13742624
OMNY-A tOMography Nano crYo stage.
For many scientific questions gaining three-dimensional insight into a specimen can provide valuable information. We here present an instrument called "tOMography Nano crYo (OMNY)," dedicated to high resolution 3D scanning x-ray microscopy at cryogenic conditions via hard X-ray ptychography. Ptychography is a lens-less imaging method requiring accurate sample positioning. In OMNY, this in achieved via dedicated laser interferometry and closed-loop position control reaching sub-10 nm positioning accuracy. Cryogenic sample conditions are maintained via conductive cooling. 90 K can be reached when using liquid nitrogen as coolant, and 10 K is possible with liquid helium. A cryogenic sample-change mechanism permits measurements of cryogenically fixed specimens. We compare images obtained with OMNY with older measurements performed using a nitrogen gas cryo-jet of stained, epoxy-embedded retina tissue and of frozen-hydrated Chlamydomonas cells.
2449015
Ptychographic X-ray computed tomography at the nanoscale
X-ray tomography is an invaluable tool in biomedical imaging. It can deliver the three-dimensional internal structure of entire organisms as well as that of single cells, and even gives access to quantitative information, crucially important both for medical applications and for basic research. Most frequently such information is based on X-ray attenuation. Phase contrast is sometimes used for improved visibility but remains significantly harder to quantify. Here we describe an X-ray computed tomography technique that generates quantitative high-contrast three-dimensional electron density maps from phase contrast information without reverting to assumptions of a weak phase object or negligible absorption. This method uses a ptychographic coherent imaging approach to record tomographic data sets, exploiting both the high penetration power of hard X-rays and the high sensitivity of lensless imaging. As an example, we present images of a bone sample in which structures on the 100 nm length scale such as the osteocyte lacunae and the interconnective canalicular network are clearly resolved. The recovered electron density map provides a contrast high enough to estimate nanoscale bone density variations of less than one per cent. We expect this high-resolution tomography technique to provide invaluable information for both the life and materials sciences.
16228958
X-ray ptychographic computed tomography at 16 nm isotropic 3D resolution
X-ray ptychography is a scanning variant of coherent diffractive imaging with the ability to image large fields of view at high resolution. It further allows imaging of non-isolated specimens and can produce quantitative mapping of the electron density distribution in 3D when combined with computed tomography. The method does not require imaging lenses, which makes it dose efficient and suitable to multi-keV X-rays, where efficient photon counting, pixelated detectors are available. Here we present the first highly resolved quantitative X-ray ptychographic tomography of an extended object yielding 16 nm isotropic 3D resolution recorded at 2 Å wavelength. This first-of-its-kind demonstration paves the way for ptychographic X-ray tomography to become a promising method for X-ray imaging of representative sample volumes at unmatched resolution, opening tremendous potential for characterizing samples in materials science and biology by filling the resolution gap between electron microscopy and other X-ray imaging techniques.
4448836
High-resolution non-destructive three-dimensional imaging of integrated circuits
Modern nanoelectronics has advanced to a point at which it is impossible to image entire devices and their interconnections non-destructively because of their small feature sizes and the complex three-dimensional structures resulting from their integration on a chip. This metrology gap implies a lack of direct feedback between design and manufacturing processes, and hampers quality control during production, shipment and use. Here we demonstrate that X-ray ptychography—a high-resolution coherent diffractive imaging technique—can create three-dimensional images of integrated circuits of known and unknown designs with a lateral resolution in all directions down to 14.6 nanometres. We obtained detailed device geometries and corresponding elemental maps, and show how the devices are integrated with each other to form the chip. Our experiments represent a major advance in chip inspection and reverse engineering over the traditional destructive electron microscopy and ion milling techniques. Foreseeable developments in X-ray sources, optics and detectors, as well as adoption of an instrument geometry optimized for planar rather than cylindrical samples, could lead to a thousand-fold increase in efficiency, with concomitant reductions in scan times and voxel sizes.
