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DNA molecules are atomic-scale information storage molecules that promote reliable information transfer via fault-free repetitions of replications and transcriptions. Remarkable accuracy of compacting a few-meters-long DNA into a micrometer-scale object, and the reverse, makes the chromosome one of the most intriguing structures from both physical and biological viewpoints. However, its three-dimensional (3D) structure remains elusive with challenges in observing native structures of specimens at tens-of-nanometers resolution. Here, using cryogenic coherent X-ray diffraction imaging, we succeeded in obtaining nanoscale 3D structures of metaphase chromosomes that exhibited a random distribution of electron density without characteristics of high-order folding structures. Scaling analysis of the chromosomes, compared with a model structure having the same density profile as the experimental results, has discovered the fractal nature of density distributions. Quantitative 3D density maps, corroborated by molecular dynamics simulations, reveal that internal structures of chromosomes conform to diffusion-limited aggregation behavior, which indicates that 3D chromatin packing occurs via stochastic processes.
Assuntos
Cromatina/genética , Cromossomos/genética , Linhagem Celular Tumoral , DNA/genética , Células HCT116 , Humanos , Metáfase/genética , Difração de Raios X/métodos , Raios XRESUMO
We demonstrate a propagation-based phase-contrast imaging method for full-field X-ray microscopy based on advanced Kirkpatrick-Baez (AKB) mirrors to achieve high-contrast observations of weak phase objects and correct field curvature aberrations. Through a demonstration performed at SPring-8, the phase contrast of weak phase objects such as polystyrene spheres and chemically fixed cells was successfully observed with high sensitivity (â¼0.03 rad). Furthermore, the field of view of the AKB mirrors was expanded to the full area of the obtained images (25 × 30 µm) by correcting the field curvature aberration using reconstructed complex wavefields.
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Characterizing interactions between microbial cells and their specific inhibitory drugs is essential for developing effective drugs and understanding the therapeutic mechanism. Functional metal nanoclusters can be effective inhibitory agents against microorganisms according to various characterization methods, but quantitative three-dimensional (3D) spatial structural analysis of intact cells is lacking. Herein, using coherent X-ray diffraction imaging, we performed in situ 3D visualization of unstained Staphylococcus aureus cells treated with peptide-mineralized Au-cluster probes at a resolution of â¼47 nm. Subsequent 3D mass-density mapping and quantitative structural analyses of S. aureus in different degrees of destruction showed that the bacterial cell wall was damaged and cytoplasmic constituents were released from cells, confirming the significant antibacterial effects of the Au-cluster probe. This study provides a promising nondestructive approach for quantitative imaging and paves the way for further research into microbe-inhibitor drug interactions.
Assuntos
Imageamento Tridimensional , Staphylococcus aureus , Antibacterianos/química , Antibacterianos/farmacologia , Imageamento Tridimensional/métodos , Testes de Sensibilidade Microbiana , Peptídeos/farmacologia , Difração de Raios XRESUMO
The new Brain Imaging Beamline (BIB) of the Taiwan Photon Source (TPS) has been commissioned and opened to users. The BIB and in particular its endstation are designed to take advantage of bright unmonochromatized synchrotron X-rays and target fast 3D imaging, â¼1â ms exposure time plus very high â¼0.3â µm spatial resolution. A critical step in achieving the planned performances was the solution to the X-ray induced damaging problems of the detection system. High-energy photons were identified as their principal cause and were solved by combining tailored filters/attenuators and a high-energy cut-off mirror. This enabled the tomography acquisition throughput to reach >1â mm3â min-1, a critical performance for large-animal brain mapping and a vital mission of the beamline.
Assuntos
Encéfalo/diagnóstico por imagem , Imageamento Tridimensional , Lesões por Radiação/prevenção & controle , Microtomografia por Raio-X/instrumentação , Animais , Desenho de Equipamento , Fótons , Síncrotrons , TaiwanRESUMO
We propose an extended X-ray adaptive zoom condenser that can form an intermediate virtual focus. The system comprises two deformable mirrors for focusing within a single dimension and can vary its numerical aperture (NA) without changing the positions of the light source, mirrors, or final focus. The desired system NA is achieved simply by controlling the mirror surfaces, which enables conversion between convex and concave forms, by varying the position of the intermediate virtual focus. A feasibility test at SPring-8 under a photon energy of 10 keV demonstrated that the beam size can be varied between 134 and 1010 nm.
