To devise new strategies to treat bone disease in an ageing society, a more detailed characterisation of the process by which bone mineralises is needed. In vitro studies have suggested that carbonated mineral might be a precursor for deposition of bone apatite. Increased carbonate content in bone may also have significant implications in altering the mechanical properties, for example in diseased bone. However, information about the chemistry and coordination environment of bone mineral, and their spatial distribution within healthy and diseased tissues, is lacking. Spatially resolved analytical transmission electron microscopy is the only method available to probe this information at the length scale of the collagen fibrils in bone. In this study, scanning transmission electron microscopy combined with electron energy-loss spectroscopy (STEM-EELS) was used to differentiate between calcium-containing biominerals (hydroxyapatite, carbonated hydroxyapatite, beta-tricalcium phosphate and calcite). A carbon K-edge peak at 290 eV is a direct marker of the presence of carbonate. We found that the oxygen K-edge structure changed most significantly between minerals allowing discrimination between calcium phosphates and calcium carbonates. The presence of carbonate in carbonated HA (CHA) was confirmed by the formation of peak at 533 eV in the oxygen K-edge. These observations were confirmed by simulations using density functional theory. Finally, we show that this method can be utilised to map carbonate from the crystallites in bone. We propose that our calibration library of EELS spectra could be extended to provide spatially resolved information about the coordination environment within bioceramic implants to stimulate the development of structural biomaterials.

Lithium iron phosphate (LiFePO 4) has considerable potential for automotive applications due to its high rate capability, reasonable energy density and environmentally benign nature [1]. However, performance degradation seen after thousands of cycles at high charging-rates (C-rates) has been a point of major concern [2]. Studies of the aging mechanism suggest that phases (LiFePO 4 /FePO 4) formed in the cathode during discharge influence the aging profile [3]. These phases have been investigated recently using x-ray and neutron diffraction [4, 5]. While these methods provide insights into crystal structure and unit cell volume changes associated with phase changes between LiFePO 4 and FePO 4 , their ability to provide spatially resolved measurements of quantities such as Li and Fe concentration at the nanoscale is severely limited. Such measurements are key to confirming the presence or absence of the phases mentioned above and solid solution phases (LixFePO 4). With recent advances in aberration corrected electron microscopy, electron energy-loss spectroscopy (EELS) performed in the scanning transmission electron microscope (STEM) has emerged as a leading technique to obtain spatially resolved measurements of chemistry, structure and bonding. Previously, researchers have used shifts observed in core-loss peaks of iron and oxygen to study the delithiation of FePO 4 host lattice [6-8]. However, these ionization edges have relatively small inelastic scattering cross sections, and consequently, a relatively high electron dose is required to achieve acceptable signal-to-noise ratios in the data resulting in significant electron beam damage to the specimen. The low energy-loss region of the spectrum (0-40 eV) can be very useful since (i) the intensity is much larger compared to the coreloss spectrum and (ii) lesser collection time is required which significantly lowers the risk of beam damage to the material. In this study, we explore the use of low loss EELS to characterize the nominally pure LiFePO 4 powder with trace amounts of FePO 4 which is typical of phase mixtures that is present during phase transformation within the LiFePO 4 battery cathode at the nanoscale during aging.