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Effect of plate orientation on apparent thickness of mineral plates by transmission electron microscopy

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Abstract

Purpose

Transmission electron microscopy (TEM) is widely used to study the ultrastructure of bone. The mineral of bone occurs as polycrystalline mineral plates about 3 to 6 nm in thickness. A problem in using TEM to make quantitative analyses of bone is that the orientation of the plates with respect to the plane of the section being imaged is expected to affect their apparent thickness. The purpose of this study was to test if this was true, if the apparent thickness of plates changed substantially as a result of tilt of the section.

Methods

We prepared TEM sections of samples of cortical human bone by ion beam milling, orienting one section parallel to the collagen fibril axes and one perpendicular to them. We obtained TEM bright field and HAADF images of these sections, tilting the sections up to ± 20° at 2° intervals and measuring the apparent thickness of individual mineral platelets at each angle of tilt.

Results

Thickness appears to double as section is tilted ± 20°. True thickness of plates is determined by tilting the section along an axis parallel to the plate orientation and determining the minimum apparent thickness. However, as plates are tilted away from minimum-thickness orientation, they become less well-resolved, disappearing when tilted more than 20°. We therefore also measured apparent thickness of only the darkest (most electron scattering) plate images in an untilted section and obtained the same average thickness as that obtained by tilting.

Conclusion

We conclude that tilting of the section is not necessary to obtain an accurate measurement of the thickness of mineral plates.

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Acknowledgements

We are grateful to Ivan Strakhov and an unknown referee of a previous paper for having suggested the problem illustrated in Fig. 2 of this paper.

Funding

This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

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Authors and Affiliations

  1. School of Geography and Earth Sciences, McMaster University, Hamilton, ON, L8S 4K1, Canada

    Henry Schwarcz

  2. School of Biomedical Engineering, McMaster University, Hamilton, ON, L8S 4L7, Canada

    Henry Schwarcz & Kathryn Grandfield

  3. Department of Materials Science and Engineering, McMaster University, Hamilton, ON, L8S 4L7, Canada

    Chiara Micheletti & Kathryn Grandfield

  4. Department of Biomaterials, Sahlgrenska Academy, University of Gothenburg, Göteborg, 40530, Sweden

    Chiara Micheletti

Authors
  1. Henry Schwarcz
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  2. Chiara Micheletti
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Correspondence to Henry Schwarcz.

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Schwarcz, H., Micheletti, C. & Grandfield, K. Effect of plate orientation on apparent thickness of mineral plates by transmission electron microscopy. J Bone Miner Metab 42, 344–351 (2024). https://doi.org/10.1007/s00774-024-01507-5

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  • DOI: https://doi.org/10.1007/s00774-024-01507-5

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