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The distribution of topological charges on X-ray vortices was measured by differential Fourier space filtering microscope, differential radial Hilbert transform microscope. It was experimentally verified for the first time using a Spiral Fresnel zone plate objective lens. This X-ray microscope is highly sensitive to X-ray topological defects, such as edges and vortices, at the exit-face wave field of objects. Its efficient use is also discussed.
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We experimentally studied the shear effect on dynamical heterogeneity near glass transition temperature. X-ray photon correlation spectroscopy was utilized to study the dynamics of polyvinyl acetate with tracer particles near its glass transition temperature, to determine the local shear rate from the anisotropic behavior of the time autocorrelation function and to calculate the dynamical heterogeneity using higher-order correlation function. The obtained results show a decrease in the dynamical heterogeneity and faster dynamics with increasing shear rate. This is the first experimental result that proved the predictions of previous molecular dynamics simulations.
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A novel full-field X-ray fluorescence microscope based on total-reflection advanced Kirkpatrick-Baez mirror optics was developed. The total-reflection imaging mirror optics arrangement, with four reflections, has the advantage of being able to function both as a powerful low-pass energy filter, completely rejecting incident excitation X-rays, and as an achromatic optical imaging system. Isolated X-ray fluorescence signals can be imaged, avoiding imaging-detector saturation, with low background noise. A prototype fluorescence microscope constructed at SPring-8 demonstrated the capability to simultaneously image elemental distributions using various X-ray fluorescence signals (Ni, Cu, Zn, Ge, and Bi). A half-period spatial resolution of ~0.5-1 µm (1000-500 LP/mm) was achieved, owing to the achromaticity of the imaging mirrors and the photon-counting scheme of the CCD camera used for fluorescence detection.
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We demonstrated that the combination of a hyperbolic convex and elliptical concave mirrors works as a compact reflective X-ray imaging system with a short optical focal length and large magnification factor. We performed an experiment to form a one-dimensional demagnified image with a demagnification factor of 321 within an approximately 2-m-long optical setup at an X-ray energy of 10 keV. The results showed that this imaging optics system is capable of providing a resolution of ~40 nm. From wavefront analysis, it was confirmed that the optics possessed a wide field-of-view with a significant reduction of comatic aberration.
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A high-resolution lens-coupled X-ray imaging detector equipped with a thin-layer transparent ceramics scintillator has been developed. The scintillator consists of a 5 µm thick Ce-doped Lu3Al5O12 layer (LuAG:Ce) bonded onto the support substrate of the non-doped LuAG ceramics by using a solid-state diffusion technique. Secondary electron microscopy of the bonded interface indicated that the crystal grains were densely packed without any pores in the optical wavelength scale, indicating a quasi-uniform refractive index across the interface. This guarantees high transparency and minimum reflection, which are essential properties for X-ray imaging detectors. The LuAG:Ce scintillator was incorporated into an X-ray imaging detector coupled with an objective lens with a numerical aperture of 0.85 and an optical magnification of 100. The scintillation light was imaged onto a complementary metal-oxide-semiconductor image sensor. The effective pixel size on the scintillator plane was 65 nm. X-ray transmission images of 200 nm line-and-space patterns were successfully resolved. The high spatial resolution was demonstrated by X-ray transmission images of large integrated circuits with the wiring patterns clearly visualized.
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Biominerals such as bones and teeth have elaborate nanostructures composed of aligned anisotropic hydroxyapatite (HAp) nanocrystals, which results in excellent mechanical properties. Construction of such ordered structures of HAp nanocrystals in synthetic materials is challenging. Recently, we reported that HAp-nanorod-based colloidal liquid crystals could be obtained. In the present study, the static structure and dynamics of liquid-crystalline (LC) colloidal dispersions of HAp nanorods are investigated by using small-angle X-ray scattering (SAXS) and X-ray photon correlation spectroscopy (XPCS). The SAXS results reveal that the interparticle distance decreases with increasing HAp concentration, φHAp, and the decrease of the interparticle distance for the short-axis direction is significantly smaller in the LC phase than the interparticle distance in the isotropic phase. In the dynamical studies of the LC phase using XPCS, we observe the diffusive motion of the HAp colloids, with the diffusion coefficient being dependent on the wave number. The diffusive motion slows down with increasing φHAp. We observe anisotropic dynamics after long-term storage (160 days after sealing), whereas only isotropic dynamics are observed in the initial XPCS measurements after short-term storage (14 days after sealing). Moreover, we have found that the dynamics slows down with increasing storage time.