30125688
High-Resolution Scanning X-ray Diffraction Microscopy
Coherent diffractive imaging (CDI) and scanning transmission x-ray microscopy (STXM) are two popular microscopy techniques that have evolved quite independently. CDI promises to reach resolutions below 10 nanometers, but the reconstruction procedures put stringent requirements on data quality and sample preparation. In contrast, STXM features straightforward data analysis, but its resolution is limited by the spot size on the specimen. We demonstrate a ptychographic imaging method that bridges the gap between CDI and STXM by measuring complete diffraction patterns at each point of a STXM scan. The high penetration power of x-rays in combination with the high spatial resolution will allow investigation of a wide range of complex mesoscopic life and material science specimens, such as embedded semiconductor devices or cellular networks.
6714282
Hard-x-ray lensless imaging of extended objects.
We demonstrate a hard-x-ray microscope that does not use a lens and is not limited to a small field of view or an object of finite size. The method does not suffer any of the physical constraints, convergence problems, or defocus ambiguities that often arise in conventional phase-retrieval diffractive imaging techniques. Calculation times are about a thousand times shorter than in current iterative algorithms. We need no a priori knowledge about the object, which can be a transmission function with both modulus and phase components. The technique has revolutionary implications for x-ray imaging of all classes of specimen.
1807257
Phase retrieval with transverse translation diversity: a nonlinear optimization approach.
We develop and test a nonlinear optimization algorithm for solving the problem of phase retrieval with transverse translation diversity, where the diverse far-field intensity measurements are taken after translating the object relative to a known illumination pattern. Analytical expressions for the gradient of a squared-error metric with respect to the object, illumination and translations allow joint optimization of the object and system parameters. This approach achieves superior reconstructions, with respect to a previously reported technique [H. M. L. Faulkner and J. M. Rodenburg, Phys. Rev. Lett. 93, 023903 (2004)], when the system parameters are inaccurately known or in the presence of noise. Applicability of this method for samples that are smaller than the illumination pattern is explored.
122763600
Maximum-likelihood refinement for coherent diffractive imaging
We introduce the application of maximum-likelihood (ML) principles to the image reconstruction problem in coherent diffractive imaging. We describe an implementation of the optimization procedure for ptychography, using conjugate gradients and including preconditioning strategies, regularization and typical modifications of the statistical noise model. The optimization principle is compared to a difference map reconstruction algorithm. With simulated data important improvements are observed, as measured by a strong increase in the signal-to-noise ratio. Significant gains in resolution and sensitivity are also demonstrated in the ML refinement of a reconstruction from experimental x-ray data. The immediate consequence of our results is the possible reduction of exposure, or dose, by up to an order of magnitude for a reconstruction quality similar to iterative algorithms currently in use.
62617596
Principles of computerized tomographic imaging
Tomography refers to the cross-sectional imaging of an object from either transmission or reflection data collected by illuminating the object from many different directions. The impact of tomography in diagnostic medicine has been revolutionary, since it has enabled doctors to view internal organs with unprecedented precision and safety to the patient. There are also numerous nonmedical imaging applications which lend themselves to methods of computerized tomography, such as mapping of underground resources...cross-sectional imaging of for nondestructive testing...the determination of the brightness distribution over a celestial sphere...three-dimensional imaging with electron microscopy. Principles of Computerized Tomographic Imaging provides a tutorial overview of topics in tomographic imaging covering mathematical principles and theory...how to apply the theory to problems in medical imaging and other fields...several variations of tomography that are currently being researched.
205172982
An instrument for 3D x-ray nano-imaging.
We present an instrument dedicated to 3D scanning x-ray microscopy, allowing a sample to be precisely scanned through a beam while the angle of x-ray incidence can be changed. The position of the sample is controlled with respect to the beam-defining optics by laser interferometry. The instrument achieves a position stability better than 10 nm standard deviation. The instrument performance is assessed using scanning x-ray diffraction microscopy and we demonstrate a resolution of 18 nm in 2D imaging of a lithographic test pattern while the beam was defined by a pinhole of 3 μm in diameter. In 3D on a test object of copper interconnects of a microprocessor, a resolution of 53 nm is achieved.
117009115
Error motion compensating tracking interferometer for the position measurement of objects with rotational degree of freedom
Abstract. The nonaxial interferometric position measurement of rotating objects can be performed by imaging the laser beam of the interferometer to a rotating mirror which can be a sphere or a cylinder. This, however, requires such rotating mirrors to be centered on the axis of rotation as a wobble would result in loss of the interference signal. We present a tracking-type interferometer that performs such measurement in a general case where the rotating mirror may wobble on the axis of rotation, or even where the axis of rotation may be translating in space. Aside from tracking, meaning to measure and follow the position of the rotating mirror, the interferometric measurement errors induced by the tracking motion of the interferometer itself are optically compensated, preserving nanometric measurement accuracy. As an example, we show the application of this interferometer in a scanning x-ray tomography instrument.
