Tubulin participates in establishing protoxylem vessel reinforcement patterns and hydraulic conductivity in maize.

Water transportation to developing tissues relies on the structure and function of plant xylem cells. Plant microtubules govern the direction of cellulose microfibrils and guide secondary cell wall formation and morphogenesis. However, the relevance of microtubule-determined xylem wall thickening patterns in plant hydraulic conductivity remains unclear. In the present study, we identified a maize (Zea mays) semi-dominant mutant, designated drought-overly-sensitive1 (ZmDos1), the upper leaves of which wilted even when exposed to well-watered conditions during growth; the wilting phenotype was aggravated by increased temperatures and decreased humidity. Protoxylem vessels in the stem and leaves of the mutant showed altered thickening patterns of the secondary cell wall (from annular to spiral), decreased inner diameters, and limited water transport efficiency. The causal mutation for this phenotype was found to be a G-to-A mutation in the maize gene α-tubulin4, resulting in a single amino acid substitution at position 196 (E196K). Ectopic expression of the mutant α-tubulin4 in Arabidopsis (Arabidopsis thaliana) changed the orientation of microtubule arrays, suggesting a determinant role of this gene in microtubule assembly and secondary cell wall thickening. Our findings suggest that the spiral wall thickenings triggered by the α-tubulin mutation are stretched during organ elongation, causing a smaller inner diameter of the protoxylem vessels and affecting water transport in maize. This study underscores the importance of tubulin-mediated protoxylem wall thickening in regulating plant hydraulics, improves our understanding of the relationships between protoxylem structural features and functions, and offers candidate genes for the genetic enhancement of maize.

Full-field hard X-ray nano-tomography at SSRF.

An in-house designed transmission X-ray microscopy (TXM) instrument has been developed and commissioned at beamline BL18B of the Shanghai Synchrotron Radiation Facility (SSRF). BL18B is a hard (5-14 keV) X-ray bending-magnet beamline recently built with sub-20 nm spatial resolution in TXM. There are two kinds of resolution mode: one based on using a high-resolution-based scintillator-lens-coupled camera, and the other on using a medium-resolution-based X-ray sCMOS camera. Here, a demonstration of full-field hard X-ray nano-tomography for high-Z material samples (e.g. Au particles, battery particles) and low-Z material samples (e.g. SiO2 powders) is presented for both resolution modes. Sub-50 nm to 100 nm resolution in three dimensions (3D) has been achieved. These results represent the ability of 3D non-destructive characterization with nano-scale spatial resolution for scientific applications in many research fields.

Real-time X-ray imaging of mouse cerebral microvessels in vivo using a pixel temporal averaging method.

Rodents are used extensively as animal models for the preclinical investigation of microvascular-related diseases. However, motion artifacts in currently available imaging methods preclude real-time observation of microvessels in vivo. In this paper, a pixel temporal averaging (PTA) method that enables real-time imaging of microvessels in the mouse brain in vivo is described. Experiments using live mice demonstrated that PTA efficiently eliminated motion artifacts and random noise, resulting in significant improvements in contrast-to-noise ratio. The time needed for image reconstruction using PTA with a normal computer was 250 ms, highlighting the capability of the PTA method for real-time angiography. In addition, experiments with less than one-quarter of photon flux in conventional angiography verified that motion artifacts and random noise were suppressed and microvessels were successfully identified using PTA, whereas conventional temporal subtraction and averaging methods were ineffective. Experiments performed with an X-ray tube verified that the PTA method could also be successfully applied to microvessel imaging of the mouse brain using a laboratory X-ray source. In conclusion, the proposed PTA method may facilitate the real-time investigation of cerebral microvascular-related diseases using small animal models.

Comprehensive characterization of TSV etching performance with phase-contrast X-ray microtomography.