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X-ray diffraction imaging is a technique for visualizing the structure of biological cells. In X-ray diffraction imaging experiments using synchrotron radiation, cryogenic conditions are necessary in order to reduce radiation damage in the biological cells. Frozen-hydrated biological specimens kept at cryogenic temperatures are also free from drying and bubbling, which occurs in wet specimens under vacuum conditions. In a previous study, the diffraction apparatus KOTOBUKI-1 [Nakasako et al. (2013), Rev. Sci. Instrum. 84, 093705] was constructed for X-ray diffraction imaging at cryogenic temperatures by utilizing a cryogenic pot, which is a cooling device developed in low-temperature physics. In this study a new cryogenic pot, suitable for tomography experiments, has been developed. The pot can rotate a biological cell over an angular range of ±170° against the direction of the incident X-ray beam. Herein, the details and the performance of the pot and miscellaneous devices are reported, along with established experimental procedures including specimen preparation. The apparatus has been used in tomography experiments for visualizing the three-dimensional structure of a Cyanidioschyzon merolae cell with an approximate size of 5â µm at a resolution of 136â nm. Based on the experimental results, the necessary improvements for future experiments and the resolution limit achievable under experimental conditions within a maximum tolerable dose are discussed.
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We have developed the new hybrid adaptive X-ray mirror based on mechanical and piezo-driven deformation to realize precise shape controllability on a long-length mirror. The mechanical bender approximately provides the required ellipse, while the piezoelectric actuators attached to the mirror correct very small residual errors to satisfy the diffraction-limited condition. The mechanical bender significantly reduces the role of the piezoelectric actuator, resulting in the suppression of accuracy degradation due to the drift and/or junction effect of the piezoelectric actuators. In addition, line focusing was demonstrated with two different numerical apertures at SPring-8, and the obtained beam sizes were 127 and 253 nm (FWHM), which agree well with the diffraction-limited sizes.
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X-ray science has greatly benefited from the progress in X-ray optics. Advances in the design and the manufacturing techniques of X-ray optics are key to the success of various microscopic and spectroscopic techniques practiced today. Here the generation of apodized X-ray illumination using a two-stage deformable Kirkpatrick-Baez mirror system is presented. Such apodized illumination is marked by the suppression of the side-lobe intensities of the focused beam. Thus generated apodized illumination was employed to improve the image quality in scanning X-ray fluorescence microscopy. Imaging of a non-isolated object by coherent X-ray diffractive imaging with apodized illumination in a non-scanning mode is also presented.
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Fatty acids are taken up by cells and incorporated into complex lipids such as neutral lipids and glycerophospholipids. Glycerophospholipids are major constituents of cellular membranes. More than 1000 molecular species of glycerophospholipids differ in their polar head groups and fatty acid compositions. They are related to cellular functions and diseases and have been well analyzed by mass spectrometry. However, intracellular imaging of fatty acids and glycerophospholipids has not been successful due to insufficient resolution using conventional methods. Here, we developed a method for labeling fatty acids with bromine (Br) and applied scanning X-ray fluorescence microscopy (SXFM) to obtain intracellular Br mapping data with submicrometer resolution. Mass spectrometry showed that cells took up Br-labeled fatty acids and metabolized them mainly into glycerophospholipids in CHO cells. Most Br signals observed by SXFM were in the perinuclear region. Higher resolution revealed a spot-like distribution of Br in the cytoplasm. The current method enabled successful visualization of intracellular Br-labeled fatty acids. Single-element labeling combined with SXFM technology facilitates the intracellular imaging of fatty acids, which provides a new tool to determine dynamic changes in fatty acids and their derivatives at the single-cell level.-Shimura, M., Shindou, H., Szyrwiel, L., Tokuoka, S. M., Hamano, F., Matsuyama, S., Okamoto, M., Matsunaga, A., Kita, Y., Ishizaka, Y., Yamauchi, K., Kohmura, Y., Lobinski, R., Shimizu, I., Shimizu, T. Imaging of intracellular fatty acids by scanning X-ray fluorescence microscopy.
