Tomography of a Cryo-immobilized Yeast Cell Using Ptychographic Coherent X-Ray Diffractive Imaging.

The structural investigation of noncrystalline, soft biological matter using x-rays is of rapidly increasing interest. Large-scale x-ray sources, such as synchrotrons and x-ray free electron lasers, are becoming ever brighter and make the study of such weakly scattering materials more feasible. Variants of coherent diffractive imaging (CDI) are particularly attractive, as the absence of an objective lens between sample and detector ensures that no x-ray photons scattered by a sample are lost in a limited-efficiency imaging system. Furthermore, the reconstructed complex image contains quantitative density information, most directly accessible through its phase, which is proportional to the projected electron density of the sample. If applied in three dimensions, CDI can thus recover the sample's electron density distribution. As the extension to three dimensions is accompanied by a considerable dose applied to the sample, cryogenic cooling is necessary to optimize the structural preservation of a unique sample in the beam. This, however, imposes considerable technical challenges on the experimental realization. Here, we show a route toward the solution of these challenges using ptychographic CDI (PCDI), a scanning variant of coherent imaging. We present an experimental demonstration of the combination of three-dimensional structure determination through PCDI with a cryogenically cooled biological sample--a budding yeast cell (Saccharomyces cerevisiae)--using hard (7.9 keV) synchrotron x-rays. This proof-of-principle demonstration in particular illustrates the potential of PCDI for highly sensitive, quantitative three-dimensional density determination of cryogenically cooled, hydrated, and unstained biological matter and paves the way to future studies of unique, nonreproducible biological cells at higher resolution.

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

The propagation of hard X-ray synchrotron beams in waveguides with guiding layer diameters in the 9-35 nm thickness range has been studied. The planar waveguide structures consist of an optimized two-component cladding. The presented fabrication method is suitable for short and leak-proof waveguide slices with lengths (along the optical axis) in the sub-500 µm range, adapted for optimized transmission at photon energies of 11.5-18 keV. A detailed comparison between finite-difference simulations of waveguide optics and the experimental results is presented, concerning transmission, divergence of the waveguide exit beam, as well as the angular acceptance. In a second step, two crossed waveguides have been used to create a quasi-point source for propagation-based X-ray imaging at the new nano-focus endstation of the P10 coherence beamline at Petra III. By inverting the measured Fraunhofer diffraction pattern by an iterative error-reduction algorithm, a two-dimensional focus of 10 nm × 10 nm is obtained. Finally, holographic imaging of a lithographic test structure based on this optical system is demonstrated.

Hard X-ray imaging of bacterial cells: nano-diffraction and ptychographic reconstruction.

Ptychographic coherent X-ray diffractive imaging (PCDI) has been combined with nano-focus X-ray diffraction to study the structure and density distribution of unstained and unsliced bacterial cells, using a hard X-ray beam of 6.2keV photon energy, focused to about 90nm by a Fresnel zone plate lens. While PCDI provides images of the bacteria with quantitative contrast in real space with a resolution well below the beam size at the sample, spatially resolved small angle X-ray scattering using the same Fresnel zone plate (cellular nano-diffraction) provides structural information at highest resolution in reciprocal space up to 2nm(-1). We show how the real and reciprocal space approach can be used synergistically on the same sample and with the same setup. In addition, we present 3D hard X-ray imaging of unstained bacterial cells by a combination of ptychography and tomography.

Ptychographic coherent x-ray diffractive imaging in the water window.

Coherent x-ray diffractive microscopy enables full reconstruction of the complex transmission function of an isolated object to diffraction-limited resolution without relying on any optical elements between the sample and detector. In combination with ptychography, also specimens of unlimited lateral extension can be imaged. Here we report on an application of ptychographic coherent diffractive imaging (PCDI) in the soft x-ray regime, more precisely in the so-called water window of photon energies where the high scattering contrast between carbon and oxygen is well-suited to image biological samples. In particular, we have reconstructed the complex sample transmission function of a fossil diatom at a photon energy of 517 eV. In imaging a lithographically fabricated test sample a resolution on the order of 50 nm (half-period length) has been achieved. Along with this proof-of-principle for PCDI at soft x-ray wavelengths, we discuss the experimental and technical challenges which can occur especially for soft x-ray PCDI.

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.

Experimental 3D coherent diffractive imaging from photon-sparse random projections.

The routine atomic resolution structure determination of single particles is expected to have profound implications for probing structure-function relationships in systems ranging from energy-storage materials to biological molecules. Extremely bright ultrashort-pulse X-ray sources - X-ray free-electron lasers (XFELs) - provide X-rays that can be used to probe ensembles of nearly identical nanoscale particles. When combined with coherent diffractive imaging, these objects can be imaged; however, as the resolution of the images approaches the atomic scale, the measured data are increasingly difficult to obtain and, during an X-ray pulse, the number of photons incident on the 2D detector is much smaller than the number of pixels. This latter concern, the signal 'sparsity', materially impedes the application of the method. An experimental analog using a conventional X-ray source is demonstrated and yields signal levels comparable with those expected from single biomolecules illuminated by focused XFEL pulses. The analog experiment provides an invaluable cross check on the fidelity of the reconstructed data that is not available during XFEL experiments. Using these experimental data, it is established that a sparsity of order 1.3 × 10-3 photons per pixel per frame can be overcome, lending vital insight to the solution of the atomic resolution XFEL single-particle imaging problem by experimentally demonstrating 3D coherent diffractive imaging from photon-sparse random projections.