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Tofu is a toolkit for processing large amounts of images and for tomographic reconstruction. Complex image processing tasks are organized as workflows of individual processing steps. The toolkit is able to reconstruct parallel and cone beam as well as tomographic and laminographic geometries. Many pre‐ and post‐processing algorithms needed for high‐quality 3D reconstruction are available, e.g. phase retrieval, ring removal and de‐noising. Tofu is optimized for stand‐alone GPU workstations on which it achieves reconstruction speed comparable with costly CPU clusters. It automatically utilizes all GPUs in the system and generates 3D reconstruction code with minimal number of instructions given the input geometry (parallel/cone beam, tomography/laminography), hence yielding optimal run‐time performance. In order to improve accessibility for researchers with no previous knowledge of programming, tofu contains graphical user interfaces for both optimization of 3D reconstruction parameters and batch processing of data with pre‐configured workflows for typical computed tomography reconstruction. The toolkit is open source and extensive documentation is available for both end‐users and developers. Thanks to the mentioned features, tofu is suitable for both expert users with specialized image processing needs (e.g. when dealing with data from custom‐built computed tomography scanners) and for application‐specific end‐users who just need to reconstruct their data on off‐the‐shelf hardware. The versatile and user‐friendly image processing toolkit tofu, optimized for 3D reconstruction of parallel beam, cone beam, tomography and laminography data, is presented.

We present Biomedisa, a free and easy-to-use open-source online platform developed for semi-automatic segmentation of large volumetric images. The segmentation is based on a smart interpolation of sparsely pre-segmented slices taking into account the complete underlying image data. Biomedisa is particularly valuable when little a priori knowledge is available, e.g. for the dense annotation of the training data for a deep neural network. The platform is accessible through a web browser and requires no complex and tedious configuration of software and model parameters, thus addressing the needs of scientists without substantial computational expertise. We demonstrate that Biomedisa can drastically reduce both the time and human effort required to segment large images. It achieves a significant improvement over the conventional approach of densely pre-segmented slices with subsequent morphological interpolation as well as compared to segmentation tools that also consider the underlying image data. Biomedisa can be used for different 3D imaging modalities and various biomedical applications. Manual segmentation of biological images is a time-consuming task. Here the authors present Biomedisa, an open-source online platform for segmentation of large volumetric images starting from sparsely presegmented slices.

X-ray phase-contrast microtomography (XPCμT) is a label-free, high-resolution imaging modality for analyzing early development of vertebrate embryos in vivo by using time-lapse sequences of 3D volumes. Here we provide a detailed protocol for applying this technique to study gastrulation in Xenopus laevis (African clawed frog) embryos. In contrast to μMRI, XPCμT images optically opaque embryos with subminute temporal and micrometer-range spatial resolution. We describe sample preparation, culture and suspension of embryos, tomographic imaging with a typical duration of 2 h (gastrulation and neurulation stages), intricacies of image pre-processing, phase retrieval, tomographic reconstruction, segmentation and motion analysis. Moreover, we briefly discuss our present understanding of X-ray dose effects (heat load and radiolysis), and we outline how to optimize the experimental configuration with respect to X-ray energy, photon flux density, sample-detector distance, exposure time per tomographic projection, numbers of projections and time-lapse intervals. The protocol requires an interdisciplinary effort of developmental biologists for sample preparation and data interpretation, X-ray physicists for planning and performing the experiment and applied mathematicians/computer scientists/physicists for data processing and analysis. Sample preparation requires 9–48 h, depending on the stage of development to be studied. Data acquisition takes 2–3 h per tomographic time-lapse sequence. Data processing and analysis requires a further 2 weeks, depending on the availability of computing power and the amount of detail required to address a given scientific problem.

