Coherent X-ray Diffraction Microscopy Research Articles

Multi-beam X-ray ptychography using coded probes for rapid non-destructive high resolution imaging of extended samples

Imaging large areas of a sample non-destructively and with high resolution is of great interest for both science and industry. For scanning coherent X-ray diffraction microscopy, i. e., ptychography, the achievable scan area at a given spatial resolution is limited by the coherent photon flux of modern X-ray sources. Multibeam X-ray ptychography can improve the scanning speed by scanning the sample with several parallel mutually incoherent beams, e. g., generated by illuminating multiple focusing optics in parallel by a partially coherent beam. The main difficulty with this scheme is the robust separation of the superimposed signals from the different beams, especially when the beams and the illuminated sample areas are quite similar. We overcome this difficulty by encoding each of the probing beams with its own X-ray phase plate. This helps the algorithm to robustly reconstruct the multibeam data. We compare the coded multibeam scans to uncoded multibeam and single beam scans, demonstrating the enhanced performance on a microchip sample with regular and repeating structures.

Phase separated bi-metallic PtNi nanoparticles formed by pulsed laser dewetting

We present morphological and compositional analysis of phase-separated Pt–Ni alloy nanoparticles (NPs) formed by ns pulsed laser dewetting. The PtNi NPs obtained by the pulsed laser dewetting consist of phase-separated multiple domains including Pt3Ni, PtNi and PtNi3 phases with various crystal orientations as revealed by transmission electron microscopy, which is in contrast to thermal dewetting resulting NPs of a uniform composition. A three-dimensional (3D) electron density map of a dewetted PtNi NP obtained using the coherent x-ray diffraction microscopy elucidates the 3D morphology of Pt- and Ni-rich regions together with a nano-cavity formed during the pulsed laser irradiation.

Introduction of Coherent X-Ray Diffraction Microscopy to Analyze Manganese-Based Olivine Cathode Materials in Lithium-Ion Batteries

The carbon-coated LiMn0.8Fe0.2PO4 (LMFP) mesocrystals were designed to be favorable for kinetics of electron and lithium-ion conductivity due to its well-developed internal path composed of ~50-nm-sized nanocrystallites. The carbon-coated LMFP mesocrystals exhibited reasonable electrochemical cycle performance as well as superior rate capability, ensuring a proper model material to systematic study of reaction mechanism upon battery cycling. In order to investigate the reversibility of charge/discharge reaction on carbon-coated LMFP mesocrystals, we performed coherent x-ray diffraction microscopy to confirm the electronic-structure effects of each transition metal (Mn and Fe) and morphological changes. In addition, the electrochemical impedance spectroscopy at quasi-steady state has been analyzed as a supplementary tool. We believe that our systematic approaches as well as complementary analyses can provide clear experimental inspection on the origin of homogeneity/ inhomogeneity of charge/ discharge in LMFP electrodes via coherent x-ray diffraction microscopy and electrochemical impedance spectroscopy.

Lensless Tomographic Imaging of Near Surface Structures of Frozen Hydrated Malaria-Infected Human Erythrocytes by Coherent X-Ray Diffraction Microscopy

Lensless, coherent X-ray diffraction microscopy has been drawing considerable attentions for tomographic imaging of whole human cells. In this study, we performed cryogenic coherent X-ray diffraction imaging of human erythrocytes with and without malaria infection. To shed light on structural features near the surface, “ghost cells” were prepared by the removal of cytoplasm. From two-dimensional images, we found that the surface of erythrocytes after 32 h of infection became much rougher compared to that of healthy, uninfected erythrocytes. The Gaussian roughness of an infected erythrocyte surface (69 nm) is about two times larger than that of an uninfected one (31 nm), reflecting the formation of protein knobs on infected erythrocyte surfaces. Three-dimensional tomography further enables to obtain images of the whole cells with no remarkable radiation damage, whose accuracy was estimated using phase retrieval transfer functions to be as good as 64 nm for uninfected and 80 nm for infected erythrocytes, respectively. Future improvements in phase retrieval algorithm, increase in degree of coherence, and higher flux in combination with complementary X-ray fluorescence are necessary to gain both structural and chemical details of mesoscopic architectures, such as cytoskeletons, membraneous structures, and protein complexes, in frozen hydrated human cells, especially under diseased states.

