X-Ray Holographic Imaging of Hydrated Biological Cells in Solution

We demonstrate nanoscale x-ray holographic imaging using optimized illumination wave fronts emitted by x-ray waveguide channels. Mode filtering minimizes wave-front distortions and artifacts encountered in most hard x-ray focusing schemes, enabling quantitative reconstruction of the projected density, as evidenced by a test pattern imaged with a field of view of about $20\times 40\mu \mathrm{m}$ and at 22 nm resolution. The dose efficiency and contrast sensitivity make the optical scheme compatible with samples of intrinsically low contrast, typical for hydrated soft matter. This is demonstrated by imaging bacteria in the hydrated and living state, with quantitative phase contrast revealing dense structures of the bacterial nucleoids associated with compactified DNA. In response to continued irradiation, characteristic changes in these dense structures are observed.

Figure 1

A monochromatic hard x-ray beam is focused by Kirkpatrick-Baez (KB) mirrors onto a waveguide (WG) system. The sample (S) is illuminated by the waveguide beam and magnified Fresnel diffraction patterns are recorded at the detection plane (D).

Figure 2

Silicon channel waveguide fabricated by electron-beam lithography, followed by wafer bonding. (a) Scanning electron micrograph of the exit surface. The channel cross section is enclosed by the dashed rectangle ($97\times 73\mathrm{nm}$). (b) Logarithmic far-field intensity distribution, as measured in photon numbers by a pixel detector about 5 m behind the waveguide. The smooth central part (the dashed rectangle) is used for imaging. (c) Reconstructed near-field intensity, linear color coding. (d) Simulated intensity distribution along the beam direction ($z$) within and right behind a two-dimensional waveguide with channel dimensions $d_x=97\mathrm{nm}$ and $d_y=73\mathrm{nm}$, logarithmic color coding. Scale bars (a) 100 nm, (b) 10 mm, (c) 20 nm.

Figure 3

(a) Normalized hologram of a test structure milled into 200 nm thick gold. (b) Holographic phase reconstruction. (c) Phase reconstruction based on the contrast transfer functions (CTF). (d) Iterative mHIO phase reconstruction, the support information (the dashed line) was obtained from the reconstruction shown in (c). Scale bars, $2\mu \mathrm{m}$.

Figure 4

(a) Normalized hologram of freeze-dried Deinococcus radiodurans cells, obtained in a single recording with 8 s dwell time along with (b) the iterative mHIO phase reconstruction. (c) mHIO reconstruction of (initially) living cells in solution. Each frame was accumulated for $8\times 10$ seconds (every other frame is shown). Pronounced changes in the densities are observed after successive irradiation, as quantified in (d), showing the normalized electron density in the high density nucleoid regions indicated by the corresponding colors as a function of dose. The images in (c) are reconstructions corresponding to averages over the colored columns in (d). Scale bars, $4\mu \mathrm{m}$.