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

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.


Results
X-ray ptychography. Ptychography is an X-ray imaging technique with spatial resolution limited in principle by the spatial wavelength of the incident beam and the maximum angle at which diffracted signal can be measured with sufficient signal-to-noise ratio, although in practice the resolution can be also limited by scanning precision or radiation induced damage on the specimen under study (Fig. 1). In living samples, intracellular motions are happening during a single scan which can blur the images. A ptychography setup with a pinhole-defined illumination was chosen in order to achieve good contrast and high resolution with a reduced dosage of radiation (Fig. 1A) due to the broad spatial spectrum 23 . We obtained reconstructed images with pixel sizes of 45 × 45 nm 2 and an estimated resolution in the range of 100-200 nm of live cells in aqueous environment and reduced the radiation doses down to about 10 3 -10 4 Gy per scan. These doses are two to three orders of magnitude less than 1.8 × 10 6 Gy recorded for recent X-ray images of (initially) alive eukaryotic cells 18 and close to the dose of 8.9 × 10 3 Gy used for holographic imaging of living bacteria 19 .
Here, we explore the dynamics of living fission yeast Schizosaccharomyces pombe cells during meiosis in natural, aqueous environment in situ. Fission yeast cells are ideal eukaryotic model organisms, because many of basic cellular principles and cell regulators are conserved from yeast to humans 8 . Meiosis in fission yeast is induced by depleting nitrogen sources from the culture medium and haploid cells of the opposite mating type conjugate and form a diploid "banana-shaped" zygote 6 (Fig. 1b,c). At the horsetail stage of meiosis strong oscillations of chromosomes can be observed with extended periods of chromosomal back and forth motions along the cell axis 25 . 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 brightfield 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 24 . Here, only one of two zygotes was at the horsetail stage.
SCIentIfIC REPORTS | 7: 13775 | DOI:10.1038/s41598-017-13175-9 The period of an individual oscillation is about 10-15 min 6 . After several hours, at the end of the horsetail stage, the oscillations slow down and finally stop. X-ray induced autophagy in fission yeast cells. X-ray ptychography micrographs of a successive image sequence of a fission yeast zygote and an analysis of the impact of ionizing radiation on the cell are shown in Fig. 2. During the first four scans, no structural changes -almost homogenous density within the entire cellof a zygote were observed. A further exposure of X-rays in the successive scans led to the appearance of clear, light and rounded structures in the zygote. These observed cellular structures may be a signature of a radiation induced formation of vacuoles 26 and autophagic bodies 27 , which were described for fission yeast cells and might be a visual indication of autophagy [28][29][30] . These structural changes coincided with an overall positive shift in the phase shift histograms of zygote images showing autophagic vacuoles in comparison to zygote images without vacuoles (Fig. 2b). Increasing the radiation dose further, a bursting of the membrane and shrinkage of the cell was observed ( Fig. 2a-ix,x), which demonstrates that after accumulating a certain amount of radiation the zygote perished. To characterize the dynamics of the autophagic vacuole formation and cell lysis, changes of the projected zygote area and of the projected area of individual vacuoles were analyzed (Fig. 2c). In the first four ptychography scans, a slight increase of the projected area of the zygote was observed. At an accumulated dose of about 2.2 × 10 4 Gy (sixth scan) for the particular scan shown in Fig. 2c, the area decreased back to its initial size and the autophagic vacuole formation set in, a first critical dose for live fission yeast cells can be defined. Applying more X-ray radiation, the initial number of vacuoles did not change, whereas their volume (projected area) increased until the cell burst. At a dose of about 9.2 × 10 4 Gy for this particular cell, the bursting of the cell membrane was concurred by a strong decrease of the projected zygote area. The observed formation of vacuoles can be attributed to ionizing effects of the X-ray radiation, which induce radiolysis of water and cause hydrogen peroxide (H 2 O 2 ) and hydroxyl radical (OH . ) formation 27 . These very reactive oxygen species (ROS) activate protein kinases in yeast 9 and can cause autophagy 31 . Apart from the fact that the zygote was exposed to X-rays, yeast cells grew under nitrogen depletion, which might be a stimulus to facilitate autophagy if cells starve for several hours 32 . However, the autophagic vacuole formation was observed only after several ptychography scans, and was not observed in comparable experiments using optical microscopy. In total, nine individual live fission yeast zygotes were analyzed. The average radiation dose at which autophagic vacuole formation occurred was about (3.30 ± 0.74) × 10 4 Gy. When the radiation dosages accumulated to (9.6 ± 3.2) × 10 4 Gy, zygotes were lysed, which can be defined as the lethal radiation dose for fission yeast. Interestingly, non-meiotic cells were more resistant to X-ray radiation, where vacuole formation occurred at higher doses of (8.7 ± 3.8) × 10 4 Gy and cell death at about (1.20 ± 0.19) × 10 5 Gy, indicating that the lethal radiation dose of non-meiotic cells is similar to zygotes, whereas the vacuole formation in non-meiotic cells occurs at much higher radiation doses.