Three-dimensional reconstruction for coherent diffraction patterns obtained by XFEL.

The three-dimensional (3D) structural analysis of single particles using an X-ray free-electron laser (XFEL) is a new structural biology technique that enables observations of molecules that are difficult to crystallize, such as flexible biomolecular complexes and living tissue in the state close to physiological conditions. In order to restore the 3D structure from the diffraction patterns obtained by the XFEL, computational algorithms are necessary as the orientation of the incident beam with respect to the sample needs to be estimated. A program package for XFEL single-particle analysis based on the Xmipp software package, that is commonly used for image processing in 3D cryo-electron microscopy, has been developed. The reconstruction program has been tested using diffraction patterns of an aerosol nanoparticle obtained by tomographic coherent X-ray diffraction microscopy.

Quantitative Imaging of Single Unstained Magnetotactic Bacteria by Coherent X-ray Diffraction Microscopy.

Novel coherent diffraction microscopy provides a powerful lensless imaging method to obtain a better understanding of the microorganism at the nanoscale. Here we demonstrated quantitative imaging of intact unstained magnetotactic bacteria using coherent X-ray diffraction microscopy combined with an iterative phase retrieval algorithm. Although the signal-to-noise ratio of the X-ray diffraction pattern from single magnetotactic bacterium is weak due to low-scattering ability of biomaterials, an 18.6 nm half-period resolution of reconstructed image was achieved by using a hybrid input-output phase retrieval algorithm. On the basis of the quantitative reconstructed images, the morphology and some intracellular structures, such as nucleoid, polyβ-hydroxybutyrate granules, and magnetosomes, were identified, which were also confirmed by scanning electron microscopy and energy dispersive spectroscopy. With the benefit from the quantifiability of coherent diffraction imaging, for the first time to our knowledge, an average density of magnetotactic bacteria was calculated to be ∼1.19 g/cm(3). This technique has a wide range of applications, especially in quantitative imaging of low-scattering biomaterials and multicomponent materials at nanoscale resolution. Combined with the cryogenic technique or X-ray free electron lasers, the method could image cells in a hydrated condition, which helps to maintain their natural structure.

Analytic 3D Imaging of Mammalian Nucleus at Nanoscale Using Coherent X-Rays and Optical Fluorescence Microscopy

Despite the notable progress that has been made with nano-bio imaging probes, quantitative nanoscale imaging of multistructured specimens such as mammalian cells remains challenging due to their inherent structural complexity. Here, we successfully performed three-dimensional (3D) imaging of mammalian nuclei by combining coherent x-ray diffraction microscopy, explicitly visualizing nuclear substructures at several tens of nanometer resolution, and optical fluorescence microscopy, cross confirming the substructures with immunostaining. This demonstrates the successful application of coherent x-rays to obtain the 3D ultrastructure of mammalian nuclei and establishes a solid route to nanoscale imaging of complex specimens.

Imaging Fully Hydrated Whole Cells by Coherent X-Ray Diffraction Microscopy

Nanoscale imaging of biological specimens in their native condition is of long-standing interest, in particular with direct, high resolution views of internal structures of intact specimens, though as yet progress has been limited. Here we introduce wet coherent x-ray diffraction microscopy capable of imaging fully hydrated and unstained biological specimens. Whole cell morphologies and internal structures better than 25nm can be clearly visualized without contrast degradation.