Pulsed-laser assisted nanoparticle synthesis in liquids (PLAL) is a versatile tool for nanoparticle synthesis. However, fundamental aspects of structure formation during PLAL are presently poorly understood. We analyse the spatio-temporal kinetics during PLAL by means of fast X-ray radiography (XR) and scanning small-angle X-ray scattering (SAXS), which permits us to probe the process on length scales from nanometers to millimeters with microsecond temporal resolution. We find that the global structural evolution, such as the dynamics of the vapor bubble can be correlated to the locus and evolution of silver nanoparticles. The bubble plays an important role in particle formation, as it confines the primary particles and redeposits them to the substrate. Agglomeration takes place for the confined particles in the second bubble. Additionally, upon the collapse of the second bubble a jet of confined material is ejected perpendicularly to the surface. We hypothesize that these kinetics influence the final particle size distribution and determine the quality of the resulting colloids, such as polydispersity and modality through the interplay between particle cloud compression and particle release into the liquid.

Scientific cinematography using ultrafast optical imaging is a common tool to study motion. In opaque organisms or structures, X-ray radiography captures sequences of 2D projections to visualize morphological dynamics, but for many applications full four-dimensional (4D) spatiotemporal information is highly desirable. We introduce in vivo X-ray cine-tomography as a 4D imaging technique developed to study real-time dynamics in small living organisms with micrometer spatial resolution and sub-second time resolution. The method enables insights into the physiology of small animals by tracking the 4D morphological dynamics of minute anatomical features as demonstrated in this work by the analysis of fast-moving screw-and-nut—type weevil hip joints. The presented method can be applied to a broad range of biological specimens and biotechnological processes.

Vertebrate models provide indispensable paradigms to study development and disease. Their analysis requires a quantitative morphometric study of the body, organs and tissues. This is often impeded by pigmentation and sample size. X-ray micro-computed tomography (micro-CT) allows high-resolution volumetric tissue analysis, largely independent of sample size and transparency to visual light. Importantly, micro-CT data are inherently quantitative. We report a complete pipeline of high-throughput 3D data acquisition and image analysis, including tissue preparation and contrast enhancement for micro-CT imaging down to cellular resolution, automated data processing and organ or tissue segmentation that is applicable to comparative 3D morphometrics of small vertebrates. Applied to medaka fish, we first create an annotated anatomical atlas of the entire body, including inner organs as a quantitative morphological description of an adult individual. This atlas serves as a reference model for comparative studies. Using isogenic medaka strains we show that comparative 3D morphometrics of individuals permits identification of quantitative strain-specific traits. Thus, our pipeline enables high resolution morphological analysis as a basis for genotype-phenotype association studies of complex genetic traits in vertebrates.

Atomistic processes during pulsed-laser deposition (PLD) growth influence the physical properties of the resulting films. We investigated the PLD of epitaxial layers of hexagonal LuFeO 3 by measuring the X-ray diffraction intensity in the quasiforbidden reflection 0003 in situ during deposition. From measured X-ray diffraction intensities we determined coverages of each layer and studied their time evolution which is described by scaling exponent β directly connected to the surface roughness. Subsequently we modelled the growth using kinetic Monte Carlo simulations. While the experimentally obtained scaling exponent β decreases with the laser frequency, the simulations provided the opposite behaviour. We demonstrate that the increase of the surface temperature caused by impinging ablated particles satisfactorily explains the recorded decrease in the scaling exponent with the laser frequency. This phenomena is often overlooked during the PLD growth.

