Alexandra Pacureanu

Bone canalicular network segmentation in 3D nano-CT images through geodesic voting and image tessellation

Recent studies emphasized the role of the bone lacuno-canalicular network (LCN) in the understanding of bone diseases such as osteoporosis. However, suitable methods to investigate this structure are lacking. The aim of this paper is to introduce a methodology to segment the LCN from three-dimensional (3D) synchrotron radiation nano-CT images. Segmentation of such structures is challenging due to several factors such as limited contrast and signal-to-noise ratio, partial volume effects and huge number of data that needs to be processed, which restrains user interaction. We use an approach based on minimum-cost paths and geodesic voting, for which we propose a fully automatic initialization scheme based on a tessellation of the image domain. The centroids of pre-segmented lacunæ are used as Voronoi-tessellation seeds and as start-points of a fast-marching front propagation, whereas the end-points are distributed in the vicinity of each Voronoi-region boundary. This initialization scheme was devised to cope with complex biological structures involving cells interconnected by multiple thread-like, branching processes, while the seminal geodesic-voting method only copes with tree-like structures. Our method has been assessed quantitatively on phantom data and qualitatively on real datasets, demonstrating its feasibility. To the best of our knowledge, presented 3D renderings of lacunæ interconnected by their canaliculi were achieved for the first time.

High-energy cryo x-ray nano-imaging at the ID16A beamline of ESRF

The ID16A beamline at ESRF offers unique capabilities for X-ray nano-imaging, and currently produces the worlds brightest high energy diffraction-limited nanofocus. Such a nanoprobe was designed for quantitative characterization of the morphology and the elemental composition of specimens at both room and cryogenic temperatures. Billions of photons per second can be delivered in a diffraction-limited focus spot size down to 13 nm. Coherent X-ray imaging techniques, as magnified holographic-tomography and ptychographic-tomography, are implemented as well as X-ray fluorescence nanoscopy. We will show the latest developments in coherent and spectroscopic X-ray nanoimaging implemented at the ID16A beamline

Image based in situ sequencing for RNA analysis in tissue

Profiling of gene expression is necessary to study the function of cells, organs and ultimately organisms, in health and disease. However, the currently available mRNA sequencing methods are limited to homogenized tissue samples. Therefore, the obtained information represents either the average expression profile of the full tissue sample or expression profiles from isolated single cells extracted from the tissue. We have developed an image-based approach for sequencing of mRNA fragments directly in fixed tissue. As opposed to current routine techniques, this allows gene expression profiling in relation to the preserved morphological and spatial information of cells and tissue. We present here the details of the image analysis and demonstrate its application to breast cancer tissue samples.

Assessment of imaging quality in magnified phase CT of human bone tissue at the nanoscale

Bone properties at all length scales have a major impact on the fracture risk in disease such as osteoporosis. However, quantitative 3D data on bone tissue at the cellular scale are still rare. Here we propose to use magnified X-ray phase nano-CT to quantify bone ultra-structure in human bone, on the new setup developed on the beamline ID16A at the ESRF, Grenoble. Obtaining 3D images requires the application of phase retrieval prior to tomographic reconstruction. Phase retrieval is an ill-posed problem for which various approaches have been developed. Since image quality has a strong impact on the further quantification of bone tissue, our aim here is to evaluate different phase retrieval methods for imaging bone samples at the cellular scale. Samples from femurs of female donors were scanned using magnified phase nano-CT at voxel sizes of 120 and 30 nm with an energy of 33 keV. Four CT scans at varying sample-to-detector distances were acquired for each sample. We evaluated three phase retrieval methods adapted to these conditions: Paganin’s method at single distance, Paganin’s method extended to multiple distances, and the contrast transfer function (CTF) approach for pure phase objects. These methods were used as initialization to an iterative refinement step. Our results based on visual and quantitative assessment show that the use of several distances (as opposed to single one) clearly improves image quality and the two multi-distance phase retrieval methods give similar results. First results on the segmentation of osteocyte lacunae and canaliculi from such images are presented.

Nanopositioning for the ESRF ID16A Nano-Imaging Beamline

Synchrotron radiation newS, Vol. 31, No. 5, 2018 9 Introduction New scientific frontiers in biomedicine, materials science, and nanotechnology make increasing use of characterization methods at the mesoscopic and nanometer scale. These studies of heterogeneous samples are most often three-dimensional and require multiple and stable acquisitions where the spatial and/or angular position of the sample is changed in a well-controlled way. The high average brightness of synchrotron sources, which will be further enhanced by the future diffraction-limited storage rings [1], is invaluable in this context. Several nanoscale characterization methods have become available using highly coherent probes, nanofocused probes, or both [2–8]. However, the required nanopositioning and stability (absence of vibrations and long-term drifts) remain huge challenges, especially when the environment of the specimen needs to be controlled. The European Synchrotron has developed a number of specialized nanoprobe endstations covering energies from the tender X-ray regime (the X-ray microscopy beamline ID21 under refurbishment [9]), over moderate X-ray energies (the micro-diffraction imaging beamline ID01 [10], the microfocus beamline ID13 [11], the nano-imaging beamline ID16A [12], and the nano-analysis beamline ID16B [13]), to the high-energy regime (the materials science beamline ID11 [14] and the future endstation of the high-energy beamline ID31 [15]). In this article, we develop the specific case of the Nano-Imaging beamline ID16A that provides routine imaging on the nanoscale with a sub-13 nm X-ray beam with a very high flux. At the expense of flexibility, it exploits one of the most advanced nanopositioning endstations. We introduce its specifications, discuss the specific design choices, and illustrate the performance with metrology and X-ray experimental results.

Nanoscale three-dimensional imaging of biological tissue with x-ray holographic tomography

Enabling exploration of biological tissue in three-dimensions at sub-cellular scale is instrumental for advancing our understanding of biological systems and for finding better ways to cope with diseases. Over the last few years, remarkable advances in microscopy facilitated probing cells and tissues at the nanometer scale but many limitations are yet to be overcome. Here we present a novel technique which enables label-free volume imaging of biological tissue with pixel sizes down to 25 nm while maintaining extensive sample coverage. X-ray holographic nanotomography is a full-field 3D imaging technique which benefits from the deep penetration of X-rays and the powerful mechanism of phase contrast. By using cryogenic sample preservation, the tissue can be investigated close to the native state. The unprecedented data created by this technique opens new avenues in life sciences research.