Characterization of Ca-phosphate biological materials by scanning transmission X-ray microscopy (STXM) at the Ca L2,3-, P L2,3- and C K-edges

Abstract

Several naturally occurring biological materials, including bones and teeth, pathological calcifications, microbial mineral deposits formed in marine phosphogenesis areas, as well as bio-inspired cements used for bone and tooth repair are composed of Ca-phosphates. These materials are usually identified and characterized using bulk-scale analytical tools such as X-ray diffraction, Fourier transform infrared spectroscopy or nuclear magnetic resonance. However, there is a need for imaging techniques that provide information on the spatial distribution and chemical composition of the Ca-phosphate phases at the micrometer- and nanometer scales. Such analyses provide insightful indications on how the materials may have formed, e.g. through transient precursor phases that eventually remain spatially separated from the mature phase. Here, we present scanning transmission X-ray microscopy (STXM) analyses of Ca-phosphate reference compounds, showing the feasibility of fingerprinting Ca-phosphate-based materials. We calibrate methods to determine important parameters of Ca-phosphate phases, such as their Ca/P ratio and carbonate content at the ∼25nm scale, using X-ray absorption near-edge spectra at the C K-, Ca L2,3- and P L2,3-edges. As an illustrative case study, we also perform STXM analyses on hydroxyapatite precipitates formed in a dense fibrillar collagen matrix. This study paves the way for future research on Ca-phosphate biomineralization processes down to the scale of a few tens of nanometers.

Biomineralization Patterns of Intracellular Carbonatogenesis in Cyanobacteria: Molecular Hypotheses

“…They showed peaks at 136.7, 137.7 and 139 eV as well as a wider peak at 146.8 eV, similarly to the measured reference polyphosphate compound ( Figure 3). These XANES spectra could be assigned to polyphosphates based on existing libraries of P-containing compounds [40][41][42]. When transferred to MS-2 medium, both strains lost their polyphosphate granules, and produced Ca-carbonates only ( STEM-EDXS microanalyses showed that polyphosphate granules generally contained about 53%-63% of phosphorus (atomic proportion of P over P + Ca + K + Mg), ~13% to ~29% of calcium, ~2% to ~18% of magnesium and ~7% to ~15% of potassium (Figures 1d and 2d, Tables A1 and A3).…”

“…Alignment of images of stacks and extraction of XANES spectra were done using the aXis2000 Software [39]. Carbonate inclusions were discriminated based on their different spectral signature at the C K-edge and the Ca L 2,3 -edges following the approach detailed by Cosmidis et al [40].…”

Characterization of fossil remains using XRF, XPS and XAFS spectroscopies

“…SEM-energy dispersive X-ray spectra (EDS) were used for the determination of the chemical composition. The dominant phase was identified as a non-apatitic calcium phosphate matrix with average Ca/P atomic ratio of 1 were also identified throughout the specimen. The latter inclusions are related to minerals introduced from the deposition environment.…”

“…More specifically, Ca L 2,3 -edge near edge X-ray absorption fine structure (NEXAFS) and X-ray photoelectron (XPS) spectroscopies are applied in combination with extended X-ray absorption fine structure (EXAFS) spectroscopy, X-ray fluorescence (XRF) mapping and more commonly used techniques such as optical microscopy (OM) and scanning electron microscopy (SEM) for the assessment of diagenesis of two different types of paleontological remains: a hyena coprolite and a giant tortoise costal plate. The Ca L 2,3 -edge NEXAFS and Ca 2p photoelectron spectra provide quantitative information concerning the bone apatite and Ca-rich secondary phases [1][2]. OM and SEM are used to assess the morphology and chemical composition of the coprolite matrix and inclusions, whereas XRF maps were recorded to acquire complementary information on the distribution of P, Ca, Mn and Fe in the specimens [3][4].…”

Analytical transmission electron microscopy at organic interfaces

“…To the best of our knowledge, high spectral resolution ELNES from collagen alone have not been reported in the literature. However XANES measurements, which probe analogous transitions using soft X-rays rather than fast electrons, are available for both collagen alone [30], as well as collagen within bone samples [31] (figure 6b,d). In the X-ray microscope, collagen exhibits a major peak at ~288 eV assigned to a 1s → π* C=O transition in carbonyl groups, along with a minor peak at 285 eV and the broad resonance at ~300 eV from the 1s →σ* transition in C-C bonds [30].…”