Comprehensive evaluation of through-silicon via (TSV) reliability often requires deterministic and 3D descriptions of local morphological and statistical features of via formation with the Bosch process. Here, a highly sensitive phase-contrast X-ray microtomography approach is presented based on recorrection of abnormal projections, which provides comprehensive and quantitative characterization of TSV etching performance. The key idea is to replace the abnormal projections at specific angles in principles of linear interpolation of neighboring projections, and to distinguish the interface between silicon and air by using phase-retrieval algorithms. It is demonstrated that such a scheme achieves high accuracy in obtaining the etch profile based on the 3D microstructure of the vias, including diameter, bottom curvature radius, depth and sidewall angle. More importantly, the 3D profile error of the via sidewall and the consistency of parameters among all the vias are achieved and analyzed statistically. The datasets in the results and the 3D microstructure can be applied directly to a reference and model for further finite element analysis. This method is general and has potentially broad applications in 3D integrated circuits.

High-efficiency fast X-ray imaging detector development at SSRF.

Indirect X-ray imaging detectors consisting of scintillator screens, long-working-distance microscope lenses and scientific high-speed complementary metal-oxide semiconductor (CMOS) cameras are usually used to realize fast X-ray imaging with white-beam synchrotron radiation. However, the detector efficiency is limited by the coupling efficiency of the long-working-distance microscope lenses, which is only about 5%. A long-working-distance microscope lenses system with a large numerical aperture (NA) is designed to increase the coupling efficiency. It offers an NA of 0.5 at 8× magnification. The Mitutoyo long-working-distance microscope lenses system offers an NA of 0.21 at 7.5× magnification. Compared with the Mitutoyo system, the developed long-working-distance microscope lenses system offers about twice the NA and four times the coupling efficiency. In the indirect X-ray imaging detector, a 50 µm-thick LuAG:Ce scintillator matching with the NA, and a high-speed visible-light CMOS FastCAM SAZ Photron camera are used. Test results show that the detector realized fast X-ray imaging with a frame rate of 100000 frames s-1 and fast X-ray microtomography with a temporal sampling rate up to 25 Hz (25 tomograms s-1).

Synchrotron radiation micro-tomography for high-resolution neurovascular network morphology investigation.

There has been increasing interest in using high-resolution micro-tomography to investigate the morphology of neurovascular networks in the central nervous system, which remain difficult to characterize due to their microscopic size as well as their delicate and complex 3D structure. Synchrotron radiation X-ray imaging, which has emerged as a cutting-edge imaging technology with a high spatial resolution, provides a novel platform for the non-destructive imaging of microvasculature networks at a sub-micrometre scale. When coupled with computed tomography, this technique allows the characterization of the 3D morphology of vasculature. The current review focuses on recent progress in developing synchrotron radiation methodology and its application in probing neurovascular networks, especially the pathological changes associated with vascular abnormalities in various model systems. Furthermore, this tool represents a powerful imaging modality that improves our understanding of the complex biological interactions between vascular function and neuronal activity in both physiological and pathological states.

Origin of Noncubic Scaling Law in Disordered Granular Packing.

Recent diffraction experiments on metallic glasses have unveiled an unexpected noncubic scaling law between density and average interatomic distance, which led to the speculation of the presence of fractal glass order. Using x-ray tomography we identify here a similar noncubic scaling law in disordered granular packing of spherical particles. We find that the scaling law is directly related to the contact neighbors within the first nearest neighbor shell, and, therefore, is closely connected to the phenomenon of jamming. The seemingly universal scaling exponent around 2.5 arises due to the isostatic condition with a contact number around 6, and we argue that the exponent should not be universal.

Improving the efficiency of small-angle x-ray scattering computed tomography using the OSEM algorithm.

