X-Ray Structural Analysis of Single Adult Cardiomyocytes: Tomographic Imaging and Microdiffraction.

We present a multiscale imaging approach to characterize the structure of isolated adult murine cardiomyocytes based on a combination of full-field three-dimensional coherent x-ray imaging and scanning x-ray diffraction. Using these modalities, we probe the structure from the molecular to the cellular scale. Holographic projection images on freeze-dried cells have been recorded using highly coherent and divergent x-ray waveguide radiation. Phase retrieval and tomographic reconstruction then yield the three-dimensional electron density distribution with a voxel size below 50 nm. In the reconstruction volume, myofibrils, sarcomeric organization, and mitochondria can be visualized and quantified within a single cell without sectioning. Next, we use microfocusing optics by compound refractive lenses to probe the diffraction signal of the actomyosin lattice. Comparison between recordings of chemically fixed and untreated, living cells indicate that the characteristic lattice distances shrink by ∼10% upon fixation.

Radiation damage studies in cardiac muscle cells and tissue using microfocused X-ray beams: experiment and simulation.

Soft materials are easily affected by radiation damage from intense, focused synchrotron beams, often limiting the use of scanning diffraction experiments to radiation-resistant samples. To minimize radiation damage in experiments on soft tissue and thus to improve data quality, radiation damage needs to be studied as a function of the experimental parameters. Here, the impact of radiation damage in scanning X-ray diffraction experiments on hydrated cardiac muscle cells and tissue is investigated. It is shown how the small-angle diffraction signal is affected by radiation damage upon variation of scan parameters and dose. The experimental study was complemented by simulations of dose distributions for microfocused X-ray beams in soft muscle tissue. As a simulation tool, the Monte Carlo software package EGSnrc was used that is widely used in radiation dosimetry research. Simulations also give additional guidance for a more careful planning of dose distribution in tissue.

A beamline-compatible STED microscope for combined visible-light and X-ray studies of biological matter.

A dedicated stimulated emission depletion (STED) microscope had been designed and implemented into the Göttingen Instrument for Nano-Imaging with X-rays (GINIX) at the synchrotron beamline P10 of the PETRA III storage ring (DESY, Hamburg). The microscope was installed on the same optical table used for X-ray holography and scanning small-angle X-ray scattering (SAXS). Scanning SAXS was implemented with the Kirkpatrick-Baez (KB) nano-focusing optics of GINIX, while X-ray holography used a combined KB and X-ray waveguide optical system for full-field projection recordings at a defocus position of the object. The STED optical axis was aligned (anti-)parallel to the focused synchrotron beam and was laterally displaced from the KB focus. This close proximity between the STED and the X-ray probe enabled in situ combined recordings on the same biological cell, tissue or any other biomolecular sample, using the same environment and mounting. Here, the instrumentation and experimental details of this correlative microscopy approach are described, as first published in our preceding work [Bernhardt et al. (2018), Nat. Commun. 9, 3641], and the capabilities of correlative STED microscopy, X-ray holography and scanning SAXS are illustrated by presenting additional datasets on cardiac tissue cells with labeled actin cytoskeleton.

The optical stretcher as a tool for single-particle X-ray imaging and diffraction.

For almost half a century, optical tweezers have successfully been used to micromanipulate micrometre and sub-micrometre-sized particles. However, in recent years it has been shown experimentally that, compared with single-beam traps, the use of two opposing and divergent laser beams can be more suitable in studying the elastic properties of biological cells and vesicles. Such a configuration is termed an optical stretcher due to its capability of applying high deforming forces on biological objects such as cells. In this article the experimental capabilities of an optical stretcher as a potential sample delivery system for X-ray diffraction and imaging studies at synchrotrons and X-ray free-electron laser (FEL) facilites are demonstrated. To highlight the potential of the optical stretcher its micromanipulation capabilities have been used to image polymer beads and label biological cells. Even in a non-optimized configuration based on a commercially available optical stretcher system, X-ray holograms could be recorded from different views on a biological cell and the three-dimensional phase of the cell could be reconstructed. The capability of the setup to deform cells at higher laser intensities in combination with, for example, X-ray diffraction studies could furthermore lead to interesting studies that couple structural parameters to elastic properties. By means of high-throughput screening, the optical stretcher could become a useful tool in X-ray studies employing synchrotron radiation, and, at a later stage, femtosecond X-ray pulses delivered by X-ray free-electron lasers.

Scanning X-ray diffraction on cardiac tissue: automatized data analysis and processing.

A scanning X-ray diffraction study of cardiac tissue has been performed, covering the entire cross section of a mouse heart slice. To this end, moderate focusing by compound refractive lenses to micrometer spot size, continuous scanning, data acquisition by a fast single-photon-counting pixel detector, and fully automated analysis scripts have been combined. It was shown that a surprising amount of structural data can be harvested from such a scan, evaluating the local scattering intensity, interfilament spacing of the muscle tissue, the filament orientation, and the degree of anisotropy. The workflow of data analysis is described and a data analysis toolbox with example data for general use is provided. Since many cardiomyopathies rely on the structural integrity of the sarcomere, the contractile unit of cardiac muscle cells, the present study can be easily extended to characterize tissue from a diseased heart.

Elemental quantification and analysis of structural abnormalities in neurons from Parkinson's-diseased brains by X-ray fluorescence microscopy and diffraction.