19041673
Evolutionary-Optimized Photonic Network Structure in White Beetle Wing Scales.
Most studies of structural color in nature concern periodic arrays, which through the interference of light create color. The "color" white however relies on the multiple scattering of light within a randomly structured medium, which randomizes the direction and phase of incident light. Opaque white materials therefore must be much thicker than periodic structures. It is known that flying insects create "white" in extremely thin layers. This raises the question, whether evolution has optimized the wing scale morphology for white reflection at a minimum material use. This hypothesis is difficult to prove, since this requires the detailed knowledge of the scattering morphology combined with a suitable theoretical model. Here, a cryoptychographic X-ray tomography method is employed to obtain a full 3D structural dataset of the network morphology within a white beetle wing scale. By digitally manipulating this 3D representation, this study demonstrates that this morphology indeed provides the highest white retroreflection at the minimum use of material, and hence weight for the organism. Changing any of the network parameters (within the parameter space accessible by biological materials) either increases the weight, increases the thickness, or reduces reflectivity, providing clear evidence for the evolutionary optimization of this morphology.
43391045
MISTRAL: a transmission soft X-ray microscopy beamline for cryo nano-tomography of biological samples and magnetic domains imaging.
The performance of MISTRAL is reported, the soft X-ray transmission microscopy beamline at the ALBA light source (Barcelona, Spain) which is primarily dedicated to cryo soft X-ray tomography (cryo-SXT) for three-dimensional visualization of whole unstained cells at spatial resolutions down to 30 nm (half pitch). Short acquisition times allowing for high-throughput and correlative microscopy studies have promoted cryo-SXT as an emerging cellular imaging tool for structural cell biologists bridging the gap between optical and electron microscopy. In addition, the beamline offers the possibility of imaging magnetic domains in thin magnetic films that are illustrated here with an example.
834619
The Bionanoprobe: hard X-ray fluorescence nanoprobe with cryogenic capabilities
The Bionanoprobe has been developed to study trace elements in frozen-hydrated biological systems with sub-100 nm spatial resolution. Here its performance is demonstrated and first results reported.
19244169
Three-dimensional cellular ultrastructure resolved by X-ray microscopy
We developed an X-ray microscope using partially coherent object illumination instead of previously used quasi-incoherent illumination. The design permitted the incorporation of a cryogenic tilt stage, enabling tomography of frozen-hydrated, intact adherent cells. We obtained three-dimensional reconstructions of mouse adenocarcinoma cells at ∼36-nm (Rayleigh) and ∼70-nm (Fourier ring correlation) resolution, which allowed us to visualize the double nuclear membrane, nuclear pores, nuclear membrane channels, mitochondrial cristae and lysosomal inclusions.
139730228
High-energy cryo x-ray nano-imaging at the ID16A beamline of ESRF
The ID16A beamline at ESRF offers unique capabilities for X-ray nano-imaging, and currently produces the worlds brightest high energy diffraction-limited nanofocus. Such a nanoprobe was designed for quantitative characterization of the morphology and the elemental composition of specimens at both room and cryogenic temperatures. Billions of photons per second can be delivered in a diffraction-limited focus spot size down to 13 nm. Coherent X-ray imaging techniques, as magnified holographic-tomography and ptychographic-tomography, are implemented as well as X-ray fluorescence nanoscopy. We will show the latest developments in coherent and spectroscopic X-ray nanoimaging implemented at the ID16A beamline
206494562
Three-dimensional mass density mapping of cellular ultrastructure by ptychographic X-ray nanotomography.