Small-angle x-ray scattering computed tomography (SAXS-CT) is a nondestructive method for the nanostructure analysis of heterogeneous materials. However, the limits of a long data acquisition time and vast amounts of data prevent SAXS-CT from becoming a routine experimental method in the applications of synchrotron radiation. In this study, the ordered subsets expectation maximization (OSEM) algorithm is introduced to improve the efficiency of SAXS-CT. To demonstrate the practicability of this method, a systematic simulation and experiments were carried out. The simulation results on a numerical phantom show that the OSEM-based SAXS-CT can effectively eliminate streaking artifacts and improve the efficiency of data acquisition by at least 3 times compared with the filter backprojection algorithm. By compromising the reconstruction speed and image quality, the optimal reconstruction parameters are also given for the image reconstruction in the OSEM-based SAXS-CT experiments. An experiment on a bamboo sample verified the validity of the proposed method with limited projection data. A further experiment on polyethylene demonstrated that the OSEM-based SAXS-CT is able to reveal the local nanoscale information about the crystalline structure and distributional difference inside the sample. In conclusion, the OSEM-based SAXS-CT can significantly improve experimental efficiency, which may promote SAXS-CT becoming a conventional method.

Monochromatic-beam-based dynamic X-ray microtomography based on OSEM-TV algorithm.

Monochromatic-beam-based dynamic X-ray computed microtomography (CT) was developed to observe evolution of microstructure inside samples. However, the low flux density results in low efficiency in data collection. To increase efficiency, reducing the number of projections should be a practical solution. However, it has disadvantages of low image reconstruction quality using the traditional filtered back projection (FBP) algorithm. In this study, an iterative reconstruction method using an ordered subset expectation maximization-total variation (OSEM-TV) algorithm was employed to address and solve this problem. The simulated results demonstrated that normalized mean square error of the image slices reconstructed by the OSEM-TV algorithm was about 1/4 of that by FBP. Experimental results also demonstrated that the density resolution of OSEM-TV was high enough to resolve different materials with the number of projections less than 100. As a result, with the introduction of OSEM-TV, the monochromatic-beam-based dynamic X-ray microtomography is potentially practicable for the quantitative and non-destructive analysis to the evolution of microstructure with acceptable efficiency in data collection and reconstructed image quality.

A robust method for high-precision quantification of the complex three-dimensional vasculatures acquired by X-ray microtomography.

The quantification of micro-vasculatures is important for the analysis of angiogenesis on which the detection of tumor growth or hepatic fibrosis depends. Synchrotron-based X-ray computed micro-tomography (SR-µCT) allows rapid acquisition of micro-vasculature images at micrometer-scale spatial resolution. Through skeletonization, the statistical features of the micro-vasculature can be extracted from the skeleton of the micro-vasculatures. Thinning is a widely used algorithm to produce the vascular skeleton in medical research. Existing three-dimensional thinning methods normally emphasize the preservation of topological structure rather than geometrical features in generating the skeleton of a volumetric object. This results in three problems and limits the accuracy of the quantitative results related to the geometrical structure of the vasculature. The problems include the excessively shortened length of elongated objects, eliminated branches of blood vessel tree structure, and numerous noisy spurious branches. The inaccuracy of the skeleton directly introduces errors in the quantitative analysis, especially on the parameters concerning the vascular length and the counts of vessel segments and branching points. In this paper, a robust method using a consolidated end-point constraint for thinning, which generates geometry-preserving skeletons in addition to maintaining the topology of the vasculature, is presented. The improved skeleton can be used to produce more accurate quantitative results. Experimental results from high-resolution SR-µCT images show that the end-point constraint produced by the proposed method can significantly improve the accuracy of the skeleton obtained using the existing ITK three-dimensional thinning filter. The produced skeleton has laid the groundwork for accurate quantification of the angiogenesis. This is critical for the early detection of tumors and assessing anti-angiogenesis treatments.

Fourier-Transform Ghost Imaging with Hard X Rays.

Knowledge gained through x-ray crystallography fostered structural determination of materials and greatly facilitated the development of modern science and technology in the past century. However, it is only applied to crystalline structures and cannot resolve noncrystalline materials. Here we demonstrate a novel lensless Fourier-transform ghost imaging method with pseudothermal hard x rays that extends x-ray crystallography to noncrystalline samples. By measuring the second-order intensity correlation function of the light, Fourier-transform diffraction pattern of a complex amplitude sample is achieved at the Fresnel region in our experiment and the amplitude and phase distributions of the sample in the spatial domain are retrieved successfully. For the first time, ghost imaging is experimentally realized with x rays. Since a highly coherent x-ray source is not required, the method can be implemented with laboratory x-ray sources and it also provides a potential solution for lensless diffraction imaging with fermions, such as neutrons and electrons where intensive coherent sources usually are not available.

