qsSAXSI

Synchrotron quantitative scanning-SAXS imaging

This project goes back to my PhD (2000-04) on the development of synchrotron X-ray scattering analysis for nanoscale structural studies of materials at the European Synchrotron Radiation Facility (ESRF) under supervision of Christian Riekel. The imaging aspect was started during my postdoc at the Max-Planck Institute of Colloids and Interfaces (2004-06), under supervision of Oskar Paris and Peter Fratzl. The theoretical and experimental developments involved many people over time, but the most important are Manfred Burghammer (ID13, ESRF), Oliver Bunk (SLS, PSI) and Jean Doucet (LPS, Orsay) who supported my CNRS project.

Keywords: X-ray scattering, SAXS, WAXS, Synchrotron radiation, Imaging, scanning-X-ray imaging, qsSAXSI.

The aim is to develop a quantitative imaging method to map nanoscale structural components in materials using small-angle X-ray scattering contrast .

In simple terms

All biological materials and many synthetic ones are built from well-identifiable nanoscale building blocks. Understanding how those building blocks are organized over multiple length scales is key to deciphering how those materials achieve exceptional properties. This strongly resonates, for example, in the fields of biomineralization, bio-inspired materials design, and nanomedicine. Nanoscale imaging and analysis is therefore an extremely important aspect for such matters.

The main problem when zooming in at the nanoscale, is that the information is obtained within a very small region, typically some tens of square microns. This is fine for homogeneous materials, but all biological materials are intrinsically heterogeneous, so what you see in a microscopic region might be very different from a neighbouring one. Since nanoscale imaging require relatively sophisticated instruments, repeating measurements can become extremely costly and burdensome.

An interesting alternative to nanoscale imaging is to use structural analysis methods such as spectroscopy, scattering, diffraction etc. The main difference is that with imaging, you actually “see” the atoms or nano-objects of interest, while other methods provide indirect structural information resulting from the physical interaction between the probe and the object. Using photons, electrons, neutrons (amongst others) allows acquiring information on atoms or molecules structure and their collective organization. By mapping, one can therefore derive images of structural parameters of interest over much larger regions than those accessible with standard nanoscale imaging.

More precisely

A detailed account of the qsSAXSI method applied to bone studies can be found in this book chapter: (Wagermaier et al., 2013)

In brief, X-ray scattering arises from the presence of nanoscale inhomogeneities such as pores, inclusions, interphases, etc., and is therefore widely used in materials science. The intensity of the scattered signal is directly proportional to the electron density contrast between the matrix and inhomoteneities, but remains with low probability (typically a few percent of the incident beam). So X-ray scattering studies strongly benefit from very bright synchrotron sources which are typically 3 to 9 orders of magnitude more intense. Synchrotrons also allow tunning the X-ray wavelength (and thus the energy) and efficient beam focusing, which is essential for mapping.

Scanning X-ray scattering measurements involve recording the 2D scattered signal by an X-ray microbeam as a function of 2D scan position. Each 2D scattering patterns is analyzed to extract scalar values to form 2D structural maps, i.e. parametric images. So my contribution to the field was essentially to develop efficient data reduction or analysis strategies.

Principle of qsSAXSI from (Gourrier et al., 2007). The black rectangle in (a) indicates the scanned region of an osteon in cortical bone. The image in (b) shows a homogenous sample transmission. Image (c) reveals mineral nanoparticle orientation fluctuations. Each pixel represents a reduced SAXS pattern as shown in (e),(f).

The general problem with X-ray scattering, is that the measured signal is proportional to the square-Fourier transform (FT) of the electron density contrast. The “square” means that the phase is lost and only the modulus of the FT can be retrieved. This “phase problem” means that the FT cannot be mathematically inverted in a simple way, i.e. one cannot directly reconstruct the information without some a priori information. So Small-Angle X-ray scattering (SAXS) data analysis can be performed in two ways: either by extracting generic parameters assuming very simple approximations, e.g. biphasic material, dilute suspension of nano-objects etc., or by fitting a relatively precise structural model. The second case is therefore very case-specific and should rely on other types of measurements providing sufficient clues to what the model should be (although this sounds obvious, this is not always the case…).

My work on qsSAXSI was mainly focused on bone and other mineralized tissue as well as biominerals. So only those will be discussed here, although part of the methods could be extended to other very different materials.