We demonstrate absolute quantitative mass density mapping in three dimensions of frozen-hydrated biological matter with an isotropic resolution of 180 nm. As model for a biological system we use Chlamydomonas cells in buffer solution confined in a microcapillary. We use ptychographic X-ray computed tomography to image the entire specimen, including the 18 μm-diameter capillary, thereby providing directly an absolute mass density measurement of biological matter with an uncertainty of about 6%. The resulting maps have sufficient contrast to distinguish cells from the surrounding ice and several organelles of different densities inside the cells. Organelles are identified by comparison with a stained, resin-embedded specimen, which can be compared with established transmission electron microscopy results. For some identified organelles, the knowledge of their elemental composition reduces the uncertainty of their mass density measurement down to 1% with values consistent with previous measurements of dry weight concentrations in thin cellular sections by scanning transmission electron microscopy. With prospects of improving the spatial resolution in the near future, we expect that the capability of non-destructive three-dimensional mapping of mass density in biological samples close to their native state becomes a valuable method for measuring the packing of organic matter on the nanoscale.
68448
Recreating the synthesis of starch granules in yeast
Starch, as the major nutritional component of our staple crops and a feedstock for industry, is a vital plant product. It is composed of glucose polymers that form massive semi-crystalline granules. Its precise structure and composition determine its functionality and thus applications; however, there is no versatile model system allowing the relationships between the biosynthetic apparatus, glucan structure and properties to be explored. Here, we expressed the core Arabidopsis starch-biosynthesis pathway in Saccharomyces cerevisiae purged of its endogenous glycogen-metabolic enzymes. Systematic variation of the set of biosynthetic enzymes illustrated how each affects glucan structure and solubility. Expression of the complete set resulted in dense, insoluble granules with a starch-like semi-crystalline organization, demonstrating that this system indeed simulates starch biosynthesis. Thus, the yeast system has the potential to accelerate starch research and help create a holistic understanding of starch granule biosynthesis, providing a basis for the targeted biotechnological improvement of crops. DOI: http://dx.doi.org/10.7554/eLife.15552.001
136106762
Cavitation in strained polyethylene/aluminium oxide nanocomposites
The incorporation of metal oxide (e.g. Al2O3) nanoparticles has a pronounced positive effect on low-density polyethylene (LDPE) as an insulating material for high-voltage direct-current (HVDC) cabl ...
32588638
Three-Dimensional Nanometer Features of Direct Current Electrical Trees in Low-Density Polyethylene.
Electrical trees are one reason for the breakdown of insulating materials in electrical power systems. An understanding of the growth of electrical trees plays a crucial role in the development of reliable high voltage direct current (HVDC) power grid systems with transmission voltages up to 1 MV. A section that contained an electrical tree in low-density polyethylene (LDPE) has been visualized in three dimensions (3D) with a resolution of 92 nm by X-ray ptychographic tomography. The 3D imaging revealed prechannel-formations with a lower density with the width of a couple of hundred nanometers formed around the main branch of the electrical tree. The prechannel structures were partially connected with the main tree via paths through material with a lower density, proving that the tree had grown in a step-by-step manner via the prestep structures formed in front of the main channels. All the prechannel structures had a size well below the limit of the Paschen law and were thus not formed by partial discharges. Instead, it is suggested that the prechannel structures were formed by electro-mechanical stress and impact ionization, where the former was confirmed by simulations to be a potential explanation with electro-mechanical stress tensors being almost of the same order of magnitude as the short-term modulus of low-density polyethylene.
10218404
High-efficiency Fresnel zone plates for hard X-rays by 100 keV e-beam lithography and electroplating
The efficiencies of several Fresnel zone plates, that were fabricated using a direct-write method with high-energy electrons, were measured over a wide range of photon energies.
136614951
High‐Efficiency Gold Fresnel Zone Plates for Multi‐keV X‐rays
We report a direct e‐beam writing process of Fresnel zone plates (FZPs) using thick layers of PMMA resist as electroplating molds. We used 100‐kV electron beam lithography to directly expose thick PMMA layers, which were later used as plating molds without intermediate etching steps. High‐quality 500‐nm‐ and 1‐μm‐thick Au FZPs with outermost zone widths down to 50‐nm and 70‐nm, respectively, and with diameters up to 600 μm were fabricated. In this paper we present the details of the optimized fabrication process, such as development times, developer, dose tables, and line shrinkages required to obtain the desired zone widths and gaps between the zones. The diffraction efficiencies of the fabricated FZPs were measured for a wide range of x‐ray energies (2.8–13.2 keV) showing excellent values up to 65–75% of the theoretical maximum, reflecting the good quality of the FZPs. Spatially resolved diffraction efficiency measurements indicate the uniformity of the zone plates and lack of defects.
