Soft X-Ray Diffraction Microscopy of a Frozen Hydrated Yeast Cell

We report the first image of an intact, frozen hydrated eukaryotic cell using x-ray diffraction microscopy, or coherent x-ray diffraction imaging. By plunge freezing the specimen in liquid ethane and maintaining it below $-170°\mathrm{C}$, artifacts due to dehydration, ice crystallization, and radiation damage are greatly reduced. In this example, coherent diffraction data using 520 eV x rays were recorded and reconstructed to reveal a budding yeast cell at a resolution better than 25 nm. This demonstration represents an important step towards high resolution imaging of cells in their natural, hydrated state, without limitations imposed by x-ray optics.

Figure 1

Demonstration of the stability of frozen hydrated yeast cells to 520 eV x-ray exposure for various absorbed doses in Gray [34]. Shown here are the fractional radial shifts of the centers of speckles in coherent diffraction patterns of yeast cells. The top curve is of cells that were viewed while frozen hydrated at $-170°\mathrm{C}$, while the bottom curve is of cells that were freeze-dried and then exposed at room temperature. For the case of freeze-dried, room temperature cells, the center positions of speckles moved outwards from the diffraction pattern center, indicating a shrinkage of the object in real space.

Figure 2

Images of Saccharomyces cerevisiae yeast cells. At left is shown a visible light DIC image of a budding yeast taken using a $100\times$ $\mathrm{NA}=1.3$ immersion objective. The arrow indicates the assumed beam direction for the x-ray diffraction micrograph at right, which is of a different yeast cell. The x-ray diffraction micrograph is a complex wave reconstruction at $0\mu \mathrm{m}$ defocus, where the magnitude is represented by brightness and the phase by hue. A possible mitochondrion is indicated with the red arrow.

Figure 3

Through-focus imaging using the complex wave front reconstructed in XDM. Four selected transverse lines are shown on the magnitude-only representation of the $0\mu \mathrm{m}$ defocus reconstructed wave front at left. The image at right shows transverse line profiles of the reconstructed image magnitude as the wave field is defocused by propagation from the reconstruction plane. In-focus edges look like the waist of an hourglass in such a representation; the line profiles from the inner and outer spherical objects in the reconstruction appear to be at different focal planes, consistent with the interpretation that the larger parent cell is at lines B1 and B2, and the bud is at lines T1 and T2.

Figure 4

Estimation of the resolution of the x-ray diffraction micrograph. At top is shown both the azimuthal average of the diffraction intensity signal $I_{\mathrm{data}}$ (red), and the magnitude ratio (or PRTF) $M_{\mathrm{recon}}/M_{\mathrm{data}}$ (black). The PRTF measures the reproducibility of reconstructed pixel values as a function of spatial frequency; its decline at higher frequency provides an indication of the resolution. The resolution cutoff is estimated at the spatial frequency where the PRTF drops below a value of 0.6. This provides one estimate of the half-period resolution of 20 nm. At bottom is shown a line scan across T2 in Fig. 3 at the plane of sharpest focus of this object. Also shown is a Gaussian-smoothed fit which indicates a half width at half maximum of 25 nm (or $\sigma =21\mathrm{nm}$ in $\exp [-0.5x^2/{\sigma }^2]$) for the combination of feature size and imaging resolution.