Humidity-controlled preparation of frozen-hydrated biological samples for cryogenic coherent x-ray diffraction microscopy

Coherent x-ray diffraction microscopy (CXDM) has the potential to visualize the structures of micro- to sub-micrometer-sized biological particles, such as cells and organelles, at high resolution. Toward advancing structural studies on the functional states of such particles, here, we developed a system for the preparation of frozen-hydrated biological samples for cryogenic CXDM experiments. The system, which comprised a moist air generator, microscope, micro-injector mounted on a micromanipulator, custom-made sample preparation chamber, and flash-cooling device, allowed for the manipulation of sample particles in the relative humidity range of 20%-94%rh at 293 K to maintain their hydrated and functional states. Here, we report the details of the system and the operation procedure, including its application to the preparation of a frozen-hydrated chloroplast sample. Sample quality was evaluated through a cryogenic CXDM experiment conducted at BL29XUL of SPring-8. Taking the performance of the system and the quality of the sample, the system was suitable to prepare frozen-hydrated biological samples for cryogenic CXDM experiments.

Coherent X-ray diffraction imaging and its applications in materials science and biology

In site quantitative, high-contrast and high-resolution imaging of micro/nanoscale material is an important goal of the X-ray microscopy and imaging. A novel method which is called lensless imaging or coherent X-ray diffraction imaging, is a promising approach to solving these problems. In this review, a brief introduction to imaging theory and development of coherent X-ray diffraction imaging, and some typical applications in material science and biology are presented. For instance, two-dimensional (2D) imaging of Bi dopant distribution in a Si crystal, quantitative three-dimensional (3D) imaging of a GaN quantum dot with core shell structure, 2D imaging of stained Escherichia coli bacteria, nanoscale imaging and mechanisms of biomineralization of fish bones, 2D high-contrast imaging of an unstained herpes virus, 3D high-resolution imaging of an unstained yeast cell and in situ quantitative analysis are illuminated. Finally, the future prospect of coherent X-ray diffraction imaging is given. With the development of X-ray free electron lasers and combining cryogenic techniques with coherent X-ray diffraction microscopy, coherent diffraction imaging will be a powerful tool and widely used in materials science and biology.

Application of a real-space three-dimensional image reconstruction method in the structural analysis of noncrystalline biological macromolecules enveloped by water in coherent x-ray diffraction microscopy

Coherent x-ray diffraction microscopy is a novel technique in the structural analyses of particles that are difficult to crystallize, such as the biological particles composing living cells. As water is indispensable for maintaining particles in functional structures, sufficient hydration of targeted particles is required during sample preparation for diffraction microscopy experiments. However, the water enveloping particles also contributes significantly to the diffraction patterns and reduces the electron-density contrast of the sample particles. In this study, we propose a protocol for the structural analyses of particles in water by applying a three-dimensional reconstruction method in real space for the projection images phase-retrieved from diffraction patterns, together with a developed density modification technique. We examined the feasibility of the protocol through three simulations involving a protein molecule in a vacuum, and enveloped in either a droplet or a cube-shaped water. The simulations were carried out for the diffraction patterns in the reciprocal planes normal to the incident x-ray beam. This assumption and the simulation conditions corresponded to experiments using x-ray wavelengths of shorter than 0.03Å. The analyses demonstrated that our protocol provided an interpretable electron-density map. Based on the results, we discuss the advantages and limitations of the proposed protocol and its practical application for experimental data. In particular, we examined the influence of Poisson noise in diffraction patterns on the reconstructed three-dimensional electron density in the proposed protocol.

2N1548 コヒーレントX線回折顕微鏡法実験の為の湿度制御下生体粒子凍結水和試料作製装置の開発(バイオイメージング2,第49回日本生物物理学会年会)

2N1548 コヒーレントX線回折顕微鏡法実験の為の湿度制御下生体粒子凍結水和試料作製装置の開発(バイオイメージング2,第49回日本生物物理学会年会)

Coherent X-ray diffraction microscopy for structural studies of biological particles

Coherent X-ray diffraction microscopy for structural studies of biological particles

1SI-02 非結晶生体粒子のに対する低温コヒーレントX線回折顕微鏡法実験(1SI 第4世代光源、X線自由電子レーザーが拓く生物物理学,日本生物物理学会第49回年会(2011年度))

1SI-02 非結晶生体粒子のに対する低温コヒーレントX線回折顕微鏡法実験(1SI 第4世代光源、X線自由電子レーザーが拓く生物物理学,日本生物物理学会第49回年会(2011年度))