119377591
Hard x-ray nanofocusing by multilayer Laue lenses
Multilayer Laue lens (MLL) is a new class of x-ray optics that offer great promise for achieving nanometre-level spatial resolution by focusing hard x-rays. Fabricating an MLL via thin-film deposition provides the means to achieve a linear Fresnel-zone plate structure with zone widths below 1?nm, while retaining a virtually limitless aspect ratio. Despite its similarity to the Fresnel-zone plate, MLL exhibits categorically distinctive focusing properties and their fabrication comes with a wide array of challenges. This article provides a comprehensive review of advances in MLLs, and includes extensive theoretical modelling on focusing performance, discussion on fabrication challenges, their current capabilities and notable results from x-ray focusing experiments.
17755711
Formation of optical images by X-rays.
Several conceivable methods for the formation of optical images by x-rays are considered, and a method employing concave mirrors is adopted as the most promising. A concave spherical mirror receiving radiation at grazing incidence (a necessary arrangement with x-rays) images a point into a line in accordance with a focal length f=Ri/2 where R is the radius of curvature and i the grazing angle. The image is subject to an aberration such that a ray reflected at the periphery of the mirror misses the focal point of central rays by a distance given approximately by S=1.5Mr2/R, where M is the magnification of the image and r is the radius of the mirror face. The theoretically possible resolving power is such as to resolve point objects separated by about 70A, a limit which is independent of the wave-length used. Point images of points and therefore extended images of extended objects may be produced by causing the radiation to reflect from two concave mirrors in series. Sample results are presented.
51943425
Breaking the 10 nm barrier in hard-X-ray focusing
X-ray sources such as free-electron lasers offer the potential to study matter at unprecedented spatial and temporal resolution. But that potential is limited by the poor quality of conventional X-ray optical elements. An in situ technique that corrects for wavefront aberrations and allows X-rays to be focused to a spot just 7 nm wide could provide a solution.
46824821
OMNY PIN-A versatile sample holder for tomographic measurements at room and cryogenic temperatures.
Nowadays ptychographic tomography in the hard x-ray regime, i.e., at energies above about 2 keV, is a well-established measurement technique. At the Paul Scherrer Institut, currently two instruments are available: one is measuring at room temperature and atmospheric pressure, and the other, the so-called OMNY (tOMography Nano crYo) instrument, is operating at ultra-high vacuum and offering cryogenic sample temperatures down to 10 K. In this manuscript, we present the sample mounts that were developed for these instruments. Aside from excellent mechanical stability and thermal conductivity, they also offer highly reproducible mounting. Various types were developed for different kinds of samples and are presented in detail, including examples of how specimens can be mounted on these holders. We also show the first hard x-ray ptychographic tomography measurements of high-pressure frozen biological samples, in the present case Chlamydomonas cells, the related sample pins and preparation steps. For completeness, we present accessories such as transportation containers for both room temperature and cryogenic samples and a gripper mechanism for automatic sample changing. The sample mounts are not limited to x-ray tomography or hard x-ray energies, and we believe that they can be very useful for other instrumentation projects.
207319951
Phase tomography from x-ray coherent diffractive imaging projections.
Coherent diffractive imaging provides accurate phase projections that can be tomographically combined to yield detailed quantitative 3D reconstructions with a resolution that is not limited by imaging optics. We present robust algorithms for post-processing and alignment of these tomographic phase projections. A simple method to remove undesired constant and linear phase terms on the reconstructions is given. Also, we provide an algorithm for automatic alignment of projections that has good performance even for samples with no fiducial markers. Currently applied to phase projections, this alignment algorithm has proven to be robust and should also be useful for lens-based tomography techniques that pursue nanoscale 3D imaging. Lastly, we provide a method for tomographic reconstruction that works on phase projections that are known modulo 2π, such that the phase unwrapping step is avoided. We demonstrate the performance of these algorithms by 3D imaging of bacteria population in legume root-nodule cells.
