital biomedical cases. We quantified the iron distributions in a population of macrophages treated with Mycobacterium-tuberculosis-targeting iron-oxide nanocontainers. Our work allowed to visualize the internalization of the nanocontainer agglomerates in the cytosol. From the iron areal mass maps, we obtained a distribution of antibiotic load per agglomerate and an average areal concentration of nanocontainers in the agglomerates. In the second application we mapped the calcium content in a human bone matrix in close proximity to osteocyte lacunae (perilacunar matrix). A concurrently acquired ptychographic image was used to remove the mass-thickness effect from the raw calcium map. The resulting ptychography-enhanced calcium distribution allowed then to observe a locally lower degree of mineralization of the perilacunar matrix

Ptychographic X-ray speckle tracking with multi-layer Laue lens systems

The ever-increasing brightness of synchrotron radiation sources demands improved X-ray optics to utilize their capability for imaging and probing biological cells, nano-devices and functional matter on the nanometre scale with chemical sensitivity. Hard X-rays are ideal for high-resolution imaging and spectroscopic applications owing to their short wavelength, high penetrating power and chemical sensitivity. The penetrating power that makes X-rays useful for imaging also makes focusing them technologically challenging. Recent developments in layer deposition techniques have enabled the fabrication of a series of highly focusing X-ray lenses, known as wedged multi-layer Laue lenses. Improvements to the lens design and fabrication technique demand an accurate, robust, in situ and at-wavelength characterization method. To this end, a modified form of the speckle tracking wavefront metrology method has been developed. The ptychographic X-ray speckle tracking method is capable of operating with highly divergent wavefields. A useful by-product of this method is that it also provides high-resolution and aberration-free projection images of extended specimens. Three separate experiments using this method are reported, where the ray path angles have been resolved to within 4 nrad with an imaging resolution of 45 nm (full period). This method does not require a high degree of coherence, making it suitable for laboratory-based X-ray sources. Likewise, it is robust to errors in the registered sample positions, making it suitable for X-ray free-electron laser facilities, where beam-pointing fluctuations can be problematic for wavefront metrology