First, we showed that images of the integrated scattered intensity (similar to what would be recorded using a photodiode - but not strictly equal though) of anisotripic nano-objects (e.g. mineral nanoparticles in bone) contains orientation information that can be quantified under certain assumptions (Gourrier et al., 2007). This allows visualizing differences in collagen/mineral orientataion in osteonal bone and was used to show that micro-elasticity fluctuations detected with GHz acoustic microscopy are mainly determined by collagen/mineral orientation - and not so much by mineral nanocrystal size (Granke et al., 2013). This is an example based on the use of integral SAXS parameters which only rely on the assumption that the material is biphasic and was therefore shown to also work on as diverse materials as egg-shells or even hair (Gourrier et al., 2007).

Second, I worked on refining a 1D stack-of-cards model to fit the radial SAXS profile which was initially proposed by Peter Fratzl. This model allowed better quantifying changes in the size and organization of mineral nanoparticles in bone in a case of Skeletal Fluorosis (Gourrier et al., 2010) and confirmed that bone formed during NaF treatment is strongly disorganized at the tissue level, which is totally inefficient biomechanically speaking.

Those developments were used in a broad range of medical (Fratzl-Zelman et al., 2009), biomechanics (Groetsch et al., 2019; Groetsch et al., 2023) and even archaeological studies (Gourrier et al., 2017; Albéric et al., 2014).

References

2023

ActaBiomater
The elasto-plastic nano-and microscale compressive behaviour of rehydrated mineralised collagen fibres

Alexander Groetsch, Aurélien Gourrier, Daniele Casari, and 5 more authors

Acta biomaterialia, 2023

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The hierarchical design of bio-based nanostructured materials such as bone enables them to combine unique structure-mechanical properties. As one of its main components, water plays an important role in bone’s material multiscale mechanical interplay. However, its influence has not been quantified at the length-scale of a mineralised collagen fibre. Here, we couple in situ micropillar compression, and simultaneous synchrotron small angle X-ray scattering (SAXS) and X-ray diffraction (XRD) with a statistical constitutive model. Since the synchrotron data contain statistical information on the nanostructure, we establish a direct connection between experiment and model to identify the rehydrated elasto-plastic micro- and nanomechanical fibre behaviour. Rehydration led to a decrease of 65%-75% in fibre yield stress and compressive strength, and 70% in stiffness with a 3x higher effect on stresses than strains. While in agreement with bone extracellular matrix, the decrease is 1.5-3x higher compared to micro-indentation and macro-compression. Hydration influences mineral more than fibril strain with the highest difference to the macroscale when comparing mineral and tissue levels. The effect of hydration seems to be strongly mediated by ultrastructural interfaces while results provide insights towards mechanical consequences of reported water-mediated structuring of bone apatite. The missing reinforcing capacity of surrounding tissue for an excised fibril array is more pronounced in wet than dry conditions, mainly related to fibril swelling. Differences leading to higher compressive strength between mineralised tissues seem not to depend on rehydration while the lack of kink bands supports the role of water as an elastic embedding influencing energy-absorption mechanisms.
@article{groetsch2023elasto, title = {The elasto-plastic nano-and microscale compressive behaviour of rehydrated mineralised collagen fibres}, author = {Groetsch, Alexander and Gourrier, Aur{\'e}lien and Casari, Daniele and Schwiedrzik, Jakob and Shephard, Jonathan D and Michler, Johann and Zysset, Philippe K and Wolfram, Uwe}, journal = {Acta biomaterialia}, volume = {164}, pages = {332--345}, year = {2023}, publisher = {Elsevier}, url = {https://doi.org/10.1016/j.actbio.2023.03.041}, doi = {10.1016/j.actbio.2023.03.041} }

2019

ActaBiomater
Compressive behaviour of uniaxially aligned individual mineralised collagen fibres at the micro-and nanoscale

