Correlative Spectromicroscopy and Tomography
hallmark of Alzheimer’s disease (AD). Tere is evidence that metal ions, including Fe, play a crucial role [7]. Te co-existence of iron and Aβ1−42
may enhance toxicity through redox-based chemistry and the generation of harmful free radicals, which
can cause neuronal injury [8]. Tus, visualization of Fe-Aβ1−42 complexes and analysis of the Fe oxidation states therein, as well as analysis of the peptide, could provide insights into the mecha- nism of AD and possibly suggest approaches for treatment. Until recently, the type of iron associated with plaques was not well characterized. Using advanced electron microscopy techniques, several groups observed mineralized iron in the form of magne- tite nanoparticles in Aβ plaque cores from post-mortem human cases of Alzheimer’s disease [7,9,10]. Magnetite is not a normal feature of the human brain, so its presence suggests that aber- rant iron redox chemistry might be involved [7,9]. Our work [11] extended this important study by using correlative elec- tron microscopy and scanning transmission X-ray microscopy (STXM) to investigate relationships between iron biochemistry and AD pathology in the intact cortex from the brain of APP/ PS1 transgenic mice, an established mouse model which over- produces Aβ peptide and reproduces the amyloid deposition characteristics of AD. We found a direct correlation of amyloid plaque morphology with iron type and levels, determined the oxidation states of nanoscale iron and their distribution in corti- cal tissue, supported the prior observation of magnetite involve- ment, and showed that Aβ-induced chemical reduction of iron in iron-amyloid complexes could occur in vivo.
Materials and Methods Transmission electron microscopy. (TEM) is a versa-
tile tool for probing the structure and composition of materi- als at near atomic resolution. In this work, TEM was used for imaging with conventional bright-field images based on mass- thickness contrast and also for high-angle annular dark-field (HAADF) imaging in scanning TEM (STEM), which provides an image intensity roughly proportional to the atomic number squared, enabling compositional contrast. Both approaches are highly useful for probing biomedical materials. In addition to producing 2D images, TEM can be used to
determine the three-dimensional morphology of objects using a technique called electron tomography (ET). By tilting a sample through a range of tilt angles and acquiring projection images at each tilt, one can use computer algorithms to reconstruct a 3D image with the resolution of the reconstruction dependent on the resolution of the individual images, the number of projections, and the range of tilt angles. In fact, HAADF-STEM tomography is an efficient technique to visualize the inhomogeneous microstruc- tures of biomaterials and biointerfaces in three dimensions (3D) since it provides both nano-scale resolution and compositional contrast [12]. However, for a conventional thin lamellae TEM sample, the “missing wedge,” a limitation in tilt range as a result of sample thickness and shadowing at high angles, leads to two main issues: artifacts and elongation in the final reconstruction, and limited ability to combine the tilt-series with spectral information (for example, electron energy loss spectrometry, EELS). On-axis ET of a cylindrical sample removes the missing wedge and allows acquisition of high-fidelity, quantitative reconstructions of osseo- integration [13]. Furthermore, if a cylindrical or needle-shaped sample geometry is used, on-axis ET becomes compatible with
2019 March •
www.microscopy-today.com
the collection of spectral information (for example, EELS or X-ray) since the specimen thickness remains constant at all tilt angles. TEM, STEM, and EELS analyses were done using an FEI Titan 80-300 TEM/STEM at McMaster University, operated at 300 keV. Atom probe tomography (APT) is a 4D tomographic tech-
nique capable of mapping element distributions with atomic spatial resolution and parts per million chemical sensitivity. Tis technique is based on the evaporation of surface atoms triggered by a pulsed electric field [14]. With the incorporation of laser pulsing and improvements in sample preparation, APT has expanded its application base from metal and semicon- ductor materials to non-conductive biomaterials and minerals [15,16]. Te ions of various elements removed by laser pulsing are identified by a time-of-flight mass spectrometer via time- gated position-sensitive detectors. Te resulting APT spectra (number of ions versus mass-to-charge ratios) enable the iden- tification of chemical species. Tese are reconstructed into a 3D point cloud, where every point represents an elementally identified ion in the sample volume. Tis reconstructed volume can be further analyzed to extract and visualize the nanoscale chemical features of samples in 3D with Integrated Visualiza- tion and Analysis Soſtware from the instrument manufacturer Cameca. However, the lack of chemical state information has always been a limitation of APT. By correlating APT with elec- tron microscopy (i.e., ET) and spectroscopy (i.e. TEM-EELS and STXM), complementary analyses provide chemical state and crystallinity information [17]. APT was done using a Cam- eca LEAP 4000X HR Atom Probe at McMaster University. Scanning transmission X-ray microscopy (STXM) is a
synchrotron-based technique that routinely achieves 30 nm spatial resolution, with state-of-the art performance of 10 nm [18]. By imaging at different photon energies, 3D (x,y,z) and 4D (x,y,z,E) data sets can be acquired, providing X-ray tomo- graphic imaging (X-ray CT) and elemental maps through X-ray absorption spectroscopy (XAS), respectively. Details of the X-ray absorption near edge structure (XANES) spectra can provide information about the chemical state of the ele- ments present. Multivariate statistical methods or forward fit- ting using reference X-ray absorption spectra on quantitative intensity scales can be used to generate quantitative maps of the chemical species present from “stacks” of full-area images at a sequence of photon energies. In this case the mapping was done by converting the measured 3D (x,y,E) data cube to opti- cal density (OD), then fitting the OD stack to a set of reference spectra {i} extracted from distinct regions in the area measured. Te (x,y,i) fitting coefficients are assembled into maps of the individual components (i), which can then be combined into a color-coded composite [18]. In addition to identifying and mapping iron species from X-ray absorption spectra (XAS), magnetic properties can be characterized by X-ray magnetic circular dichroism (XMCD), where changes in XAS intensities are recorded with leſt and right circularly polarized X-rays. Te XMCD signal arises from preferential excitation of electrons from core levels with orbital angular momentum >0 (e.g. Fe L23
) into partly filled majority or minority spin valence energy
bands. XMCD is sensitive to the direction and magnitude of the magnetic vector, and in the case of magnetite, also to the crystal site [19]. For the work described here photon energies from 280 eV to 730 eV were used. Te XAS and XMCD studies
13
Page 1 |
Page 2 |
Page 3 |
Page 4 |
Page 5 |
Page 6 |
Page 7 |
Page 8 |
Page 9 |
Page 10 |
Page 11 |
Page 12 |
Page 13 |
Page 14 |
Page 15 |
Page 16 |
Page 17 |
Page 18 |
Page 19 |
Page 20 |
Page 21 |
Page 22 |
Page 23 |
Page 24 |
Page 25 |
Page 26 |
Page 27 |
Page 28 |
Page 29 |
Page 30 |
Page 31 |
Page 32 |
Page 33 |
Page 34 |
Page 35 |
Page 36 |
Page 37 |
Page 38 |
Page 39 |
Page 40 |
Page 41 |
Page 42 |
Page 43 |
Page 44 |
Page 45 |
Page 46 |
Page 47 |
Page 48 |
Page 49 |
Page 50 |
Page 51 |
Page 52 |
Page 53 |
Page 54 |
Page 55 |
Page 56