X-ray imaging of the chromosome motion. To further demonstrate the potential of ptychography for studying cellular dynamics, we imaged meiotic yeast zygote at the horsetail stage. During this stage, an extended movement of the whole chromatin happens, and at the same time, the motion matches the time scales of the X-ray imaging technique. Freshly prepared samples were firstly analyzed with a fluorescence microscope to find a zygote at the horsetail stage in the sample and then mounted on the X-ray ptychography setup. A temporal sequence of six X-ray ptychography images of a live fission yeast zygote in the horsetail stage is presented in Fig. 3a. Interestingly, a darker (a more negative phase shift) and denser structure in the top part of the zygote was observed. This structure was moving during one scan to another and furthermore changed its shape. We identified this densified structure as moving chromatin, which has the same appearance as the chromosomes in the fluorescence micrograph taken before the X-ray ptychography scans (Fig. 3b). Image processed contours of the chromosomes overlaid on the original ptychography images are shown in Fig. 3c. The motion of the chromosomes was analyzed by calculating their center of mass. Starting from the initial position of the chromosomes the subsequent center of mass positions showed a movement of several hundreds of nanometer away from the upper cell end in the direction of the lower part of the cell (Fig. 3d). For further analysis, we compared the shape changes of the chromosomes over time by calculating the radius of gyration R r r ( ) , where N is the number of pixels of the chromatin, r i  are the position vectors and → r CM is the center of mass of the chromosomes. The radius of gyration, which characterizes the packing and shape of the chromosomes, versus time is plotted in Fig. 3f. Firstly, a looser packing of the chromosomes, bigger R Gg , was observed, reaching a maximum of R G = (1.32 ± 0.07) µm after 10 min during their motion to the lower cell end. Afterwards, R G steadily decreased to a minimum of (0.85 ± 0.04) µm, which characterizes a strong compaction of the chromosomes. This chromosome compaction was found in conjunction with the formation of autophagic vacuoles, which could be observed after about 20 min (Fig. 3a). The motion of the chromosomes was slowed down in comparison to the observed period of the oscillation of about 10-15 min 6 . This observed deceleration of the chromosome motion is an indication of an impact of X-ray radiation, which most probably damages the cell at the molecular level; especially the appearance of autophagic vacuoles in concurrence with the chromosomes compaction had a strong impact on the oscillating chromosomes. Moreover, due to a time-consuming sample preparation and mounting procedure, the X-ray ptychography images were presumably taken at the end of the horsetail period, when the oscillations slowed down. The appearance of autophagic vacuoles was observed, when the radiation dose accumulated to about 1.2 × 10 4 Gy. This dose of the vacuole formation is less than half of the dose, which was found for normal yeast zygotes, which might be an indication that zygotes in the horsetail stage are more sensitive to X-ray radiation.

