Sebastian Kalbfleisch

Quantitative biological imaging by ptychographic x-ray diffraction microscopy

Recent advances in coherent x-ray diffractive imaging have paved the way to reliable and quantitative imaging of noncompact specimens at the nanometer scale. Introduced a year ago, an advanced implementation of ptychographic coherent diffractive imaging has removed much of the previous limitations regarding sample preparation and illumination conditions. Here, we apply this recent approach toward structure determination at the nanoscale to biological microscopy. We show that the projected electron density of unstained and unsliced freeze-dried cells of the bacterium Deinococcus radiodurans can be derived from the reconstructed phase in a straightforward and reproducible way, with quantified and small errors. Thus, the approach may contribute in the future to the understanding of the highly disputed nucleoid structure of bacterial cells. In the present study, the estimated resolution for the cells was 85 nm (half-period length), whereas 50-nm resolution was demonstrated for lithographic test structures. With respect to the diameter of the pinhole used to illuminate the samples, a superresolution of about 15 was achieved for the cells and 30 for the test structures, respectively. These values should be assessed in view of the low dose applied on the order of ≃1.3·105 Gy, and were shown to scale with photon fluence.

Sub-10 nm beam confinement by X-ray waveguides: design, fabrication and characterization of optical properties

Optimized X-ray waveguides have been fabricated and characterized in terms of transmission, angular acceptance, farfield pattern and imaging applications. Beam confinement down to sub-10 nm in two orthogonal directions has been demonstrated, at the nano-focus endstation at P10 of PETRA III at HASYLAB/DESY.

Compound focusing mirror and X-ray waveguide optics for coherent imaging and nano-diffraction

A compound optical system for coherent focusing and imaging at the nanoscale is reported, realised by high-gain fixed-curvature elliptical mirrors in combination with X-ray waveguide optics or different cleaning apertures. The key optical concepts are illustrated, as implemented at the Göttingen Instrument for Nano-Imaging with X-rays (GINIX), installed at the P10 coherence beamline of the PETRA III storage ring at DESY, Hamburg, and examples for typical applications in biological imaging are given. Characteristic beam configurations with the recently achieved values are also described, meeting the different requirements of the applications, such as spot size, coherence or bandwidth. The emphasis of this work is on the different beam shaping, filtering and characterization methods.

Low-dose three-dimensional hard x-ray imaging of bacterial cells

We have imaged the three-dimensional density distribution of unstained and unsliced, freeze-dried cells of the gram-positive bacterium Deinococcus radiodurans by tomographic x-ray propagation microscopy, i.e. projection tomography with phase contrast formation by free space propagation. The work extends previous x-ray imaging of biological cells in the simple in-line holography geometry to full three-dimensional reconstruction, based on a fast iterative phase reconstruction algorithm which circumvents the usual twin-image problem. The sample is illuminated by the highly curved wave fronts emitted from a virtual quasi-point source with 10 nm cross section, realized by two crossed x-ray waveguides. The experimental scheme allows for a particularly dose efficient determination of the 3D density distribution in the cellular structure.

Holographic and diffractive x-ray imaging using waveguides as quasi-point sources

We report on lensless nanoscale imaging using x-ray waveguides as ultra-small sources for quasi-point-like illumination. We first give a brief account of the basic optical setup, an overview of the progress in waveguide fabrication and characterization, as well as the basics of image formation. We then compare one-step holographic and iterative ptychographic reconstruction, both for simulated and experimental data collected on samples illuminated by waveguided beams. We demonstrate that scanning the sample with partial overlap can substantially improve reconstruction quality in holographic imaging, and that divergent beams make efficient use of the limited dynamic range of current detectors, regardless of the reconstruction scheme. Among different experimental settings presented, smallest source dimensions of 29 nm (horizontal) ×17 nm have been achieved, using multi-modal interference effects. These values have been determined by ptychographic reconstruction of a Ta test structure at 17.5 keV and have been corroborated by simulations of field propagation inside the waveguide.