Alexander Groetsch, Aurélien Gourrier, Jakob Schwiedrzik, and 6 more authors

Acta biomaterialia, 2019

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The increasing incidence of osteoporotic bone fractures makes fracture risk prediction an important clinical challenge. Computational models can be utilised to facilitate such analyses. However, they critically depend on bone’s underlying hierarchical material description. To understand bone’s irreversible behaviour at the micro- and nanoscale, we developed an in situ testing protocol that allows us to directly relate the experimental data to the mechanical behaviour of individual mineralised collagen ﬁbres and its main constitutive phases, the mineralised collagen ﬁbrils and the mineral nanocrystals, by combining micropillar compression of single ﬁbres with small angle X-ray scattering (SAXS) and X-ray diffraction (XRD). Failure modes were assessed by SEM. Strain ratios in the elastic region at ﬁbre, ﬁbril and mineral levels were found to be approximately 22:5:2 with strain ratios at the point of compressive strength of 0.23 Æ 0.11 for ﬁbril-to-ﬁbre and 0.07 Æ 0.01 for mineral-to-ﬁbre levels. Mineral-to-ﬁbre levels showed highest strain ratios around the apparent yield point, ﬁbril-to-ﬁbre around apparent strength. The mineralised collagen ﬁbrils showed a delayed mechanical response, contrary to the mineral phase, which points towards preceding deformations of mineral nanocrystals in the extraﬁbrillar matrix. No damage was measured at the level of the mineralised collagen ﬁbre which indicates an incomplete separation of the mineral and collagen, and an extraﬁbrillar interface failure. The formation of kink bands and the gradual recruitment of ﬁbrils upon compressive loading presumably led to localised strains. Our results from a well-controlled ﬁbrillar architecture provide valuable input for micromechanical models and computational non-linear bone strength analyses that may provide further insights for personalised diagnosis and treatment as well as bio-inspired implants for patients with bone diseases.
@article{groetsch2019compressive, title = {Compressive behaviour of uniaxially aligned individual mineralised collagen fibres at the micro-and nanoscale}, author = {Groetsch, Alexander and Gourrier, Aur{\'e}lien and Schwiedrzik, Jakob and Sztucki, Michael and Beck, Rainer J and Shephard, Jonathan D and Michler, Johann and Zysset, Philippe K and Wolfram, Uwe}, journal = {Acta biomaterialia}, volume = {89}, pages = {313--329}, year = {2019}, publisher = {Elsevier}, url = {https://doi.org/10.1016/j.actbio.2019.03.043}, doi = {10.1016/j.actbio.2019.02.053} }

2017

PLOSOne
Nanoscale modifications in the early heating stages of bone are heterogeneous at the microstructural scale

Aurélien Gourrier, Céline Chadefaux, Estelle Lemaitre, and 8 more authors

PloS one, 2017

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Nanoscale studies of bone provide key indicators to evidence subtle structural changes that may occur in the biomedical, forensic and archaeological contexts. One specific problem encountered in all those disciplines, for which the identification of nanostructural cues could prove useful, is to properly monitor the effect of heating on bone tissue. In particular, the mechanisms at work at the onset of heating are still relatively unclear. Using a multiscale approach combining Raman microspectroscopy, transmission electron microscopy (TEM), synchrotron quantitative scanning small-angle X-ray scattering imaging (qsSAXSI) and polarized light (PL) microscopy, we investigate the ultrastructure of cortical bovine bone heated at temperatures < 300 ̊C, from the molecular to the macroscopic scale. We show that, despite limited changes in crystal structure, the mineral nanoparticles increase in thickness and become strongly disorganized upon heating. Furthermore, while the nanostructure in distinct anatomical quadrants appears to be statistically different, our results demonstrate this stems from the tissue histology, i.e. from the high degree of heterogeneity of the microstructure induced by the complex cellular processes involved in bone tissue formation. From this study, we conclude that the analysis of bone samples based on the structure and organization of the mineral nanocrystals requires performing measurements at the histological level, which is an advantageous feature of qsSAXSI. This is a critical aspect that extends to a much broader range of questions relating to nanoscale investigations of bone, which could also be extended to other classes of nanostructured heterogeneous materials.
@article{gourrier2017nanoscale, title = {Nanoscale modifications in the early heating stages of bone are heterogeneous at the microstructural scale}, author = {Gourrier, Aur{\'e}lien and Chadefaux, C{\'e}line and Lemaitre, Estelle and Bellot-Gurlet, Ludovic and Reynolds, Michael and Burghammer, Manfred and Plazanet, Marie and Boivin, Georges and Farlay, Delphine and Bunk, Oliver and others}, journal = {PloS one}, volume = {12}, number = {4}, pages = {e0176179}, year = {2017}, publisher = {Public Library of Science San Francisco, CA USA}, url = {https://doi.org/10.1371/journal.pone.0176179}, doi = {10.1371/journal.pone.0176179} }