Assuntos
Membrana Celular/metabolismo , Ácidos Graxos/metabolismo , Glicerofosfolipídeos/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Citoplasma/metabolismo , Metabolismo dos Lipídeos , Lipídeos , Microscopia de Fluorescência/métodos , Raios XRESUMO
A combination of plane and threefold-shape X-ray mirrors was installed in SPring-8 BL29XUL. The second mirror has parabolic cylinder surfaces that collimate X-rays in the vertical direction. A performance test was conducted, yielding highly collimated 8â keV photon beams with an effective angular divergence of 0.4â µrad, below only 5% of that of the original beams. The double-mirror system preserved 70% of the total incident flux and nearly tripled the flux density at 988â m from the light source. The values of the observations were almost similar to those of our ray-tracing simulation. Based on the results a discussion of future prospects of the mirror system is included.
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We show momentum-space characteristics of X-rays affected by Berry's phase in a deformed crystal, allowing a 15 keV beam inside a silicon crystal to be translated parallel to its optical axis while retaining its angular divergence and wave front. This data is the first evidence supporting the whole theoretical picture of Sawada et al., Phys. Rev. Lett. 96, 154802 (2006), consisting of two equations of motion about the X-ray propagation. An output beam was as much as 3.3% of the incident after propagating through 1.3 mm silicon along a lateral direction of the chip inclined at 17.722°. As its initial practical application we further utilized the device as an X-ray intensity modulator. Our results revealed a new aspect of the Berry phase and lead to an X-ray waveguide that can enhance the flexibility of future high-energy experiments.
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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, polyß-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 â¼1.19 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.
Assuntos
Processamento de Imagem Assistida por Computador , Magnetospirillum/química , Microscopia , Algoritmos , Magnetospirillum/isolamento & purificação , Razão Sinal-Ruído , Difração de Raios XRESUMO
We developed an achromatic and high-resolution full-field X-ray microscope based on advanced Kirkpatrick-Baez mirror optics that comprises two pairs of elliptical mirrors and hyperbolic mirrors utilizing the total reflection of X-rays. Performance tests to investigate the spatial resolution and chromatic aberration were performed at SPring-8. The microscope clearly resolved the pattern with ~100-nm feature size. Imaging the pattern by changing the X-ray energy revealed achromatism in the wide energy range of 8-11 keV.
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In order to utilize high-brilliance photon sources, such as X-ray free-electron lasers (XFELs), for advanced time-resolved photoelectron spectroscopy (TR-PES), a single-shot CCD-based data acquisition system combined with a high-resolution hemispherical electron energy analyzer has been developed. The system's design enables it to be controlled by an external trigger signal for single-shot pump-probe-type TR-PES. The basic performance of the system is demonstrated with an offline test, followed by online core-level photoelectron and Auger electron spectroscopy in 'single-shot image', 'shot-to-shot image (image-to-image storage or block storage)' and `shot-to-shot sweep' modes at soft X-ray undulator beamline BL17SU of SPring-8. In the offline test the typical repetition rate for image-to-image storage mode has been confirmed to be about 15â Hz using a conventional pulse-generator. The function for correcting the shot-to-shot intensity fluctuations of the exciting photon beam, an important requirement for the TR-PES experiments at FEL sources, has been successfully tested at BL17SU by measuring Au 4f photoelectrons with intentionally controlled photon flux. The system has also been applied to hard X-ray PES (HAXPES) in `ordinary sweep' mode as well as shot-to-shot image mode at the 27â m-long undulator beamline BL19LXU of SPring-8 and also at the SACLA XFEL facility. The XFEL-induced Ti 1s core-level spectrum of La-doped SrTiO3 is reported as a function of incident power density. The Ti 1s core-level spectrum obtained at low power density is consistent with the spectrum obtained using the synchrotron source. At high power densities the Ti 1s core-level spectra show space-charge effects which are analysed using a known mean-field model for ultrafast electron packet propagation. The results successfully confirm the capability of the present data acquisition system for carrying out the core-level HAXPES studies of condensed matter induced by the XFEL.