Opaque tissues provide a challenge for live imaging of Xenopus laevis development; a problem solved by in vivo time-lapse X-ray microtomography that is shown to provide a high-resolution three-dimensional view of structural changes and dynamics of gastrulation, and that is applied to identify and analyse new aspects of gastrulation in frog embryos. New dimensions in vertebrate embryo microtomography Xenopus laevis – the South African clawed frog – is an important model organism, and much of our understanding of the vertebrate embryology derives from this system. But the study of gastrulation, the stage at which the embryo has formed three layers arranged around a central cavity, has been hampered by the lack of high-quality live-imaging methods useable on intact Xenopus embryos, which are opaque at early stages. Ralf Hofmann, Jubin Kashef and colleagues have developed a non-invasive in vivo time-lapse phase-contrast X-ray microtomography technique that allows the observation of gastrulation. By analysing individual cell trajectories, collective tissue motion and the evolution of morphological features, the authors visualize known gastrulation movements and reveal the formation of a structure not reported on previously. This new '4D' technique should be applicable in the fields of genetics, molecular and developmental biology and medicine. An ambitious goal in biology is to understand the behaviour of cells during development by imaging— in vivo and with subcellular resolution—changes of the embryonic structure. Important morphogenetic movements occur throughout embryogenesis, but in particular during gastrulation when a series of dramatic, coordinated cell movements drives the reorganization of a simple ball or sheet of cells into a complex multi-layered organism 1 . In Xenopus laevis , the South African clawed frog and also in zebrafish, cell and tissue movements have been studied in explants 2 , 3 , in fixed embryos 4 , in vivo using fluorescence microscopy 5 , 6 or microscopic magnetic resonance imaging 7 . None of these methods allows cell behaviours to be observed with micrometre-scale resolution throughout the optically opaque, living embryo over developmental time. Here we use non-invasive in vivo , time-lapse X-ray microtomography, based on single-distance phase contrast and combined with motion analysis, to examine the course of embryonic development. We demonstrate that this powerful four-dimensional imaging technique provides high-resolution views of gastrulation processes in wild-type X. laevis embryos, including vegetal endoderm rotation, archenteron formation, changes in the volumes of cavities within the porous interstitial tissue between archenteron and blastocoel, migration/confrontation of mesendoderm and closure of the blastopore. Differential flow analysis separates collective from relative cell motion to assign propulsion mechanisms. Moreover, digitally determined volume balances confirm that early archenteron inflation occurs through the uptake of external water. A transient ectodermal ridge, formed in association with the confrontation of ventral and head mesendoderm on the blastocoel roof, is identified. When combined with perturbation experiments to investigate molecular and biomechanical underpinnings of morphogenesis, our technique should help to advance our understanding of the fundamentals of development.

Hierarchical guidance is developed for three-dimensional (3D) nanoscale X-ray imaging, enabling identification, refinement, and tracking of regions of interest (ROIs) within specimens considerably exceeding the field of view. This opens up new possibilities for in situ investigations. Experimentally, the approach takes advantage of rapid multiscale measurements based on magnified projection microscopy featuring continuous zoom capabilities. Immediate and continuous feedback on the subsequent experimental progress is enabled by suitable on-the-fly data processing. For this, by theoretical justification and experimental validation, so-called quasi-particle phase-retrieval is generalised to conical-beam conditions, being key for sufficiently fast computation without significant loss of imaging quality and resolution compared to common approaches for holographic microscopy. Exploiting 3D laminography, particularly suited for imaging of ROIs in laterally extended plate-like samples, the potential of hierarchical guidance is demonstrated by the in situ investigation of damage nucleation inside alloy sheets under engineering-relevant boundary conditions, providing novel insight into the nanoscale morphological development of void and particle clusters under mechanical load. Combined with digital volume correlation, we study deformation kinematics with unprecedented spatial resolution. Correlation of mesoscale (i.e. strain fields) and nanoscale (i.e. particle cracking) evolution opens new routes for the understanding of damage nucleation within sheet materials with application-relevant dimensions.

In this investigation, SnAgCu and SN100C solders were electromigration (EM) tested, and the 3D laminography imaging technique was employed for in-situ observation of the microstructure evolution during testing. We found that discrete voids nucleate, grow and coalesce along the intermetallic compound/solder interface during EM testing. A systematic analysis yields quantitative information on the number, volume, and growth rate of voids, and the EM parameter of DZ*. We observe that fast intrinsic diffusion in SnAgCu solder causes void growth and coalescence, while in the SN100C solder this coalescence was not significant. To deduce the current density distribution, finite-element models were constructed on the basis of the laminography images. The discrete voids do not change the global current density distribution, but they induce the local current crowding around the voids: this local current crowding enhances the lateral void growth and coalescence. The correlation between the current density and the probability of void formation indicates that a threshold current density exists for the activation of void formation. There is a significant increase in the probability of void formation when the current density exceeds half of the maximum value.