Alexey Ershov

X-ray phase-contrast in vivo microtomography probes new aspects of Xenopus gastrulation

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. In Xenopus laevis, the South African clawed frog and also in zebrafish, cell and tissue movements have been studied in explants, in fixed embryos, in vivo using fluorescence microscopy or microscopic magnetic resonance imaging. 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.

In vivo X-ray cine-tomography for tracking morphological dynamics

Significance X-ray microtomography is a well-established tool to study the three-dimensional morphology of static biological samples. To capture motion in living specimen in real time, movies of X-ray projections are frequently used. However, the resulting loss of information about the third spatial dimension has limited the applicability of such acquisition protocols. Now, by combining ultrafast X-ray microtomography and sophisticated motion analysis, we developed X-ray cine-tomography as a tool to visualize the internal dynamics of nontranslucent millimeter-sized samples in three-dimensional space. We demonstrate the technique by analyzing the fast-moving screw-and-nut–type hip joint inside a living weevil. The method may be applied to a wide range of samples and processes across materials and life sciences. 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 subsecond 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.

On the possibilities of hard X-ray imaging with high spatio-temporal resolution using polychromatic synchrotron radiation.

Time-resolved imaging with penetrating radiation has an outstanding scientific value but its realisation requires a high density of photons as well as corresponding fast X-ray image detection schemes. Bending magnets and insertion devices of third generation synchrotron light sources offer a polychromatic photon flux density which is high enough to perform hard X-ray imaging with a spatio-temporal resolution up to the μm-μs range. Existing indirect X-ray image detectors commonly used at synchrotron light sources can be adapted for fast image acquisition by employing CMOS-based digital high speed cameras already available on the market. Selected applications from life sciences and materials research underline the high potential of this high-speed hard X-ray microimaging approach.

Time-lapse X-ray phase-contrast microtomography for in vivo imaging and analysis of morphogenesis

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.

High-Speed X-ray Cineradiography for Analyzing Complex Kinematics in Living Insects

More than one hundred years ago, Lucien Bull showed, by means of his famous high-speed cinematographic movies of living species, the outstanding scientific value of time-resolved imaging, e.g. to understand the mechanisms behind insect flight [1]. For X-ray imaging, synchrotron light sources offer a photon beam that (i) propagates quasi-parallel, (ii) has fluxes that are higher by orders of magnitude than laboratory sources, and (iii) allows one to exploit more sophisticated contrast modalities. The use of synchrotron light is thus the next step in fast-imaging development: high-speed hard X-ray cineradiography employing phase contrast mechanisms [2,3]. Imaging with X-rays offers the chance to investigate complex kinematics of, for example, feeding and locomotion devices of animals, as the penetrating nature of the radiation reveals internal information.

Coalescence analysis for evolving foams via optical flow computation on projection image sequences

A novel image-processing procedure is proposed for the analysis of sequences of two-dimensional projection images. Sudden events like the merging of bubbles in an evolving foam can be detected and spatio-temporally located in a given projection image sequence. The procedure is based on optical flow computations extended by a forward-backward check for each time step. Compared with prior methods, efficient suppression of noise or false events is achieved owing to uniform foam motion, and the reliability of detection is thus increased. The applicability of the proposed procedure in combination with synchrotron radiography is illustrated by a series of characteristic studies of foams of different kind. First, the detection of single-bubble collapses in aqueous foams is considered. Second, a spatial distribution of coalescence events in metals foamed in casting molds is estimated. Finally, the structural stability of polymer foams containing admixed solid nanoparticles is examined.

syris: a flexible and efficient framework for X-ray imaging experiments simulation

