Julian Moosmann

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.

Single-distance phase retrieval at large phase shifts.

For coherent X-ray imaging of pure phase objects we study the reliability of linear relations in phase-retrieval algorithms based on a single intensity map after free-space propagation. For large phase changes and/or large propagation distances we propose two venues of working beyond linearity: Projection onto an effective, linear and local model in Fourier space and expansion of intensity contrast in powers of object-detector distance. We apply both algorithms successfully to simulated data.

Nonlinear phase retrieval from single-distance radiograph

Phase contrast in the object plane of a phase object is retrieved from intensity contrast at a {\sl single} object-detector distance. Expanding intensity contrast and phase shift in the detector plane in powers of object-detector distance, phase retrieval is performed beyond the solution to the linearized transport-of-intensity equation. The expansion coefficients are determined by the entire paraxial wave equation. The Laplacian of the phase shift in the object plane thus is written as a local expression linear in the intensity contrast and nonlinear in the phase shift in the object plane. A perturbative approach to this expression is proposed and tested with simulated phantom data.

Criticality in single-distance phase retrieval

We investigate why in free-space propagation single-distance phase retrieval based on a modified contrast-transfer function of linearized Fresnel theory yields good results for moderately strong pure-phase objects. Upscaling phase-variations in the exit plane, the growth of maxima of the modulus of the Fourier transformed intensity contrast dominates the minima. Cutting out small regions around the latter thus keeps information loss due to nonlocal, nonlinear effects negligible. This quasiparticle approach breaks down at a critical upscaling where the positions of the minima start to move rapidly. We apply our results to X-ray data of an early-stage Xenopus (frog) embryo.

Phase contrast laminography based on Talbot interferometry

Synchrotron laminography is combined with Talbot grating interferometry to address weakly absorbing specimens. Integrating both methods into one set-up provides a powerful x-ray diagnostical technique for multiple contrast screening of macroscopically large flat specimen and a subsequent non-destructive three-dimensional (3-D) inspection of regions of interest. The technique simultaneously yields the reconstruction of the 3-D absorption, phase, and the so-called dark-field contrast maps. We report on the theoretical and instrumental implementation of of this novel technique. Its broad application potential is exemplarily demonstrated for the field of cultural heritage, namely study of the historical Dead Sea parchment.

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.

TV-based conjugate gradient method and discrete L-curve for few-view CT reconstruction of X-ray in vivo data

High-resolution, three-dimensional (3D) imaging of soft tissues requires the solution of two inverse problems: phase retrieval and the reconstruction of the 3D image from a tomographic stack of two-dimensional (2D) projections. The number of projections per stack should be small to accommodate fast tomography of rapid processes and to constrain X-ray radiation dose to optimal levels to either increase the duration of in vivo time-lapse series at a given goal for spatial resolution and/or the conservation of structure under X-ray irradiation. In pursuing the 3D reconstruction problem in the sense of compressive sampling theory, we propose to reduce the number of projections by applying an advanced algebraic technique subject to the minimisation of the total variation (TV) in the reconstructed slice. This problem is formulated in a Lagrangian multiplier fashion with the parameter value determined by appealing to a discrete L-curve in conjunction with a conjugate gradient method. The usefulness of this reconstruction modality is demonstrated for simulated and in vivo data, the latter acquired in parallel-beam imaging experiments using synchrotron radiation.

Gauging low-dose X-ray phase-contrast imaging at a single and large propagationdistance

The interactions of a beam of hard and spatio-temporally coherent X-rays with a soft-matter sample primarily induce a transverse distribution of exit phase variations δϕ (retardations or advancements in pieces of the wave front exiting the object compared to the incoming wave front) whose free-space propagation over a distance z gives rise to intensity contrast gz. For single-distance image detection and |δϕ| ≪ 1 all-order-in-z phase-intensity contrast transfer is linear in δϕ. Here we show that ideal coherence implies a decay of the (shot-)noise-to-signal ratio in gz and of the associated phase noise as z(-1/2) and z(-1), respectively. Limits on X-ray dose thus favor large values of z. We discuss how a phase-scaling symmetry, exact in the limit δϕ → 0 and dynamically unbroken up to |δϕ| ∼ 1, suggests a filtering of gz in Fourier space, preserving non-iterative quasi-linear phase retrieval for phase variations up to order unity if induced by multi-scale objects inducing phase variations δϕ of a broad spatial frequency spectrum. Such an approach continues to be applicable under an assumed phase-attenuation duality. Using synchrotron radiation, ex and in vivo microtomography on frog embryos exemplifies improved resolution compared to a conventional single-distance phase-retrieval algorithm.

High-resolution X-ray phase-contrast tomography from single-distance radiographs applied to developmental stages of Xenopus laevis

Considering a pure and not necessarily weak phase object, we review a noniterative and nonlinear single-distance phase-retrieval algorithm. The latter exploits the fact that a well-known linear contrast-transfer function, which incorporates all orders in object-detector distance, can be modified to yield a quasiparticle dispersion. Accepting a small loss of information, this algorithm also retrieves the high-frequency parts of the phase in an artefact free way. We point out an extension of this highly resolving quasiparticle approach for mixed objects by assuming a global attenuation-phase duality. Tomographically reconstructing two developmental stages in Xenopus laevis, we compare our approach with a linear algorithm, based on the transport-of-intensity equation, which suppresses high-frequency information.

Optimizing structural and mechanical properties of cryogel scaffolds for use in prostate cancer cell culturing.

Prostate cancer (PCa) currently is the second most diagnosed cancer in men and the second most cause of cancer death after lung cancer in Western societies. This sets the necessity of modelling prostatic disorders to optimize a therapy against them. The conventional approach to investigating prostatic diseases is based on two-dimensional (2D) cell culturing. This method, however, does not provide a three-dimensional (3D) environment, therefore impeding a satisfying simulation of the prostate gland in which the PCa cells proliferate. Cryogel scaffolds represent a valid alternative to 2D culturing systems for studying the normal and pathological behavior of the prostate cells thanks to their 3D pore architecture that reflects more closely the physiological environment in which PCa cells develop. In this work the 3D morphology of three potential scaffolds for PCa cell culturing was investigated by means of synchrotron X-ray computed micro tomography (SXCμT) fitting the according requirements of high spatial resolution, 3D imaging capability and low dose requirements very well. In combination with mechanical tests, the results allowed identifying an optimal cryogel architecture, meeting the needs for a well-suited scaffold to be used for 3D PCa cell culture applications. The selected cryogel was then used for culturing prostatic lymph node metastasis (LNCaP) cells and subsequently, the presence of multi-cellular tumor spheroids inside the matrix was demonstrated again by using SXCμT.