Jan-David Nicolas

Combined scanning X-ray diffraction and holographic imaging of cardiomyocytes

This article presents scanning small-angle X-ray scattering (SAXS) experiments on the actomyosin assemblies in freeze-dried neo-natal rat cardiac muscle cells. By scanning the cells through a sub-micrometre focused beam, the local structure and filament orientation can be probed and quantified. To this end, SAXS data were recorded and analyzed directly in reciprocal space to generate maps of different structural parameters (scanning SAXS). The scanning SAXS experiments were complemented by full-field holographic imaging of the projected electron density, following a slight rearrangement of the instrumental setup. It is shown that X-ray holography is ideally suited to complete missing scattering data at low momentum transfer in the structure factor, extending the covered range of spatial frequencies by two orders of magnitude. Regions of interest for scanning can be easily selected on the basis of the electron density maps. Finally, the combination of scanning SAXS and holography allows for a direct verification of possible radiation-induced structural changes in the cell.

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

The time-dependent one-dimensional height profile of a standing surface acoustic wave on an LiNbO3 substrate has been reconstructed from stroboscopically recorded coherent grazing-incidence small-angle diffraction patterns.

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.

The Dual Functional Reflecting Iris of the Zebrafish

Abstract 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.

Development of High-Order Organization of Guanine-Based Reflectors Underlies the Dual Functionality of the Zebrafish Iris

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 mechanism that enables this dual functionality and the benefits of using a reflective iris have remained unclear. Using synchrotron micro-focused diffraction, cryo-SEM imaging and optical analyses on zebrafish at different stages of development, we show that the complex optical response of the iris is facilitated by the development a high-order organization of multilayered guanine-based crystal reflectors and pigments. We further demonstrates how the efficient light reflector is established during development to allow the optical functionality of the eye, already at early developmental stages. These results shed light on the evolutionary drive for developing a compact reflective iris, which are widely used by many animal species. One Sentence Summary The dual function of the zebrafish iris as a light barrier and camouflage reflector is enabled by the high-order organization of intracellular guanine crystals and pigments.

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.

Nanoscale holographic tomography of heart tissue with x-ray waveguide optics

In this work we present x-ray phase-contrast tomography of heart tissue from mouse, combining computed tomography (CT) scans with laboratory and synchrotron radiation. The work serves as a proof-of-concept that the cyto-architecture and in particular the myofibril orientation can be assessed in three dimensions (3D) by phase-contrast CT. We demonstrate the synergistic use of laboratory μ-CT and of the high resolution synchrotron setup based on waveguide optics. Details on preparation, instrumentation and analysis are given, as a state of the art reference for heart tissue tomography, and as a starting point for further progress.

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 Parkinsons 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.