Peter Cloetens

Assessing the molecular phylogeny of a near extinct group of vertebrates: the Neotropical harlequin frogs (Bufonidae; Atelopus)

Neotropical harlequin frogs, Atelopus, are a species-rich bufonid group. Atelopus monophyly has been suggested but intergeneric, interspecific and intraspecific relationships are poorly understood. One reason is that morphological characters of harlequin frogs are often difficult to interpret, making species delimitations difficult. Molecular analyses (DNA barcoding, phylogeny) may be helpful but sampling is hampered as most of the more than 100 Atelopus species have undergone severe population declines and many are possibly extinct. We processed mitochondrial DNA (12S and 16S rRNA) of 28 available ingroup samples from a large portion of the genus’ geographic range (Bayesian Inference, Maximum Likelihood). Our samples constitute a monophyletic unit, which is sister to other bufonid genera studied including the Andean genus Osornophryne. In contrast to previous morphological studies, our results suggest that Osornophryne is neither sister to Atelopus nor nested within it. Within Atelopus, we note two major clades with well supported subclades, one Amazonian–Guianan Clade (Flavescens-spumarius Clade plus Tricolor Clade) and an Andean–Chocó–Central American Clade (Varius Clade plus all other Atelopus). The first mentioned includes all species that possess a middle ear (i.e. stapes) except for A. seminiferus lacking it (like all remaining Atelopus). Previously proposed species groups based on frog-like versus toad-like overall appearance (i.e. Longirostris and Ignescens Groups) or phalangeal reduction in the thumb (i.e. Flavescens Group) are not monophyletic in our phylogeny, thus characters used to define them are not considered synapomorphies. We show that genetic divergence can be high between species belonging to different clades, in spite of their phenetic similarity (e.g. A. pulcher, Atelopus sp. 2). On the other hand, within the same clade, colour can vary tremendously, while genetic divergence is low (e.g. A. flavescens and allies). These observations demonstrate that Atelopus taxonomy is complicated and that an integrative approach is required before ‘splitting’ or ‘lumping’ nominal species.

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

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.

Dynamics and Imaging Using Coherent X-rays at the European Synchrotron

Synchrotron radiation newS, Vol. 30, No. 5, 2017 13 ESRF—The European Synchrotron—was the fi rst worldwide, third-generation synchrotron source. It began operation in 1994 and delivered an X-ray beam with unprecedented high brilliance. While previous-generation synchrotrons focused mostly on the source brightness (fl ux of photons on the sample), the use of undulators gave access not only to very intense, but also coherent, X-ray beams. Coherence is a necessary condition to observe interference effects caused by the propagation of a wavefi eld scattered by an object. The spatial coherent length of hard X-rays measures several to tens of microns and encouraged the development of novel coherence-based Xray methods for time-resolved (dynamics) and high-resolution imaging applications. The very fi rst exploitation of coherence in an X-ray experiment was the study of speckles patterns associated with critical fl uctuations in a binary alloy [1] and the introduction of phase contrast X-ray imaging [2, 3]. Since then, the use of coherence has considerably increased and numerous methods have been developed. In this article, we review the main coherence-based methods used at the ESRF today: the imaging techniques, which can either be used in the near fi eld (with the detector close to the object recording a Fresnel diffraction pattern) or in the far fi eld (where the detector will usually measure the Fraunhofer diffraction pattern of the illuminated sample) regime [4], and X-ray photon correlation spectroscopy (XPCS), which relies on a statistical analysis of the fl uctuation of speckles to probe sample dynamics.

High energy near- and far-field ptychographic tomography at the ESRF

In high-resolution tomography, one needs high-resolved projections in order to reconstruct a high-quality 3D map of a sample. X-ray ptychography is a robust technique which can provide such high-resolution 2D projections taking advantage of coherent X-rays. This technique was used in the far-field regime for a fair amount of time, but it can now also be implemented in the near-field regime. In both regimes, the technique enables not only high-resolution imaging, but also high sensitivity to the electron density of the sample. The combination with tomography makes 3D imaging possible via ptychographic X-ray computed tomography (PXCT), which can provide a 3D map of the complex-valued refractive index of the sample. The extension of PXCT to X-ray energies above 15 keV is challenging, but it can allow the imaging of object opaque to lower energy. We present here the implementation and developments of high-energy near- and far-field PXCT at the ESRF.