2014

Pal3
Early diagenesis of elephant tusk in marine environment

Marie Albéric, Aurélien Gourrier, Katharina Müller, and 4 more authors

Palaeogeography, Palaeoclimatology, Palaeoecology, 2014

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Three elephant tusks recovered from a 18th century Dutch shipwreck in Brittany seawaters were analyzed to study early diagenetic changes of ivory in a marine environment. Micro-small angle X-ray scattering (μSAXS) analysis revealed sub-microscopic structural modiﬁcations of ivory (hydroxyapatite particle thickness, mineralized collagen ﬁbers organization and orientation). Chemical compositional changes of the organic fraction (nitrogen and carbon loss) and of the mineral part (magnesium loss, carbonate, calcium, chlorine and strontium uptake) were investigated by non-invasive micro-Particle Induced X-ray Emission/Gamma-ray Emission/Rutherford and Elastic Backscattering Spectrometry analysis (μPIXE/PIGE/RBS/EBS). These chemical and structural markers can serve as proxies for the early diagenesis of elephant tusk in marine settings. The results show that the macroscopic preservation state of the tusk surface does not reﬂect its microscopic preservation state. A diagenetic sequence is proposed, depending on the local environment of each tusk. First, collagen hydrolysis and ion adsorptions from the marine environment are proposed to occur. Second, a tusk buried in the sand was protected from Ca enrichment of the surface and the formation of a ﬂaky white concretion whereas tusks out of the sand were subjected to these latter phenomena as well as sea-urchin attacks. Third, ion leaching and uptake, increased crystallinity of the hydroxyapatite crystals, and the modiﬁcations of the mineralized collagen ﬁber network are assumed to happen for all three tusks with different level of completion.
@article{alberic2014early, title = {Early diagenesis of elephant tusk in marine environment}, author = {Alb{\'e}ric, Marie and Gourrier, Aur{\'e}lien and M{\"u}ller, Katharina and Zizak, I and Wagermaier, Wolfgang and Fratzl, Peter and Reiche, Ina}, journal = {Palaeogeography, Palaeoclimatology, Palaeoecology}, volume = {416}, pages = {120--132}, year = {2014}, publisher = {Elsevier}, url = {http://dx.doi.org/10.1016/j.palaeo.2014.09.006}, doi = {10.1016/j.palaeo.2014.09.006} }

2013

BookChapt

Understanding Hierarchy and Functions of Bone Using Scanning X‐ray Scattering Methods

Wolfgang Wagermaier, Aurélien Gourrier, and Barbara Aichmayer

In Smart Materials Series, 2013

DOI Bib HTML

@incollection{wagermaier2013understanding,
  title = {Understanding Hierarchy and Functions of Bone Using Scanning X‐ray Scattering Methods},
  booktitle = {Smart Materials Series},
  author = {Wagermaier, Wolfgang and Gourrier, Aur{\'e}lien and Aichmayer, Barbara},
  editor = {Fratzl, Peter and Dunlop, John W.C. and Weinkamer, Richard},
  pages = {46--73},
  year = {2013},
  publisher = {Royal Society of Chemistry},
  url = {https://doi.org/10.1039/9781849737555-00046},
  doi = {10.1039/9781849737555-00046}
}

PLOSOne
Microfibril orientation dominates the microelastic properties of human bone tissue at the lamellar length scale

Mathilde Granke, Aurélien Gourrier, Fabienne Rupin, and 5 more authors

PLoS One, 2013

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The elastic properties of bone tissue determine the biomechanical behavior of bone at the organ level. It is now widely accepted that the nanoscale structure of bone plays an important role to determine the elastic properties at the tissue level. Hence, in addition to the mineral density, the structure and organization of the mineral nanoparticles and of the collagen microfibrils appear as potential key factors governing the elasticity. Many studies exist on the role of the organization of collagen microfibril and mineral nanocrystals in strongly remodeled bone. However, there is no direct experimental proof to support the theoretical calculations. Here, we provide such evidence through a novel approach combining several high resolution imaging techniques: scanning acoustic microscopy, quantitative scanning small-Angle X-ray scattering imaging and synchrotron radiation computed microtomography. We find that the periodic modulations of elasticity across osteonal bone are essentially determined by the orientation of the mineral nanoparticles and to a lesser extent only by the particle size and density. Based on the strong correlation between the orientation of the mineral nanoparticles and the collagen molecules, we conclude that the microfibril orientation is the main determinant of the observed undulations of microelastic properties in regions of constant mineralization in osteonal lamellar bone. This multimodal approach could be applied to a much broader range of fibrous biological materials for the purpose of biomimetic technologies.
@article{granke2013microfibril, title = {Microfibril orientation dominates the microelastic properties of human bone tissue at the lamellar length scale}, author = {Granke, Mathilde and Gourrier, Aur{\'e}lien and Rupin, Fabienne and Raum, Kay and Peyrin, Fran{\c{c}}oise and Burghammer, Manfred and Sa{\"\i}ed, Amena and Laugier, Pascal}, journal = {PLoS One}, volume = {8}, number = {3}, pages = {e58043}, year = {2013}, publisher = {Public Library of Science}, url = {http://dx.doi.org/10.1371/journal.pone.0058043}, doi = {10.1371/journal.pone.0058043} }