An open-source framework for conducting a broad range of virtual X-ray imaging experiments, syris, is presented. The simulated wavefield created by a source propagates through an arbitrary number of objects until it reaches a detector. The objects in the light path and the source are time-dependent, which enables simulations of dynamic experiments, e.g. four-dimensional time-resolved tomography and laminography. The high-level interface of syris is written in Python and its modularity makes the framework very flexible. The computationally demanding parts behind this interface are implemented in OpenCL, which enables fast calculations on modern graphics processing units. The combination of flexibility and speed opens new possibilities for studying novel imaging methods and systematic search of optimal combinations of measurement conditions and data processing parameters. This can help to increase the success rates and efficiency of valuable synchrotron beam time. To demonstrate the capabilities of the framework, various experiments have been simulated and compared with real data. To show the use case of measurement and data processing parameter optimization based on simulation, a virtual counterpart of a high-speed radiography experiment was created and the simulated data were used to select a suitable motion estimation algorithm; one of its parameters was optimized in order to achieve the best motion estimation accuracy when applied on the real data. syris was also used to simulate tomographic data sets under various imaging conditions which impact the tomographic reconstruction accuracy, and it is shown how the accuracy may guide the selection of imaging conditions for particular use cases.

Imaging Fast Processes in Liquid Metal Foams and Semi-Solid Alloys Using Synchrotron Radioscopy with Spatio-Temporal Micro-Resolution

New X-ray sources of unmatched brilliance, like the superconducting undulator device at ESRF high-energy beamline ID15A, allow for micro-radioscopic investigations with time-resolution up to the micro-second range. Here we present first results of two recent in situ experiments: the visualization of semi-solid metal flow at an acquisition speed 500 frames/s (fps) and the collapse of pore walls in liquid metallic foams investigated at 40,000 fps. Both applications reveal important qualitative and quantitative facts about the dynamic processes in liquid and/or semi-solid metals which were inaccessible until now because of either the limited spatial and/or the limited time-resolution of conventional X-ray devices. Thus, semi-solid slurry is observed to break into small particle clusters when injected at high speed. The event of cell wall collapse in metal foams is found to take ~1–2 ms time, indicating that the dynamics of this system is inertia controlled.

Synchrotron-based radioscopy with spatio-temporal micro-resolution using hard X-rays

The use of highly intense synchrotron light sources allows the next step in the fast imaging development: the use of hard X-rays. Micro-radiography as an established method to image the internal structure of an object with micrometer resolution can be extended to study its temporal evolution as well. While direct converting pixel detectors are known which can acquire images with high frame rates here detectors are needed with higher spatial resolution which can stand the highly intense synchrotron photon flux. Our approach is based on indirect pixel detectors which are already known for micro-imaging at synchrotron light sources. We combine those with CMOS cameras in order to achieve frame rates of up to 40 000 images per second, thus progressing to micro-radioscopy. Potential applications are studies of living insects with moderate frame rates up to 250 images per second (4 ms exposure time), velocity fields within a semi-solid alloy during a thixo-casting process and ruptures of individual cell walls in a liquid metal foam imaged with up to 40 000 frames per second (25 μs exposure time).

Moderne 2-D und 3-D abbildende Röntgenverfahren mit Synchrotronstrahlung

Kurzfassung Mikrotomographie erlaubt die hochauflösende Abbildung verdeckter Mikrostrukturen und den Nachweis von Mikrorissen und Mikroporen in vielfältigen Bereichen, darunter die Untersuchung von mikroporösen Materialien und Fasernetzwerken, der Mikrostruktur biokompatibler Materialien (Implantate), der Entwicklung der Struktur von Metallgefügen u.v.m. Synchrotron-Laminographie erweitert das Anwendungsspektrum der 3-D-Methoden auf die zerstörungsfreie Bauteiluntersuchung ohne Probenentnahme bzw. Probenpräparation, und erweitert die hoch auflösende Inspektion z.B. in der Mikrosystemtechnik, der Paläontologie Archäologie etc. Ultraschnelle Radiographie, Computertomographie und Laminographie erlauben die In-situ-Untersuchung von Proben und Bauteilen in Echtzeit und unter betriebsnahen Bedingungen verschiedenster Probenumgebung. Phasenkontrast erweitert die Anwendungsbreite der 2-D und 3-D abbildenden Synchrotronverfahren auf leichte Materialien und führte die Methoden damit in die Leichtbautechnologien und die Lebenswissenschaften. Die Kombination der Techniken mit Röntgenoptiken treibt die Grenzen der Ortsauflösung in den Bereich weniger 10 nm.