An experimental procedure for precise evaluation of electron density distribution of a nanostructured material by coherent x-ray diffraction microscopy

We developed a coherent x-ray diffraction microscopy (CXDM) system that enables us to precisely evaluate the electron density of an isolated sample. This system enables us to determine the dose per surface unit of x rays illuminated onto an isolated sample by combining incident x-ray intensity monitoring and the CXDM of a reference sample. By using this system, we determined the dose of x rays illuminated onto a nanostructured island fabricated by focused-ion-beam chemical vapor deposition and derived the electron density distribution of such a nanostructured island. A projection image of the nanostructured island with a spatial resolution of 24.1 nm and a contrast resolution higher than 2.3x10(7) electrons/pixel was successfully reconstructed.

Iterative phase recovery using wavelet domain constraints

Phase retrieval is a central problem in coherent x-ray diffraction microscopy. Various methods have been proposed to solve the problem with the most successful being iterative methods with a finite spatial support constraint. In this work, a new constraint is formulated in the wavelet domain using low-resolution a priori information. Experimental results indicate that the constraint is sufficient to reconstruct an object from Fourier modulus measurements.

Two-dimensional measurement of focused hard X-ray beam profile using coherent X-ray diffraction of isolated nanoparticle

A method for evaluating the two-dimensional photon density distribution in focused hard X-ray beams is proposed and demonstrated in a synchrotron experiment at SPring-8. A synchrotron X-ray beam of 11.8 keV is focused to a ∼ 1 μ m spot by Kirkpatrick–Baez mirrors. The two-dimensional intensity distribution of the focused beam is derived by monitoring the forward diffracted intensity from an isolated silver nanocube with an edge length of ∼ 150 nm positioned in the beam waist, which is two-dimensionally scanned. Furthermore, the photon density of X-rays illuminated onto the nanocube is estimated by utilizing coherent X-ray diffraction microscopy. This method is useful for evaluating the photon density distribution of hard X-ray beams focused to a spot size of less than a few micrometers.

Development of incident x-ray flux monitor for coherent x-ray diffraction microscopy

An incident x-ray flux monitor for coherent x-ray diffraction microscopy was developed. The intensities of x-rays passing through the sample were measured using an x-ray photodiode, with the simultaneous measurement of the x-ray diffraction intensities of the sample. As a result of the normalization of the x-ray diffraction intensities by the incident x-ray flux determined from the monitor, the fluctuation of the speckle intensities was successfully suppressed.

Observation of electromigration in a Cu thin line by in situ coherent x-ray diffraction microscopy

Electromigration (EM) in a 1-μm-thick Cu thin line was investigated by in situ coherent x-ray diffraction microscopy (CXDM). Characteristic x-ray speckle patterns due to both EM-induced voids and thermal deformation in the thin line were observed in the coherent x-ray diffraction patterns. Both parts of the voids and the deformation were successfully visualized in the images reconstructed from the diffraction patterns. This result not only represents the first demonstration of the visualization of structural changes in metallic materials by in situ CXDM but is also an important step toward studying the structural dynamics of nanomaterials using x-ray free-electron lasers in the near future.

Feasibility study of high-resolution coherent diffraction microscopy using synchrotron x rays focused by Kirkpatrick–Baez mirrors

High-flux coherent x rays are necessary for the improvement of the spatial resolution in coherent x-ray diffraction microscopy (CXDM). In this study, high-resolution CXDM using Kirkpatrick–Baez (KB) mirrors is proposed, and the mirrors are designed for experiments of the transmission scheme at SPring-8. Both the photon density and spatial coherence of synchrotron x rays focused by the KB mirrors are investigated by wave optical simulation. The KB mirrors can produce nearly diffraction-limited two-dimensional focusing x rays of ∼1μm in size at 8keV. When the sample size is less than ∼1μm, the sample can be illuminated with full coherent x rays by adjusting the cross-slit size set between the source and the mirrors. From the estimated photon density at the sample position, the feasibility of CXDM with a sub-1-nm spatial resolution is suggested. The present ultraprecise figuring process enables us to fabricate mirrors for carrying out high-resolution CXDM experiments.