Three-Dimensional Visualization of a Human Chromosome Using Coherent X-Ray Diffraction

Coherent x-ray diffraction microscopy is a lensless phase-contrast imaging technique with high image contrast. Although electron tomography allows intensive study of the three-dimensional structure of cellular organelles, it has inherent difficulty with thick objects. X rays have the unique benefit of allowing noninvasive analysis of thicker objects and high spatial resolution. We observed an unstained human chromosome using coherent x-ray diffraction. The reconstructed images in two or three dimensions show an axial structure, which has not been observed under unstained conditions.

Figure 1

Schematic view of coherent hard x-ray diffraction measurement of an unstained human chromosome. A 20-$\mu \mathrm{m}$-diameter pinhole $\sim 1\mathrm{m}$ upstream from the sample defined the illumination for a single chromosome. Two guard slits were aligned carefully to reduce the amount of missing data near forward-scattering angles. An x-ray CCD detector 1.32 m downstream from the sample was used to acquire coherent diffraction data. The CCD has a pixel size of $20\mu \mathrm{m}$, and an imaging array of $1340\times 1300$ pixels. A beam stop 365 mm upstream from the CCD blocked the unscattered direct x-ray beam, and shaded nearly a quadrant area of the CCD. The centrosymmetry of the diffraction data was used to recover some of the missing data behind the beam stop. The sample was rotated for 3D image reconstruction.

Figure 2

Coherent diffraction pattern of an unstained human chromosome and its reconstructed projection image. The reconstruction of coherent x-ray diffraction data (a) gave a chromosome image [(b) in gray scale and (c) in color scale]. The centromere region is indicated by an arrow in (b). The reconstructed image contains a high-intensity region resembling the chromosome axial structure near the center of the chromatids, as clearly shown in (c). (d) shows an enlarged image of the region in the red square in (b), where a different color scale was used to enhance the structure. A wavy feature in (d) is similar to the helical axis structure observed in the immunofluorescence imaging [18]. The wavy structure is also evident in the profile (e) along the magenta line in (b). (f) shows an immunofluorescence image of a different chromosome, stained by a condensin antibody (red) and DAPI (blue), showing a wavy axial structure (red). $\mathit{q}$ is defined as $|\mathit{q}|=2\sin (\Theta /2)/\lambda$, where $\Theta$ is the scattering angle.

Figure 3

Reconstructed 3D electron-density map of an unstained human chromosome. The isosurface (a) of the chromosome was drawn with a threshold value at 15% of the highest density. The planes of the cross-sectional images (b,c) include the position near the centromere (indicated by arrows) with the highest electron density. They show relatively high density near the central axis of each chromatid. The projection image (d) generated from the 3D reconstruction has a similar feature to that in the 2D reconstruction in Figs. 2b, 2c. (e) shows cross-sectional images of the chromosome at 409 nm intervals. The diffraction data sets used for the 3D analysis are at 38 incident angles: every 2.5° from $-70°$ to $-27.5°$, as well as $-7.5°$, $-5°$, 0°, 5°, 7.5°, 10°, 15°, 17.5°, 20°, 25°, 27.5°, 30°, 37.5°, 40°, 45°, 47.5°, 50°, 55°, 57.5°, and 60°.