137347696
Quantitative interior x-ray nanotomography by a hybrid imaging technique
Hierarchical structures appear often in life and materials sciences, and their characterization profits greatly from imaging methods that allow seamless probing of various length scales without sacrificing image quality. X-ray tomography is particularly adept at probing 3D structures; however, zooming in on a region of interest results in a loss of quantitativeness of image contrast and suffers from artifacts unless a priori knowledge or assumptions about the sample are used. Here, we demonstrate a hybrid technique that exploits a micrometer-resolution overview to realize ab initio nanoscale interior tomography with quantitative contrast. In a study of avian eggshell, a model for bionanoporous materials, our approach reveals a complex arrangement of vesicles with sizes ranging from hundred nanometers to a few micrometers. We anticipate that such an approach can be widely adopted and benefited from at synchrotron and laboratory sources, for instance, where such zooming capabilities are already present or can be readily realized.
121645298
On-the-fly scans for X-ray ptychography
With the increasing importance of nanotechnology, the need for reliable real-time imaging of mesoscopic objects with nanometer resolution is rising. For X-ray ptychography, a scanning microscopy technique that provides nanometric resolution on extended fields of view, and the settling time of the scanning system is one of the bottlenecks for fast imaging. Here, we demonstrate that ptychographic on-the-fly scans, i.e., collecting diffraction patterns while the sample is scanned with constant velocity, can be modelled as a state mixture of the probing radiation and allow for reliable image recovery. Characteristics of the probe modes are discussed for various scan parameters, and the application to significantly reducing the scanning time is considered.
15640757
Continuous motion scan ptychography: characterization for increased speed in coherent x-ray imaging.
Ptychography is a coherent diffraction imaging (CDI) method for extended objects in which diffraction patterns are acquired sequentially from overlapping coherent illumination spots. The object's complex transmission function can be reconstructed from those diffraction patterns at a spatial resolution limited only by the scattering strength of the object and the detector geometry. Most experiments to date have positioned the illumination spots on the sample using a move-settle-measure sequence in which the move and settle steps can take longer to complete than the measure step. We describe here the use of a continuous "fly-scan" mode for ptychographic data collection in which the sample is moved continuously, so that the experiment resembles one of integrating the diffraction patterns from multiple probe positions. This allows one to use multiple probe mode reconstruction methods to obtain an image of the object and also of the illumination function. We show in simulations, and in x-ray imaging experiments, some of the characteristics of fly-scan ptychography, including a factor of 25 reduction in the data acquisition time. This approach will become increasingly important as brighter x-ray sources are developed, such as diffraction limited storage rings.
46881491
Iterative least-squares solver for generalized maximum-likelihood ptychography.
Although ptychography does not require a precise knowledge of the illumination wavefront, common implementations rely upon assumptions such as accurate knowledge of the scan positions and constant illumination. Limited validity of these assumptions results in deterioration of the reconstruction quality. We present a generalized ptychography method that optimizes the reconstruction along multiple directions. In our manuscript, we demonstrate that the additional flexibility not only helps to amend imprecisions of the ptychography model in a self-consistent way but additionally leads to faster convergence without a significant increase of the computational cost per iteration.
7037240
Translation position determination in ptychographic coherent diffraction imaging.
Accurate knowledge of translation positions is essential in ptychography to achieve a good image quality and the diffraction limited resolution. We propose a method to retrieve and correct position errors during the image reconstruction iterations. Sub-pixel position accuracy after refinement is shown to be achievable within several tens of iterations. Simulation and experimental results for both optical and X-ray wavelengths are given. The method improves both the quality of the retrieved object image and relaxes the position accuracy requirement while acquiring the diffraction patterns.
37578952
Dense high aspect ratio hydrogen silsesquioxane nanostructures by 100 keV electron beam lithography.
We investigated the fabrication of dense, high aspect ratio hydrogen silsesquioxane (HSQ) nanostructures by 100 keV electron beam lithography. The samples were developed using a high contrast developer and supercritically dried in carbon dioxide. Dense gratings with line widths down to 25 nm were patterned in 500 nm-thick resist layers and semi-dense gratings with line widths down to 10 nm (40 nm pitch) were patterned in 250 nm-thick resist layers. The dense HSQ nanostructures were used as molds for gold electrodeposition, and the semi-dense HSQ gratings were iridium-coated by atomic layer deposition. We used these methods to produce Fresnel zone plates with extreme aspect ratio for scanning transmission x-ray microscopy that showed excellent performance at 1.0 keV photon energy.
31849721
Optimization of overlap uniformness for ptychography.
