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.
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.
X-ray diffraction from biomolecular assemblies is a powerful technique which can provide structural information about complex architectures such as the locomotor systems underlying muscle contraction. However, in its conventional form, macromolecular diffraction averages over large ensembles. Progress in x-ray optics has now enabled to probe structures on sub-cellular scales, with the beam confined to a distinct organelle. Here, we use scanning small angle x-ray scattering (scanning SAXS) to probe the diffraction from cytoskeleton networks in cardiac tissue cells. In particular, we focus on actin-myosin composites, which we identify as the dominating contribution to the anisotropic diffraction patterns, by correlation with optical fluorescence microscopy. To this end, we use a principal component analysis approach to quantify direction, degree of orientation, nematic order, and the second moment of the scattering distribution in each scan point. We compare the fiber orientation from micrographs of fluorescently labeled actin fibers to the structure orientation of the x-ray dataset and thus correlate signals of two different measurements: the native electron density distribution of the local probing area versus specifically labeled constituents of the sample. Further, we develop a robust and automated fitting approach based on a power law expansion, in order to describe the local structure factor in each scan point over a broad range of the momentum transfer q r . Finally, we demonstrate how the methodology shown for freeze dried cells in the first part of the paper can be translated to alive cell recordings.Recent progress in x-ray optics has now overcome this barrier, enabling hard x-ray spot sizes in the submicron range [24], well suited to record structural data within precise locations of a single cell. X-rays even in the multi-keV regime required for diffraction studies can nowadays be focussed by a variety of optical elements, including diffractive optics such as Fresnel zone plates [25], compound refractive lenses [26-28] and elliptical Kirkpatrick-Baez (KB) mirrors [29][30][31][32]. Similar to earlier scanning diffraction work on biomaterials such as wood and bone [33][34][35][36][37][38], we can hence now combine high resolution in reciprocal space with at least moderate resolution in real space. Scanning small angle x-ray scattering (scanning SAXS) experiments requiring a sample environment for biological cells are typically not compatible with the ultimate small spot sizes of 10 nm and below, as presented in [31,39,40], but values in the range of 80-300 nm are feasible, in particular in terms of the working distance, and readily allow structure factors to be assigned to different cellular compartments. Presently, feasibility of cellular scanning SAXS has been demonstrated for a variety of biological cells, ranging from bacterial cells D. radiodurans [6] to eukaryotes such as the amoeba D. discoideum [10], adenoma cells [7-9, 41], and human mesenchymal stem cells (hMSC) [11].Beyond these previous proof-...
We present a correlative microscopy approach for biology based on holographic X-ray imaging, X-ray scanning diffraction, and stimulated emission depletion (STED) microscopy. All modalities are combined into the same synchrotron endstation. In this way, labeled and unlabeled structures in cells are visualized in a complementary manner. We map out the fluorescently labeled actin cytoskeleton in heart tissue cells and superimpose the data with phase maps from X-ray holography. Furthermore, an array of local far-field diffraction patterns is recorded in the regime of small-angle X-ray scattering (scanning SAXS), which can be interpreted in terms of biomolecular shape and spatial correlations of all contributing scattering constituents. We find that principal directions of anisotropic diffraction patterns coincide to a certain degree with the actin fiber directions and that actin stands out in the phase maps from holographic recordings. In situ STED recordings are proposed to formulate models for diffraction data based on co-localization constraints.
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
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