X-ray propagation-based equally sloped tomography for mouse brain.

BACKGROUND: The outstanding functional importance of the brain implies a strong need for brain imaging modalities. However, the current imaging approaches that target the brain in rodents remain suboptimal. OBJECTIVE AND METHODS: In this paper, X-ray propagation-based phase contrast imaging combined with equally sloped tomography (PPCI-EST) was employed to nondestructively investigate the mouse brain. RESULTS: The grey and white matters, which have extremely small differences in electron density, were clearly discriminated. The fine structures, including the corpus callosum (cc), the optic chiasma (ox) and the caudate putamen (CPu), were revealed. Compared to the filtered back projection reconstruction, the PPCI-EST significantly reduce projection number while maintaining sufficient image quality. CONCLUSIONS: It could be a potential tool for fast and low-dose phase-contrast imaging to biomedical specimens.

3D elemental sensitive imaging by full-field XFCT.

X-ray fluorescence computed tomography (XFCT) is a stimulated emission tomography modality that maps the three-dimensional (3D) distribution of elements. Generally, XFCT is done by scanning a pencil-beam across the sample. This paper presents a feasibility study of full-field XFCT (FF-XFCT) for 3D elemental imaging. The FF-XFCT consists of a pinhole collimator and X-ray imaging detector with no energy resolution. A prototype imaging system was set up at the Shanghai Synchrotron Radiation Facility (SSRF) for imaging the phantom. The first FF-XFCT experimental results are presented. The cadmium (Cd) and iodine (I) distributions were reconstructed. The results demonstrate FF-XFCT is fit for 3D elemental imaging and the sensitivity of FF-XFCT is higher than a conventional CT system.

Optimization of reconstructed quality of hard x-ray phase microtomography.

For applications of hard x-ray propagation-based phase-contrast computed microtomography (PPCT) in high-resolution biological research, high spatial resolution and high contrast-to-noise ratio are simultaneously required for tiny structural discrimination and characterization. Most existing micro-CT techniques to improve image quality are limited by high cost, physical limitations, and complexity of the experimental hardware and setup. In this work, a novel PPCT technique, which combines a wavelet-transform-based modulation transform function compensation algorithm and a generalized phase-retrieval algorithm, is proposed to optimize the reconstruction quality of tomographic slices. Our experimental results, which compared the spatial resolutions and contrast-to-noise ratios of reconstructed images, demonstrated the validity of the proposed generalized PPCT technique. The experimental results showed that the proposed generalized PPCT technique is superior to the direct PPCT and the linearized phase-retrieval PPCT techniques. This novel PPCT technique demonstrates great potential for biological imaging, especially for applications that require high spatial resolution and limit radiation exposure.

Simulation and experimental study of aspect ratio limitation in Fresnel zone plates for hard-x-ray optics.

For acquiring high-contrast and high-brightness images in hard-x-ray optics, Fresnel zone plates with high aspect ratios (zone height/zone width) have been constantly pursued. However, knowledge of aspect ratio limits remains limited. This work explores the achievable aspect ratio limit in polymethyl methacrylate (PMMA) by electron-beam lithography (EBL) under 100 keV, and investigates the lithographic factors for this limitation. Both Monte Carlo simulation and EBL on thick PMMA are applied to investigate the profile evolution with exposure doses over 100 nm wide dense zones. A high-resolution scanning electron microscope at low acceleration mode for charging free is applied to characterize the resultant zone profiles. It was discovered for what we believe is the first time that the primary electron-beam spreading in PMMA and the proximity effect due to extra exposure from neighboring areas could be the major causes of limiting the aspect ratio. Using the optimized lithography condition, a 100 nm zone plate with aspect ratio of 15/1 was fabricated and its focusing property was characterized at the Shanghai Synchrotron Radiation Facility. The aspect ratio limit found in this work should be extremely useful for guiding further technical development in nanofabrication of high-quality Fresnel zone plates.