Conclusions
In these experiments, we optimized the sample preparation and experimental setup to successfully apply ptychography for sequential imaging -producing X-ray movies -of live meiotic yeast cells in aqueous environment and established a method to investigate intracellular nanostructures. Based on the natural electron density contrast, this label-free imaging method allowed us to visualize cellular structures in situ. We discovered autophagic cell death or type II programmed cell death 28 and cell lysis, induced by the pathological environment due to the ionizing X-ray radiation. Autophagy in the yeast and mammalian cells is similar 30 and it is considered to play an important part in the response of radiation therapy 33 . Radiation-induced damage on the molecular level most probably occurred already during the first X-ray scan, but it did not cause visible cell changes and the cell stayed alive. Thus an average radiation dose at which visible signs of autophagy occurs by formation of autophagic vacuoles 32 and a characteristic dose of membrane bursting of the eukaryotic cells can be obtained. Moreover, the dynamics of denser structures, which are most likely chromatin structures at the end of their oscillatory motion during the horsetail stage of yeast zygotes, can be imaged and analyzed. However, zygotes are most likely already damaged at the molecular level by radiation. This imaging approach also simplifies sample preparation and avoids artifact formation caused by fixation, sectioning or labeling.
We believe that improved sample environments, e.g. microfluidics setup 34 to flush fresh medium and remove free radicals 35 , modified scanning protocols and adjusted ranges of interest will further advance, as here demonstrated, the way of seeing physiological processes of individual eukaryotic cells as well as tissues with subcellular, nanoscale resolution.

Methods
In order to avoid a strong background signal and to create a cytocompatible environment, we used biocompatible and X-ray resistant 200 nm thick silicon nitride (Si 3 N 4 ) membrane windows (frame: 5 × 5 mm 2 × 200 µm, membrane: 1.5 × 1.5 mm 2 × 200 nm; Silson Ltd, Blisworth, England) 17,18,34 . The Si 3 N 4 membrane windows were coated with lectin (Sigma-Aldrich, St. Louis, MO, USA) to increase cell adhesion to the membrane surface, which is crucial for the spatial stability of the cells and thus for the reproducibility of X-ray ptychography scans. Fission yeast Schizosaccharomyces pombe cells were kept in phosphate-buffered saline (PBS) medium at room temperature. For fluorescence optical imaging, we used the genotype of the fission yeast strain h90 rec25::GFP-KanMX6. The strain was a kind gift from C. Martín-Castellanos (CM62, IBFG, Salamanca, Spain). To induce meiosis, fission yeast cells were transferred to an Eppendorf tube with 100 µl of nutrition deficient Edinburgh minimal medium (EMM-N) 7 and kept for 30 min at room temperature. Afterwards, a small droplet of medium with cells were put on a lectin-coated Si 3 N 4 membrane window and the device was covered by an uncoated Si 3 N 4 membrane window and accurately glued with UHU plus epoxy quick set adhesive at the edges of the membranes. The described procedure enables the preparation of hydrated living cell samples for X-ray experiments with an intercalated aqueous film of 5 to 10 µm in thickness. The gap was determined based on bright field optical imaging where the distance between the membranes was comparable to the size of the cell as observed by changing the focus. Since the aqueous environment drastically decreases the electron density contrast of the sample, a small sample thickness is crucial to reduce the background signal caused by the medium in the device.
