X-ray diffraction imaging of cardiac cells and tissue

Abstract

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 Structural Analysis of Single Adult Cardiomyocytes: Tomographic Imaging and Microdiffraction

“…For freezedried heart muscle cells (20,23), we have observed anisotropic diffraction patterns, and by correlating the x-ray signal to fluorescence data obtained by in situ stimulated emission depletion (STED) microscopy recordings, we have linked the signal to the actin portion of the cell (24). A diffraction signature of a sarcomeric complex was observed for the first time, to our knowledge, for hydrated and chemically fixed cardiomyocytes (22). Hence, by this technique, variations of structural parameters in single isolated cells or in muscle tissue have now become accessible, unaffected by macroscopic averaging.…”

X-ray structural analysis of single adult cardiomyocytes: tomographic imaging and micro-diffraction

“…In order to reduce absorption for the agarose embedded cells and to allow for a rotation of the sample, the cells were transferred into a capillary using a 1 mm biopsy punch. The protocol of the preparation for freeze-dried samples closely followed (20,22,23). CMs were pipetted onto SiN-windows coated with a 1% fibronectin coating and incubated for 15 min.…”

“…In view of radiation damage, a 'diffract before damage' strategy is adopted (21). Figure 6 illustrates the data collection and processing, which closely followed (22), but now applied to living cells. We also took into account the constraints of sparse sampling and reduced dose to collect a signal which is not spoiled by radiation damage (21).…”

“…For freeze-dried heart muscle cells (20,23), we have observed anisotropic diffraction patterns, and by correlating the x-ray signal to fluorescence data obtained by in situ stimulated emission depletion (STED) microscopy recordings, we have linked the signal to the actin portion of the cell (24). A diffraction signature of a sarcomeric complex was observed for the first time for hydrated and chemically fixed cardiomyocytes (22). Hence, by this technique, variations of structural parameters in single isolated cells or in muscle tissue have now become accessible, unaffected by macroscopic averaging.…”

“…Scanning diffraction from biological cells with spot sizes smaller than a single organelle are still more challenging in terms of signal-to-noise (15)(16)(17)(18)(19)(20), as well as radiation damage (21). Recently, we have used this approach to study the cytoskeletal structure of single CMs in order to compare the signal level for different preparation states (22). For freeze-dried heart muscle cells (20,23), we have observed anisotropic diffraction patterns, and by correlating the x-ray signal to fluorescence data obtained by in situ stimulated emission depletion (STED) microscopy recordings, we have linked the signal to the actin portion of the cell (24).…”