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. 2017 Oct 23;7(1):13775.
doi: 10.1038/s41598-017-13175-9.

Live cell X-ray imaging of autophagic vacuoles formation and chromatin dynamics in fission yeast

Natalja Strelnikova  1 , Nora Sauter  1 , Manuel Guizar-Sicairos  2 , Michael Göllner  1 , Ana Diaz  2 , Petrina Delivani  3 , Mariola Chacón  3 , Iva M Tolić  3   4 , Vasily Zaburdaev  5 , Thomas Pfohl  6   7   8
Affiliations

Live cell X-ray imaging of autophagic vacuoles formation and chromatin dynamics in fission yeast

Natalja Strelnikova et al. Sci Rep. .

Abstract

Seeing physiological processes at the nanoscale in living organisms without labeling is an ultimate goal in life sciences. Using X-ray ptychography, we explored in situ the dynamics of unstained, living fission yeast Schizosaccharomyces pombe cells in natural, aqueous environment at the nanoscale. In contrast to previous X-ray imaging studies on biological matter, in this work the eukaryotic cells were alive even after several ptychographic X-ray scans, which allowed us to visualize the chromatin motion as well as the autophagic cell death induced by the ionizing radiation. The accumulated radiation of the sequential scans allowed for the determination of a characteristic dose of autophagic vacuole formation and the lethal dose for fission yeast. The presented results demonstrate a practical method that opens another way of looking at living biological specimens and processes in a time-resolved label-free setting.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
Schematic representation of the experimental ptychography setup for imaging living cells. (a) For the X-ray ptychography experiments, a monochromatic (λ = 0.2 nm) beam was used to coherently illuminate a pinhole. The cell sample was scanned to collect a series of diffraction patterns from partially overlapping illuminated regions, which allow for a robust image reconstruction. The high dynamic range and count rate of the detector allows us to record the full dynamic range of the 2D diffraction patterns at the detector and avoid a loss of low spatial-frequency information that would occur if a beamstop was used. (b) A visible light bright-field optical micrograph shows three fission Schizosaccharomyces pombe yeast cells under nitrogen starvation conditions, where two of them were banana-shaped zygotes. (c) Corresponding fluorescence microscopy images of the same cells as in (b) in a time interval of 5 min are shown. In order to distinguish zygotes with moving chromosomes, ‘nuclear oscillations’, among cells with ‘non-oscillating’ ones, the rec25 gene was labeled with green fluorescent protein (GFP) and used as an indirect marker of DNA double strand breaks. Here, only one of two zygotes was at the horsetail stage.
Figure 2
Figure 2
X-ray ptychography images of a live fission yeast zygote. (a) Successive image sequence of a live fission yeast zygote obtained by ptychographic CDI scans. (b) Phase shift histograms (plot of the number of pixels with a specific phase shift) of a zygote in the image without autophagic vacuoles (a, iii) and with autophagic vacuoles (a, vi). A positive shift corresponds to a lighter color. (c) Projected cell area and projected vacuole area versus the radiation dose calculated from the ptychographic zygote images. Each set of grey circles connected by a line corresponds to a different vacuole and the filled circles correspond to the mean area of the vacuoles. A characteristic dose of the onset of vacuole formation and a lethal dose can be identified (red dashed lines).
Figure 3
Figure 3
X-ray ptychography images of chromosomes motion in a live fission yeast zygote at the horsetail stage. (a) Temporal image sequence of a live zygote in the horsetail stage obtained by X-ray ptychography. (b) Fluorescence micrograph of the zygote taken before the X-ray ptychography scans. (c) Overlay of image processed contours of the chromosomes (dark blue) on the original ptychography images. (d) Center of mass motion of chromosomes between the sequential ptychographic images. The x and y-position of the center of mass of the first scan at 0 min was set to 0,0. (e) Calculated radius of gyration of the chromosomes versus time. (f) Schematic representation of autophagic vacuoles formation and cell lysis during meiotic chromosome oscillations.
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