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Scanning Electron Microscopy of Bone

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Bone Research Protocols

Part of the book series: Methods in Molecular Biology ((MIMB,volume 816))

Abstract

This chapter described methods for Scanning Electron Microscopical imaging of bone and bone cells. Backscattered electron (BSE) imaging is by far the most useful in the bone field, followed by secondary electrons (SE) and the energy dispersive X-ray (EDX) analytical modes. This chapter considers preparing and imaging samples of unembedded bone having 3D detail in a 3D surface, topography-free, polished or micromilled, resin-embedded block surfaces, and resin casts of space in bone matrix. The chapter considers methods for fixation, drying, looking at undersides of bone cells, and coating. Maceration with alkaline bacterial pronase, hypochlorite, hydrogen peroxide, and sodium or potassium hydroxide to remove cells and unmineralised matrix is described in detail. Attention is given especially to methods for 3D BSE SEM imaging of bone samples and recommendations for the types of resin embedding of bone for BSE imaging are given. Correlated confocal and SEM imaging of PMMA-embedded bone requires the use of glycerol to coverslip. Cathodoluminescence (CL) mode SEM imaging is an alternative for visualising fluorescent mineralising front labels such as calcein and tetracyclines. Making spatial casts from PMMA or other resin embedded samples is an important use of this material. Correlation with other imaging means, including microradiography and microtomography is important. Shipping wet bone samples between labs is best done in glycerol. Environmental SEM (ESEM, controlled vacuum mode) is valuable in eliminating ­“charging” problems which are common with complex, cancellous bone samples.

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Acknowledgments

I thank Maureen Arora for her patient help in carrying out many of the procedures described in this chapter on many thousands of samples. Her employment has been funded by the Horserace Betting Levy Board.

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Authors and Affiliations

  1. Biophysics Section, Oral Growth and Development, Dental Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London, UK

    Alan Boyde

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  1. Alan Boyde
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Correspondence to Alan Boyde .

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Editors and Affiliations

  1. Institute of Medical Sciences, University of Aberdeen, Forester Hill, Aberdeen, AB25 2ZD, United Kingdom

    Miep H. Helfrich

  2. Institute of Genetics and Molecular Medi, Rheumatic Diseases Unit, University of Edinburgh, Crewe Road South, Edinburgh, EH4 2XU, United Kingdom

    Stuart H. Ralston

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Boyde, A. (2012). Scanning Electron Microscopy of Bone. In: Helfrich, M., Ralston, S. (eds) Bone Research Protocols. Methods in Molecular Biology, vol 816. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-415-5_24

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  • DOI: https://doi.org/10.1007/978-1-61779-415-5_24

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-414-8

  • Online ISBN: 978-1-61779-415-5

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