Technical developments for computed tomography on the CENBG nanobeam line

The use of ion microbeams as probes for computedtomography has proven to be a powerful tool for the three-dimensional characterization of specimens a few tens of micrometers in size. Compared to other types of probes, the main advantage is that quantitative information about mass density and composition can be obtained directly, using specific reconstruction codes. At the Centre d’Etudes Nucléaires de Bordeaux Gradignan (CENBG), this technique was initially developed for applications in cellular biology. However, the observation of the cell ultrastructure requires a sub-micron resolution. The construction of the nanobeamline at the Applications Interdisciplinaires des Faisceaux d’Ions en Region Aquitaine (AIFIRA) irradiation facility has opened new perspectives for such applications. The implementation of computedtomography on the nanobeamline of CENBG has required a careful design of the analysis chamber, especially microscopes for precise sample visualization, and detectors for scanning transmission ion microscopy (STIM) and for particle induced X-ray emission (PIXE). The sample can be precisely positioned in the three directions X, Y, Z and a stepper motor coupled to a goniometer ensures the rotational motion. First images of 3D tomography were obtained on a reference sample containing microspheres of certified diameter, showing the good stability of the beam and the sample stage, and the precision of the motion.

A digital framework to build, visualize and analyze a gene expression atlas with cellular resolution in zebrafish early embryogenesis

A gene expression atlas is an essential resource to quantify and understand the multiscale processes of embryogenesis in time and space. The automated reconstruction of a prototypic 4D atlas for vertebrate early embryos, using multicolor fluorescence in situ hybridization with nuclear counterstain, requires dedicated computational strategies. To this goal, we designed an original methodological framework implemented in a software tool called Match-IT. With only minimal human supervision, our system is able to gather gene expression patterns observed in different analyzed embryos with phenotypic variability and map them onto a series of common 3D templates over time, creating a 4D atlas. This framework was used to construct an atlas composed of 6 gene expression templates from a cohort of zebrafish early embryos spanning 6 developmental stages from 4 to 6.3 hpf (hours post fertilization). They included 53 specimens, 181,415 detected cell nuclei and the segmentation of 98 gene expression patterns observed in 3D for 9 different genes. In addition, an interactive visualization software, Atlas-IT, was developed to inspect, supervise and analyze the atlas. Match-IT and Atlas-IT, including user manuals, representative datasets and video tutorials, are publicly and freely available online. We also propose computational methods and tools for the quantitative assessment of the gene expression templates at the cellular scale, with the identification, visualization and analysis of coexpression patterns, synexpression groups and their dynamics through developmental stages.