2010

JAC
Scanning small-angle X-ray scattering analysis of the size and organization of the mineral nanoparticles in fluorotic bone using a stack of cards model

Aurélien Gourrier, Chenghao Li, Stefan Siegel, and 4 more authors

Journal of Applied Crystallography, 2010

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A model describing the size and arrangement of mineral particles in bone tissues is used to analyse the results of a scanning small-angle X-ray scattering (SAXS) experiment on a pathological bone biopsy. The overall description assumes that the nanometre-sized mineral platelets are arranged in a parallel fashion with possible fluctuations in their relative position, orientation and thickness. This method is tested on a thin sample section obtained from the biopsy of an osteoporotic patient treated with a high cumulative dose of NaF. The mineralization pattern of fluorotic bone is known to exhibit significant differences as compared to healthy bone in terms of density, particle size and organization. This is the first attempt to provide quantitative indicators of the degree of regularity in the packing of the mineral platelets in human pathological bone. Using scanning SAXS with a synchrotron microbeam of 15µm allows discrimination between pathological and healthy bone at the tissue level. Additionally, the benefits of this method are discussed with respect to the accuracy of particle size determination using SAXS.
@article{gourrier2010scanning, title = {Scanning small-angle X-ray scattering analysis of the size and organization of the mineral nanoparticles in fluorotic bone using a stack of cards model}, author = {Gourrier, Aur{\'e}lien and Li, Chenghao and Siegel, Stefan and Paris, Oskar and Roschger, Paul and Klaushofer, Klaus and Fratzl, Peter}, journal = {Journal of Applied Crystallography}, volume = {43}, number = {6}, pages = {1385--1392}, year = {2010}, publisher = {International Union of Crystallography}, url = {http://dx.doi.org/10.1107/S0021889810035090}, doi = {10.1107/S0021889810035090} }

2009

CalcifTissueInt
Combination of nanoindentation and quantitative backscattered electron imaging revealed altered bone material properties associated with femoral neck fragility

N Fratzl-Zelman, P Roschger, A Gourrier, and 6 more authors

Calcified tissue international, 2009

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Osteoporotic fragility fractures were hypothesized to be related to changes in bone material properties and not solely to reduction in bone mass. We studied cortical bone from the superior and inferior sectors of whole femoral neck sections from five female osteoporotic hip fracture cases (74–92 years) and five nonfractured controls (75–88 years). The typical calcium content (CaPeak) and the mineral particle thickness parameter (T) were mapped in large areas of the superior and inferior regions using quantitative backscattered electron imaging (qBEI) and scanning small-angle X-ray scattering, respectively. Additionally, indentation modulus (E) and hardness (H) (determined by nanoindentation) were compared at the local level to the mineral content (CaInd) at the indent positions (obtained from qBEI). CaPeak (-2.2%, P = 0.002), CaInd (-1.8%, P = 0.048), E (-5.6%, P = 0.040), and H (-6.0%, P = 0.016) were significantly lower for the superior compared to the inferior region. Interestingly, CaPeak as well as CaInd were also lower (-2.6%, P = 0.006, and –3.7%, P = 0.002, respectively) in fracture cases compared to controls, while E and H did not show any significant reduction. T values were in the normal range, independent of region (P = 0.181) or fracture status (P = 0.551). In conclusion, it appears that the observed femoral neck fragility is associated with a reduced mineral content, which was not accompanied by a reduction in stiffness and hardness of the bone material. This pilot study suggests that a stiffening process in the organic matrix component contributes to bone fragility independently of mineral content.
@article{fratzl2009combination, title = {Combination of nanoindentation and quantitative backscattered electron imaging revealed altered bone material properties associated with femoral neck fragility}, author = {Fratzl-Zelman, N and Roschger, P and Gourrier, A and Weber, M and Misof, BM and Loveridge, N and Reeve, J and Klaushofer, K and Fratzl, P}, journal = {Calcified tissue international}, volume = {85}, number = {4}, pages = {335--343}, year = {2009}, publisher = {Springer-Verlag New York}, url = {http://dx.doi.org/10.1007/s00223-009-9289-8}, doi = {10.1007/s00223-009-9289-8} }

2007

JAC

Scanning X-ray imaging with small-angle scattering contrast

Aurelien Gourrier, Wolfgang Wagermaier, Manfred Burghammer, and 6 more authors

Journal of Applied Crystallography, 2007

DOI