We demonstrate the advantages of imaging with ptychography scans that follow a Fermat spiral trajectory. This scan pattern provides a more uniform coverage and a higher overlap ratio with the same number of scan points over the same area than the presently used mesh and concentric [13] patterns. Under realistically imperfect measurement conditions, numerical simulations show that the quality of the reconstructed image is improved significantly with a Fermat spiral compared with a concentric scan pattern. The result is confirmed by the performance enhancement with experimental data, especially under low-overlap conditions. These results suggest that the Fermat spiral pattern increases the quality of the reconstructed image and tolerance to data with imperfections.
25106960
Characterization and Calibration of PILATUS Detectors
Pilatus is a silicon hybrid pixel detector system for detecting X-rays in single photon counting mode. The PILATUS II chip, fabricated in a radiation tolerant design with a standard 0.25 mum CMOS process, was used to construct multichip modules with a size of 84 times 34 mm2 comprising 94'965 pixels. All calibrations and characterizations were carried out with monochromatic X-rays from a synchrotron source. In order to set any required threshold above the noise level between 2.14 keV and 22 keV the detector was calibrated with X-rays. An algorithm to adjust thresholds pixel-by-pixel and create trim files based on X-ray flat-field images was developed. The threshold dispersion was reduced from 343 eV to 36 eV by the means of trim files. An electronic noise of 447 eV has been measured. The PILATUS modules are suitable for various X-ray applications such as diffraction and imaging techniques.
2350192
Fourier shell correlation threshold criteria.
The resolution value claimed for an electron microscopical three-dimensional reconstruction indicates the overall quality of the experiment. The Fourier shell correlation (FSC) criterion has now become the standard quality measure. However, what has continued to be controversial is the issue of the FSC threshold level at which one defines the reproducible resolution. Here, we discuss the theoretical behaviour of the FSC in conjunction with the various factors which influence it: the number of "voxels" in a given Fourier shell, the symmetry of the structure, and the size of the structure within the reconstruction volume. Both the theoretical considerations and our model experiments show that fixed-valued FSC threshold (like "0.5") may never be used in a reproducible criterion. Fixed threshold values are-as we show here-simply the result of incorrect assumptions in the basic statistics. Two families of FSC threshold curves are discussed: the sigma-factor curves and the new family of bit-based information threshold curves. Whereas sigma-factor curves indicate the resolution level at which one has collected information significantly above the noise level, the information curves indicate the resolution level at which enough information has been collected for interpretation.
15298267
3D Ultrastructural Organization of Whole Chlamydomonas reinhardtii Cells Studied by Nanoscale Soft X-Ray Tomography
The complex architecture of their structural elements and compartments is a hallmark of eukaryotic cells. The creation of high resolution models of whole cells has been limited by the relatively low resolution of conventional light microscopes and the requirement for ultrathin sections in transmission electron microscopy. We used soft x-ray tomography to study the 3D ultrastructural organization of whole cells of the unicellular green alga Chlamydomonas reinhardtii at unprecedented spatial resolution. Intact frozen hydrated cells were imaged using the natural x-ray absorption contrast of the sample without any staining. We applied different fiducial-based and fiducial-less alignment procedures for the 3D reconstructions. The reconstructed 3D volumes of the cells show features down to 30 nm in size. The whole cell tomograms reveal ultrastructural details such as nuclear envelope membranes, thylakoids, basal apparatus, and flagellar microtubule doublets. In addition, the x-ray tomograms provide quantitative data from the cell architecture. Therefore, nanoscale soft x-ray tomography is a new valuable tool for numerous qualitative and quantitative applications in plant cell biology.
4010395
X-ray ptychographic and fluorescence microscopy of frozen-hydrated cells using continuous scanning
X-ray microscopy can be used to image whole, unsectioned cells in their native hydrated state. It complements the higher resolution of electron microscopy for submicrometer thick specimens, and the molecule-specific imaging capabilites of fluorescence light microscopy. We describe here the first use of fast, continuous x-ray scanning of frozen hydrated cells for simultaneous sub-20 nm resolution ptychographic transmission imaging with high contrast, and sub-100 nm resolution deconvolved x-ray fluorescence imaging of diffusible and bound ions at native concentrations, without the need to add specific labels. By working with cells that have been rapidly frozen without the use of chemical fixatives, and imaging them under cryogenic conditions, we are able to obtain images with well preserved structural and chemical composition, and sufficient stability against radiation damage to allow for multiple images to be obtained with no observable change.