Towards automated quantitative vasculature understanding via ultra high-resolution imagery.

This chapter presents an approach to processing ultra high-resolution, large-size biomedical imaging data for the purposes of detecting and quantifying vasculature and microvasculature . Capturing early signs of any changes in vasculature may have significant values for early-diagnosis and treatment assessment due to the well understood observation that vascular changes precede cancerous growth and metastasis metastasis . With the advent of key enabling technologies for extremely high-resolution imaging, such as synchrotron radiation synchrotron radiation based computed tomography (CT) computed tomography , the required levels of detail have become accessible. However, these technologies also present challenges in data analysis. This chapter aims to offer some insights as to how these changes might be best dealt with. We argue that the necessary steps in quantitative understanding of vasculatures include targeted data enhancement enhancement , information reduction aimed at characterizing the linear structure of vessels vessels , and quantitatively describing the vessel hierarchy. We present results on cerebral and liver vasculatures of a mouse captured at the Shanghai Synchrotron Radiation Facility (SSRF). These results were achieved with a processing pipeline comprising of our empirically selected component for each of the above steps. Towards the end, we discuss how alternative and additional components may be incorporated for improved speed and robustness.

[An effective method for improving the imaging spatial resolution of terahertz time domain spectroscopy system].

Terahertz radiation is an electromagnetic radiation in the range between millimeter waves and far infrared. Due to its low energy and non-ionizing characters, THz pulse imaging emerges as a novel tool in many fields, such as material, chemical, biological medicine, and food safety. Limited spatial resolution is a significant restricting factor of terahertz imaging technology. Near field imaging method was proposed to improve the spatial resolution of terahertz system. Submillimeter scale's spauial resolution can be achieved if the income source size is smaller than the wawelength of the incoming source and the source is very close to the sample. But many changes were needed to the traditional terahertz time domain spectroscopy system, and it's very complex to analyze sample's physical parameters through the terahertz signal. A method of inserting a pinhole upstream to the sample was first proposed in this article to improve the spatial resolution of traditional terahertz time domain spectroscopy system. The measured spatial resolution of terahertz time domain spectroscopy system by knife edge method can achieve spatial resolution curves. The moving stage distance between 10 % and 90 Yo of the maximum signals respectively was defined as the, spatial resolution of the system. Imaging spatial resolution of traditional terahertz time domain spectroscopy system was improved dramatically after inserted a pinhole with diameter 0. 5 mm, 2 mm upstream to the sample. Experimental results show that the spatial resolution has been improved from 1. 276 mm to 0. 774 mm, with the increment about 39 %. Though this simple method, the spatial resolution of traditional terahertz time domain spectroscopy system was increased from millimeter scale to submillimeter scale. A pinhole with diameter 1 mm on a polyethylene plate was taken as sample, to terahertz imaging study. The traditional terahertz time domain spectroscopy system and pinhole inserted terahertz time domain spectroscopy system were applied in the imaging experiment respectively. The relative THz-power loss imaging of samples were use in this article. This method generally delivers the best signal to noise ratio in loss images, dispersion effects are cancelled. Terahertz imaging results show that the sample's boundary was more distinct after inserting the pinhole in front of, sample. The results also conform that inserting pinhole in front of sample can improve the imaging spatial resolution effectively. The theoretical analyses of the method which improve the spatial resolution by inserting a pinhole in front of sample were given in this article. The analyses also indicate that the smaller the pinhole size, the longer spatial coherence length of the system, the better spatial resolution of the system. At the same time the terahertz signal will be reduced accordingly. All the experimental results and theoretical analyses indicate that the method of inserting a pinhole in front of sample can improve the spatial resolution of traditional terahertz time domain spectroscopy system effectively, and it will further expand the application of terahertz imaging technology.

[Study of Terahertz Amplitude Imaging Based on the Mean Absorption].

