Development of protocols for the first serial block-face scanning electron microscopy (SBF SEM) studies of bone tissue
Development of protocols for the first serial block-face scanning electron microscopy (SBF SEM) studies of bone tissue
There is an unmet need for a high-resolution three-dimensional (3D) technique to simultaneously image osteocytes and the matrix in which these cells reside. In serial block-face scanning electron microscopy (SBF SEM), an ultramicrotome mounted within the vacuum chamber of a microscope repeatedly sections a resin-embedded block of tissue. Backscattered electron scans of the block face provide a stack of high-resolution two-dimensional images, which can be used to visualise and quantify cells and organelles in 3D. High-resolution 3D images of biological tissues from SBF SEM have been exploited considerably to date in the neuroscience field. However, non-brain samples, in particular hard biological tissues, have appeared more challenging to image by SBF SEM due to the difficulties of sectioning and rendering the samples conductive. We have developed and propose protocols for bone tissue preparation using SBF SEM, for imaging simultaneously soft and hard bone tissue components in 3D. We review the state of the art in high-resolution imaging of osteocytes, provide a historical perspective of SBF SEM, and we present first SBF SEM proof-of-concept studies for murine and human tissue. The application of SBF SEM to hard tissues will facilitate qualitative and quantitative 3D studies of tissue microstructure and ultrastructure in bone development, ageing and pathologies such as osteoporosis and osteoarthritis.
3D imaging, Bone, High resolution, Osteocyte, SBF SEM, Serial block-face scanning electron microscopy
Goggin, Patricia, Mary
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Ho, Elaine, Ming Li
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Gnaegi, Helmut
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Searle, Stuart
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Oreffo, Richard
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Schneider, Philipp
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February 2020
Goggin, Patricia, Mary
c2d225c4-b84d-4ead-a433-320057cb5fa9
Ho, Elaine, Ming Li
7fa9df7f-4dbf-4be4-b03f-ff79012dd44b
Gnaegi, Helmut
f5305499-8141-4197-816e-f31ee843f7b6
Searle, Stuart
18ced818-c4e0-4e1e-8898-9c4a303f10bf
Oreffo, Richard
ff9fff72-6855-4d0f-bfb2-311d0e8f3778
Schneider, Philipp
a810f925-4808-44e4-8a4a-a51586f9d7ad
Goggin, Patricia, Mary, Ho, Elaine, Ming Li, Gnaegi, Helmut, Searle, Stuart, Oreffo, Richard and Schneider, Philipp
(2020)
Development of protocols for the first serial block-face scanning electron microscopy (SBF SEM) studies of bone tissue.
Bone, 131, [115107].
(doi:10.1016/j.bone.2019.115107).
Abstract
There is an unmet need for a high-resolution three-dimensional (3D) technique to simultaneously image osteocytes and the matrix in which these cells reside. In serial block-face scanning electron microscopy (SBF SEM), an ultramicrotome mounted within the vacuum chamber of a microscope repeatedly sections a resin-embedded block of tissue. Backscattered electron scans of the block face provide a stack of high-resolution two-dimensional images, which can be used to visualise and quantify cells and organelles in 3D. High-resolution 3D images of biological tissues from SBF SEM have been exploited considerably to date in the neuroscience field. However, non-brain samples, in particular hard biological tissues, have appeared more challenging to image by SBF SEM due to the difficulties of sectioning and rendering the samples conductive. We have developed and propose protocols for bone tissue preparation using SBF SEM, for imaging simultaneously soft and hard bone tissue components in 3D. We review the state of the art in high-resolution imaging of osteocytes, provide a historical perspective of SBF SEM, and we present first SBF SEM proof-of-concept studies for murine and human tissue. The application of SBF SEM to hard tissues will facilitate qualitative and quantitative 3D studies of tissue microstructure and ultrastructure in bone development, ageing and pathologies such as osteoporosis and osteoarthritis.
Text
BONE-D-19-00771R1-1_for_Pure
- Accepted Manuscript
More information
Accepted/In Press date: 9 October 2019
e-pub ahead of print date: 24 October 2019
Published date: February 2020
Additional Information:
Funding Information:
This work was funded by the Institute for Life Sciences and the Faculty of Engineering and Physical Sciences at the University of Southampton . Funding to ROCO from the Biotechnology and Biological Sciences Research Council UK (BBSRC LO21071/ and BB/L00609X/1), the UK Regenerative Medicine Platform Acellular / Smart Materials – 3D Architecture (MR/R015651/1), and a grant from the UK Regenerative Medicine Platform (MR/L012626/1 Southampton Imaging) is gratefully acknowledged. Technical support was provided by the Biomedical Imaging Unit at the University of Southampton. We thank Vitali Gorianov, Jo McEwan and the team at Spire Hospital Southampton for human samples and Matthew McGregor-Sharp, Clinical Neurosciences, University of Southampton, for supplying murine tissue under Home Office Project License Number 30/3095. Alan Kuzirian provided the image for Fig. 2 as well as advice and encouragement. Personal communications with Lucy Collinson (Francis Crick Institute, London) and Paul Spellward (Gatan UK) are acknowledged. Appendix A
Publisher Copyright:
© 2019 The Authors
Keywords:
3D imaging, Bone, High resolution, Osteocyte, SBF SEM, Serial block-face scanning electron microscopy
Identifiers
Local EPrints ID: 435233
URI: http://eprints.soton.ac.uk/id/eprint/435233
ISSN: 8756-3282
PURE UUID: f7fe8397-90d0-4607-8162-dcf9b43ab496
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Date deposited: 28 Oct 2019 17:30
Last modified: 30 Jul 2022 04:01
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Contributors
Author:
Patricia, Mary Goggin
Author:
Helmut Gnaegi
Author:
Stuart Searle
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