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

Abstract

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 organism1. In Xenopus laevis, the South African clawed frog and also in zebrafish, cell and tissue movements have been studied in explants2,3, in fixed embryos4, in vivo using fluorescence microscopy5,6 or microscopic magnetic resonance imaging7. 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.

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

“…All these only require a clear segmentation of the associated boundaries. Because of this, optimised CGTV should be interesting for 4D livecell imaging, aspiring to the acquisition of long time-lapse series [3,4]. The in vivo data, as reconstructed in the present work, is not contaminated by a high level of statistical photon noise.…”

“…Namely, the usefulness of in vivo imaging strongly hinges on a good control of dose effects, thus enabling long time-lapse series of 3D images with weak perturbations induced by the imaging process itself. Recently, embryonic development in the vertebrate model organism Xenopus laevis (African clawed frog) was imaged in vivo via 3D time-lapse series [3,4]. In cases like these a low number of tomographic projections is essential.…”

X-Ray Near-Field Holography: Beyond Idealized Assumptions of the Probe

“…Focusing is required to generate the diverging illumination for high magnification and resolution. Note that also for parallel beam propagation imaging it is extremely common to implicitly assume perfect plane wave illumination by performing the conventional flat-field correction [128,[130][131][132][133][134]. In previous studies, we have shown that under these conditions the commonly used standard flat-field correction, i.e.…”

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

“…In vivo imaging, e.g., the development in Xenopus laevis (African clawed frog) [1,2], g z is unacceptable at such low values of z in view of the high noise-to-contrast ratio R g z , see Eq. (6) and Fig.…”

“…In particular, vertebrate model embryos such as Xenopus laevis (optically opaque) can be imaged in vivo in four dimensions and with micrometer spatial resolution, appealing to X-ray phase contrast [1,2]. As early developmental stages are composed of light elements only, essentially, such embryos act as pure-phase objects from an imaging point of view (δ /β ∼ 10 3 [3] for X-ray energies E ∼ 30 GeV, δ , β representing the projected refractive index n = 1 − δ + iβ (δ = δ (x), β (x) > 0, real).…”