A new method of terahertz (THz) imaging based on the mean absorption is proposed. Terahertz radiation is an electromagnetic radiation in the range between millimeter waves and far infrared. THz pulse imaging emerges as a novel tool in many fields because of its low energy and non-ionizing character, such as material, chemical, biological medicine and food safety. A character of THz imaging technique is it can get large amount of information. How to extract the useful parameter from the large amount of information and reconstruct sample's image is a key technology in THz imaging. Some efforts have been done for advanced visualization methods to extract the information of interest from the raw data. Both time domain and frequency domain visualization methods can be applied to extract information on the physical properties of samples from THz imaging raw data. The process of extracting useful parameter from raw data of the new method based on the mean absorption was given in this article. This method relates to the sample absorption and thickness, it delivers good signal to noise ratio in the images, and the dispersion effects are cancelled. A paper with a "THz" shape hole was taken as the sample to do the experiment. Traditional THz amplitude imaging methods in time domain and frequency domain are used to achieve the sample's image, such as relative reduction of pulse maximum imaging method, relative power loss imaging method, and relative power loss at specific frequency imaging method. The sample's information that reflected by these methods and the characteristics of these methods are discussed. The method base on the mean absorption within a certain frequency is also used to reconstruct sample's image. The experimental results show that this new method can well reflect the true information of the sample. And it can achieve a clearer image than the other traditional THz amplitude imaging methods. All the experimental results and theoretical analyses indicate that the method base on the mean absorption within a certain frequency can reflects sample absorb and thickness information, it can achieve good signal to noise ratio in the images. Because the absorption is mean absorption within in a certain frequency, so the method proposed in this article is especially suitable for samples with simple structure. And this new method can be a useful added tool for the other traditional THz amplitude imaging methods.

3D online submicron scale observation of mixed metal powder's microstructure evolution in high temperature and microwave compound fields.

In order to study the influence on the mechanical properties caused by microstructure evolution of metal powder in extreme environment, 3D real-time observation of the microstructure evolution of Al-Ti mixed powder in high temperature and microwave compound fields was realized by using synchrotron radiation computerized topography (SR-CT) technique; the spatial resolution was enhanced to 0.37 µm/pixel through the designed equipment and the introduction of excellent reconstruction method for the first time. The process of microstructure evolution during sintering was clearly distinguished from 2D and 3D reconstructed images. Typical sintering parameters such as sintering neck size, porosity, and particle size of the sample were presented for quantitative analysis of the influence on the mechanical properties and the sintering kinetics during microwave sintering. The neck size-time curve was obtained and the neck growth exponent was 7.3, which indicated that surface diffusion was the main diffusion mechanism; the reason was the eddy current loss induced by the external microwave fields providing an additional driving force for mass diffusion on the particle surface. From the reconstructed images and the curve of porosity and average particle size versus temperature, it was believed that the presence of liquid phase aluminum accelerated the densification and particle growth.

In situ investigation of the 3D mechanical microstructure at nanoscale: nano-CT imaging method of local small region in large scale sample.

To investigate the local micro-/nanoscale region in a large scale sample, an image reconstruction method for nanometer computed tomography (nano-CT) was proposed in this paper. In the algorithm, wavelets were used to localize the filtered-backprojection (FBP) algorithm because of its space-frequency localization property. After the implementation of the algorithm, two simulation local reconstruction experiments were performed to confirm its effectiveness. Three evaluation criteria were used in the experiments to judge the quality of the reconstructed images. The experimental results showed that the algorithm proposed in this paper performed best because (1) the quality of its results had improved 20%-30% compared to the results of FBP and 10%-30% compared to the results of another wavelet algorithm; (2) the new algorithm was stable under different circumstances. Besides, an actual reconstruction experiment was performed using real projection data that had been collected in a CT experiment. Two-dimensional (2D) and three-dimensional (3D) images of the sample were reconstructed. The microstructure of the sample could be clearly observed in the reconstructed images. Since much attention has been directed towards the nano-CT technique to investigate the microstructure of materials, this new wavelet-based local tomography algorithm could be considered as a meaningful effort.