Addressing K/L-edge overlap in elemental analysis from micro-X-ray fluorescence: bioimaging of tungsten and zinc in bone tissue using synchrotron radiation and laser ablation inductively coupled plasma mass spectrometry

  • Cassidy R. VanderSchee
  • David Kuter
  • Hsiang Chou
  • Brian P. Jackson
  • Koren K. Mann
  • D. Scott BohleEmail author


Synchrotron radiation micro-X-ray fluorescence (SR-μXRF) is a powerful elemental mapping technique that has been used to map tungsten and zinc distribution in bone tissue. However, the heterogeneity of the bone samples along with overlap of the tungsten L-edge with the zinc K-edge signals complicates SR-μXRF data analysis, introduces minor artefacts into the resulting element maps, and decreases image sensitivity and resolution. To confirm and more carefully delineate these SR-μXRF results, we have employed laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) to untangle the problem created by the K/L-edge overlap of the tungsten/zinc pair. While the overall elemental distribution results are consistent between the two techniques, LA-ICP-MS provides significantly higher sensitivity and image resolution compared with SR-μXRF measurements in bone. These improvements reveal tissue-specific distribution patterns of tungsten and zinc in bone, not observed using SR-μXRF. We conclude that probing elemental distribution in bone is best achieved using LA-ICP-MS, though SR-μXRF retains the advantage of being a non-destructive method with the capability of being paired with X-ray techniques, which determine speciation in situ. Since tungsten is an emerging contaminant recently found to accumulate in bone, accurately determining its distribution and speciation in situ is essential for directing toxicological studies and informing treatment regimes.

Graphical abstract

Tungsten and zinc localization and uptake in mouse femurs were imaged by synchrotron radiation, left, and by laser ablation ICP-MS, right. The increased resolution of the LA-ICP-MS technique resolves the problem of the overlap in tungsten’s L-edge and zinc’s K-edge


Tungsten LA-ICP-MS X-ray spectroscopy (XPS | XRF | EDX) Zinc Analyte Overlap 



We thank the CLS for beamtime along with Renfei Feng and Peter Blanchard for their assistance at the VESPERS beamline. Research done at the Canadian Light Source is supported by the Canada Foundation for Innovation, NSERC, the University of Saskatchewan, the Government of Saskatchewan, Western Economic Diversification Canada, the National Research Council Canada, and the CIHR. Dartmouth Trace Element Analysis Core is supported by NCI Cancer Center Support Grant 5P30CA023108-37 and NIEHS Superfund grant P42 ES007373.

Author Contributions

KKM, DSB designed the project. KKM and HC supervised sample collection from animal specimens. CRV prepared bone samples and collected BE images. CRV and DK collected, processed and analyzed SR-μ-XRF and LA-ICP-MS data. BPJ assisted with LA-ICP-MS collection and analysis. DSB, CRV and DK wrote the manuscript with input from all authors.

Funding information

This study received financial support from the Natural Sciences and Engineering Research Council of Canada (NSERC), Canadian Institutes of Health Research (CIHR), and the Canada Research Chairs Program as well as fellowship support from the CIHR to DK and CRV.

Compliance with ethical standards

Animal experiments were performed in the Lady Davis Institute Animal Care Facility following the guidelines of the McGill University Animal Care Committee–approved protocol.

Conflict of interest

The authors declare that they have no competing interests.

Supplementary material

216_2019_2244_MOESM1_ESM.pdf (1.1 mb)
ESM 1 Table S1 Close Kα1 /Lα1 Edge overlaps distinguished by differences in edge energies in keV. Energies taken from the ALS X-ray Data Booklet [5]. Particularly close overlapping pairs such as As/Pb and Ca/Sn have been discussed before [1]. Table S2 Maximum intensity values used for scaling SR-μXRF and LA-ICP-MS maps (Figure 1 and Figure S2). Intensity scale is in normalized counts for SR-μXRF and counts for LA-ICP-MS and cannot be compared between the two techniques. Fig. S1 Backscattered electron image (BEI) of bone tibia cross-section, (a) prepared using thin-sectioning method and (b) via polishing method. Scale bar = 50 μm. Fig. S2 Tungsten (a-b) and (calcium c-d) SR-μXRF maps of longitudinally sliced murine femoral knee section. Maps (a,c) are collected at a higher resolution of 5 μm step size compared to the lower resolution maps (b,d) which have a 18 μm step size. Smaller step size does not provide further elemental distribution details compared to the larger step size. Scale bar = 50 μm. Intensity values in Table S2. Fig. S3 SR-μXRF tungsten elemental distribution map corresponding to Figure 3A (indicated in b) of longitudinally sliced murine femoral head from mouse exposed to 1000 ppm of tungsten. In (a), the lower limit corresponds to upper limit from control sample, Figure 3C. White background indicates counts below the lower limit. 16 μm step size. Scale bars = 200 μm. Fig. S4 (a-b) SR-μXRF[6] and (c-d) LA-ICP-MS maps of murine tibia and femur cross-sections, respectively, from mouse exposed to 1000 ppm of tungsten for four weeks. Similar tungsten (a,c) and zinc (b,d) distribution trends are observed. Intensity scale is in normalized counts for SR-μXRF and counts for LA-ICP-MS and cannot be compared between the two techniques. Step size: (a,b) 20 μm; (c,d) 15 μm. Scale bars = 200 μm. Fig. S5 From left to right: BEI, SR-μXRF and LA-ICP-MS calcium distribution maps of the identical longitudinally sliced murine proximal tibia sample. These images are the same magnified sections as indicated in Figure 1 (PDF 1.07 MB)


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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  • Cassidy R. VanderSchee
    • 1
  • David Kuter
    • 1
  • Hsiang Chou
    • 2
  • Brian P. Jackson
    • 3
  • Koren K. Mann
    • 2
  • D. Scott Bohle
    • 1
    Email author
  1. 1.Department of ChemistryMcGill UniversityMontrealCanada
  2. 2.Lady Davis Institute for Medical ResearchMcGill UniversityMontrealCanada
  3. 3.Department of Earth SciencesDartmouth CollegeHanoverUSA

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