X-ray ptychography experiments were performed at the coherent small-angle X-ray scattering (cSAXS) beamline of the Swiss Light Source, Paul Scherrer Institut, PSI, Villigen, Switzerland. The schematic representation of the setup is shown in Fig. 1a. An X-ray beam of 6.2 keV photon energy, λ = 0.2 nm, was selected using a double crystal Si (111) monochromator. The incident beam was defined by a pinhole with a transverse diameter of about 2.5 µm in order to obtain a coherent spatially confined illumination at the sample, which was placed 3 mm downstream of the pinhole and had at the sample position approximately the same diameter. The sample of hydrated live cells was placed on a piezoelectric scanning stage to allow for nanometer precision scanning. The coherent X-ray beam diffracted by the sample propagates through a helium flushed flight tube to a photon-counting Pilatus 2D detector 36 , which is located at distance of 7.412 m from the sample. The broad angular spectrum of a pinhole-defined illumination is well suited for minimizing the radiation dose while acquiring images with good contrast and a moderately high resolution 23 . Before the X-ray ptychography experiments, the cell samples were imaged by fluorescent microscopy in order to identify oscillating zygotes. The membranes were then mounted on the setup and the identified cells were positioned using an on-stage bright field microscope.
In order to avoid the raster grid pathology 37 all scans were performed following a Fermat spiral scanning pattern 38 . In order to find optimal scanning parameters, different step sizes and exposure times were applied. For the measurements in Fig. 2a a scanning field of view of 18 × 14 µm 2 and an average step size of 0.7 µm were used with an exposure time of 0.1 s per scanning point. For these parameters the resolution was about 200 nm with an average flux of about 7.4 × 10 5 photons/µm 2 . To calculate the average flux we first normalized the reconstructed illumination intensity using the total number of counts arriving at the detector after compensating for absorbing and scattering elements in the path of the beam, then we used the scanning pattern to generate a grid of the distribution of photons incident on the sample for the whole scan. The flux in photons/µm 2 is finally calculated by integrating over an area significantly larger than the illumination and dividing by the area, in this manner we included in the calculation the total dose incident on the sample including the overlapping regions of the scan 39 . The corresponding dose of 3.9 × 10 3 Gy was estimated as described in ref. 16  In order to observe the cell behavior (death or ability to recover) between the X-ray scans, the time interval among the images was different: 5 min, 25 min, 20 min, 20 min, 50 min, 30 min, 40 min, 2 h, 3 h. This allows us to assume that the cell death is initiated by the X-ray radiation and does not occur/continue when the X-rays are switched off. The radiation doses used for single ptychography scans in Fig. 2a were different, the 1 st scan 1.96 × 10 3 Gy, 2 nd scan 4.01 × 10 3 Gy, 3 rd scan 3.87 × 10 3 Gy, 4 th scan 3.86 × 10 3 Gy, 5 th scan 4.09 × 10 3 Gy, 6 th scan 3.87 × 10 3 Gy, 7 th scan 4.14 × 10 3 Gy, and 10 th scan 8.20 × 10 3 Gy. The quality of the images in Fig. 2a(viii, ix) and resolution down to about 100 nm was improved using smaller step sizes of 0.5 µm and longer exposure times of 0.4 s, which increased the radiation dose to about 3.3 × 10 4 Gy.
Reconstructions were carried out using the maximum likelihood method through non-linear optimization 20,22 . In order to reduce the noise in the reconstructions, gradient preconditioning and regularization, as described by Thibault and Guizar-Sicairos 22 , were used. A good estimate of the incident illumination is important to facilitate the reconstruction of weak contrast specimens 41 , such as the hydrated live cells presented here. For this purpose, we characterized the incident illumination via ptychography before the experiments using a 2D test patterns similar to those used in 42 . The illumination phase and amplitude profile were stable for the duration of a single scan. For this case, the resolution of the reconstruction could not be assessed via Fourier shell correlation (FSC) 43,44 , because two identical datasets were not available due to changes or movement of the live specimens. To assess the resolution of each image we used instead a method based on the angular-averaged power spectral density (PSD) method as described in 14 .
X-ray ptychography images were analyzed using ImageJ (version 1.47k, Wayne Rasband, National Institute of Health, USA) and MATLAB (version R2012b, The MathWorks, Natick, USA) by applying custom developed scripts. The images were first denoised by conditional mean filtering resulting in an edge preserving smoothing. Further applying edge detection algorithms yield the contour of the cells, which acts as the range of interest in order to find the chromosomes by local thresholding.