In this work we use scanning X-ray microscopy to study the structure and elemental composition of neuromelanin-positive neurons in substantia nigra tissue of Parkinson patients (PD) and controls. A total of 53 neurons were analyzed with X-ray fluorescence (XRF) and diffraction using sub-µm-focused synchrotron radiation. A statistical evaluation identified copper as the most group-discriminating element and indicated that interindividual and intraindividual variations are of great relevance in tissue measurements of diseased patients and prevent from automated group clustering. XRF analyses of two Lewy bodies (LBs) highlight a heterogeneity in elemental distributions in these LBs, whereas an innovative X-ray diffraction-based method approach was used to reveal ß-sheet-rich crystalline structures in LBs. Overall, sub-µm-focus X-ray microscopy highlighted the elemental heterogeneity in PD pathology.

X-ray diffraction and second harmonic imaging reveal new insights into structural alterations caused by pressure-overload in murine hearts.

We demonstrate a label-free imaging approach to study cardiac remodeling of fibrotic and hypertrophic hearts, bridging scales from the whole organ down to the molecular level. To this end, we have used mice subjected to transverse aortic constriction and imaged adjacent cardiac tissue sections by microfocus X-ray diffraction and second harmonic generation (SHG) imaging. In this way, the acto-myosin structure was probed in a spatially resolved manner for entire heart sections. From the recorded diffraction data, spatial maps of diffraction intensity, anisotropy and orientation were obtained, and fully automated analysis depicted the acto-myosin filament spacing and direction. X-ray diffraction presented an overview of entire heart sections and revealed that in regions of severe cardiac remodeling the muscle mass is partly replaced by connective tissue and the acto-myosin lattice spacing is increased at these regions. SHG imaging revealed sub-cellular structure of cardiac tissue and complemented the findings from X-ray diffraction by revealing micro-level distortion of myofibrils, immune cell infiltration at regions of cardiac remodeling and the development of fibrosis down to the scale of a single collagen fibril. Overall, our results show that both X-ray diffraction and SHG imaging can be used for label-free and high-resolution visualization of cardiac remodeling and fibrosis progression at different stages in a cardiac pressure-overload mouse model that cannot be achieved by conventional histology.

X-ray diffraction imaging of cardiac cells and tissue.

With the development of advanced focusing optics for x-rays, we can now use x-ray beams with spot sizes in the micro- or nanometer range to scan cells and large areas of tissues and continuously record the diffraction signals. From this data, x-ray scattering maps or so-called x-ray darkfield images are computed showing how different types of cells or regions of tissues differ in their diffraction intensity. At the same time a diffraction pattern is available for each scan point which encodes the local nanostructure, averaged over many contributing constituents illuminated by the beam. In this work we have exploited these new capabilities of scanning x-ray diffraction to investigate cardiac muscle cells as well as cardiac tissue. We give examples of how cardiac cells, especially living, cultured cells, can be prepared to be compatible with the instrumentation constraints of nano- or micro-diffraction instruments. Furthermore, we show how the developmental stage, ranging from neonatal to adult cells, as well as the final preparation state of the cardiomyocytes influences the recorded scattering signal and how these diffraction signals compare to the structure of a fully developed cardiac muscle.

The Dual Functional Reflecting Iris of the Zebrafish.

Many marine organisms have evolved a reflective iris to prevent unfocused light from reaching the retina. The fish iris has a dual function, both to camouflage the eye and serving as a light barrier. Yet, the physical mechanism that enables this dual functionality and the benefits of using a reflective iris have remained unclear. Using synchrotron microfocused diffraction, cryo-scanning electron microscopy imaging, and optical analyses on zebrafish at different stages of development, it is shown that the complex optical response of the iris is facilitated by the development of high-order organization of multilayered guanine-based crystal reflectors and pigments. It is further demonstrated how the efficient light reflector is established during development to allow the optical functionality of the eye, already at early developmental stages.

Imaging of neuronal tissues by x-ray diffraction and x-ray fluorescence microscopy: evaluation of contrast and biomarkers for neurodegenerative diseases.

We have used scanning X-ray diffraction (XRD) and X-ray fluorescence (XRF) with micro-focused synchrotron radiation to study histological sections from human substantia nigra (SN). Both XRF and XRD mappings visualize tissue properties, which are inaccessible by conventional microscopy and histology. We propose to use these advanced tools to characterize neuronal tissue in neurodegeneration, in particular in Parkinson's disease (PD). To this end, we take advantage of the recent experimental progress in x-ray focusing, detection, and use automated data analysis scripts to enable quantitative analysis of large field of views. XRD signals are recorded and analyzed both in the regime of small-angle (SAXS) and wide-angle x-ray scattering (WAXS). The SAXS signal was analyzed in view of the local myelin structure, while WAXS was used to identify crystalline deposits. PD tissue scans exhibited increased amounts of crystallized cholesterol. The XRF analysis showed increased amounts of iron and decreased amounts of copper in the PD tissue compared to the control.

Time-resolved coherent X-ray diffraction imaging of surface acoustic waves.

Time-resolved coherent X-ray diffraction experiments of standing surface acoustic waves, illuminated under grazing incidence by a nanofocused synchrotron beam, are reported. The data have been recorded in stroboscopic mode at controlled and varied phase between the acoustic frequency generator and the synchrotron bunch train. At each time delay (phase angle), the coherent far-field diffraction pattern in the small-angle regime is inverted by an iterative algorithm to yield the local instantaneous surface height profile along the optical axis. The results show that periodic nanoscale dynamics can be imaged at high temporal resolution in the range of 50 ps (pulse length).