Correlative Spectromicroscopy and Tomography


Ca-containing phases, with the Ca spectrum at the interface being similar to that of amorphous calcium apatite (ACP) while that of Ca in the bone was similar to that of hydroxy- apatite (HA), based on detailed consideration of peak posi- tions and shapes of the weaker Ca L-edge signals [3]. Te Ti L2,3


but not in the interface region or the bone. Two different Ti L2,3


with one having a significantly larger Ti3+ character than the


other, based on the amplitude of the peak at 457.1 eV relative to the far Ti L23


TiOx continuum [3]. Other changes were observed


in the details of the signal between 459 and 461 eV, a region well known to be sensitive to changes in the crystal struc- ture of titanium oxides. Interestingly, a small but significant Ti L2,3


signal was observed throughout all of the bone


region which was spectrally distinct from that of the titanium oxide in the interface region. Tis might indicate that migra- tion of TiOx


into bone can occur over long time periods. Since


focused ion beam (FIB) milling can cause material redistri- bution, it would be useful to study a sample prepared without FIB to confirm that the Ti-in-bone was not an artifact of the FIB milling. Alzheimer’s Disease. Electron microscopy and STXM


were used to map protein aggregates and iron in tissue sections from APP/PS1 (Alzheimer-enhanced) mice and wild type mice. STXM was used to quantify and map iron oxidation states with nanoscale resolution. In addition, the magnetic properties of these iron deposits were studied using X-ray magnetic circu- lar dichroism (XMCD). Te latter results were found to mirror


the magnetic properties of magnetite, consistent with previous work [7,9]. Correlative microscopy of amyloid structures. We found


signal was saturated in the region of the bulk implant, signals were observed in different regions of the interface,


that the cortex of APP/PS1 transgenic mice exhibited abun- dant iron deposits that co-localized with amyloid structures, but very few iron deposits were identified in cortical sections of wild-type mice. Figure 5 shows imaging results from the correlative TEM and STXM study of brain tissues of Alzheim- er’s mice [11]. Te TEM images show the classic morphology of Aβ plaques, and the STXM Fe L2,3


map shows that Fe is


co-located with these plaques (Figure 5E). Image cross-cor- relation analysis performed on the images shown in Figures 5D and 5E confirmed a strong correlation of pixel intensity between the images with a coefficient, R = 0.91 (20 nm pixel size), suggesting that the fibrils themselves contained iron as opposed to the presence of discrete iron foci within fibril aggregates. Figure 6 reveals the Fe oxidation state distributions in some


Aβ plaques in more detail. Proximate but separate regions of high Fe(II) and high Fe(III) content are identified in a region of cortical thin section localized in Figure 6b (red box). Figure 6f shows Fe L2,3


XANES spectra of phases containing mostly


Fe(III) to mostly Fe(II) in regions labeled B1 through B4 in Figures 6c to 6e, respectively. Tese results provide a profile of the oxidation states of both magnetic and non-magnetic iron phases present in each region of the iron deposit. Further XMCD analysis of these regions (not shown) revealed that the B1 region contained only a non-magnetic Fe(III) phase consis- tent with ferrihydrite; regions B4 and B6 contained a reduced form of magnetite together with a non-magnetic Fe(II) phase; while region B5 contained a heavily oxidized form of magne- tite, which in its normal composition is a mixed Fe(II) Fe(III) species (Fe2+


Fe3+ 2O4 ). Te presence of different nanoscale iron


oxides over a small spatial scale provides evidence for a pos- sible redox cycling of the iron, possibly catalyzed by the Aβ deposit. Although the section shown in Figure 6(a–f) was too thick for TEM analysis, another similar area was identified that was sufficiently thin for correlative TEM and STXM (Figure 6 (g–i)). Here it can be seen that the dense particulate structure seen in the TEM image, correlates with iron in the Fe(II) oxi- dation state, consistent with the observation of low-oxidation state iron minerals such as magnetite and wüstite in Alzheim- er’s plaques.


Discussion Te ability to perform measurements at several length scales


with chemical sensitivity using correlative microscopy methods provides insights not available from any single method. Tus, in the bone-implant example, there are clear indications of sev- eral different Ti oxides at the bone-implant interface as well as small amounts of Ti oxide in the bone. Te TiNx


layer, which Figure 4: STXM speciation of bone-implant interface. (a) Ca L2,3 spectra from


regions near interface. (b) Color-coded composite of component maps of three Ca-containing components derived by fitting a Ca L23


stack to the reference


interface and in the bone. (d) Color-coded composite of component maps of three Ti-containing components derived by fitting a Ti L23


spectra in (c). Color coding in (b) and (d) matches spectral color coding in (a) and (c). (Adapted from [3].)


2019 March • www.microscopy-today.com


spectra in the insert to Fig. 4a. (c) Ti L2,3 spectra from two regions near the stack to the reference


was observed in a very small region by APT, was unambiguously identified by STXM to be an intrinsic part of the implant surface structure. Te correlative tomography workflow and associated TEM-EELS spectroscopy and STXM XANES spectromicros- copy has helped to visualize the inhomogeneous and hierarchi- cal bone-implant interface. While the identification of Fe in the Aβ plaques could be accomplished by STEM-EELS (which was not available at the time of the study), the higher energy electron


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