Partially coherent nano-focused x-ray radiation characterized by Talbot interferometry

We have studied the spatial coherence properties of a nano-focused x-ray beam by grating (Talbot) interferometry in projection geometry. The beam is focused by a fixed curvature mirror system optimized for high flux density under conditions of partial coherence. The spatial coherence of the divergent exit wave emitted from the mirror focus is measured by Talbot interferometry The results are compared to numerical calculations of coherence propagation. In view of imaging applications, the magnified in-line image of a test pattern formed under conditions of partial coherence is analyzed quantitatively. Finally, additional coherence filtering by use of x-ray waveguides is demonstrated. By insertion of x-ray waveguides, the beam diameter can be reduced from typical values of 200 nm to values below 15 nm. In proportion to the reduction in the focal spot size, the numerical aperture (NA) of the projection imaging system is increased, as well as the coherence length, as quantified by grating interferometry.

Sub-15 nm beam confinement by two crossed x-ray waveguides

We have combined two high transmission planar x-ray waveguides glued onto each other in a crossed geometry to form an effective quasi-point source. From measurements of the far-field diffraction pattern, the phase and amplitude of the near-field distribution is retrieved using the error-reduction algorithm. In agreement with finite difference field simulations (forward calculation), the reconstructed exit wave intensity distribution (inverse calculation) exhibits a full width at half maximum (FWHM) below 15 nm in both dimensions. Finally, holographic imaging is successfully demonstrated for the crossed waveguide device by translation of a lithographic test structure through the waveguide beam.

High aspect ratio x-ray waveguide channels fabricated by e-beam lithography and wafer bonding

We report on the fabrication and characterization of hard x-ray waveguide channels manufactured by e-beam lithography, reactive ion etching and wafer bonding. The guiding layer consists of air or vacuum and the cladding material of silicon, which is favorable in view of minimizing absorption losses. The specifications for waveguide channels which have to be met in the hard x-ray range to achieve a suitable beam confinement in two orthogonal directions are extremely demanding. First, high aspect ratios up to 106 have to be achieved between lateral structure size and length of the guides. Second, the channels have to be deeply embedded in material to warrant the guiding of the desired modes while absorbing all other (radiative) modes in the cladding material. We give a detailed report on device fabrication with the respective protocols and parameter optimization, the inspection and the optical characterization.

Versatility of a hard X-ray Kirkpatrick-Baez focus characterized by ptychography.

In the past decade Kirkpatrick-Baez (KB) mirrors have been established as powerful focusing systems in hard X-ray microscopy applications. Here a ptychographic characterization of the KB focus in the dedicated nano-imaging setup GINIX (Göttingen Instrument for Nano-Imaging with X-rays) at the P10 coherence beamline of the PETRA III synchrotron at HASLYLAB/DESY, Germany, is reported. More specifically, it is shown how aberrations in the KB beam, caused by imperfections in the height profile of the focusing mirrors, can be eliminated using a pinhole as a spatial filter near the focal plane. A combination of different pinhole sizes and illumination conditions of the KB setup makes the prepared optical setup well suited not only for high-resolution ptychographic coherent X-ray diffractive imaging but also for moderate-resolution/large-field-of-view propagation imaging in the divergent KB beam.

Orientation of biomolecular assemblies in a microfluidic jet

We have investigated multilamellar lipid assemblies in a micro- fluidic jet, operating at high shear rates of the order of 10 7 s 1 . Compared to clas- sical Couette cells or rheometers, the shear rate was increased by at least 2-3 orders of magnitude, and the sample volume was scaled down correspondingly. At the same time, the jet is characterized by high extensional stress due to elonga- tional flow. A focused synchrotron x-ray beam was used to measure the structure and orientation of the lipid assemblies in the jet. The diffraction patterns indicate conventional multilamellar phases, aligned with the membrane normals oriented along the velocity gradient of the jet. The results indicate that the setup may be well suited for coherent diffractive imaging of oriented biomolecular assemblies and macromolecules at the future x-ray free electron laser (XFEL) sources.