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A guide to analysis and reconstruction of serial block face scanning electron microscopy data

Serial block face scanning electron microscopy (SBF‐SEM) is a relatively new technique that allows the acquisition of serially sectioned, imaged and digitally aligned ultrastructural data. There is a wealth of information that can be obtained from the resulting image stacks but this presents a new c...

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Autores principales: COCKS, E., TAGGART, M., RIND, F.C., WHITE, K.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: John Wiley and Sons Inc. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947172/
https://www.ncbi.nlm.nih.gov/pubmed/29333754
http://dx.doi.org/10.1111/jmi.12676
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author COCKS, E.
TAGGART, M.
RIND, F.C.
WHITE, K.
author_facet COCKS, E.
TAGGART, M.
RIND, F.C.
WHITE, K.
author_sort COCKS, E.
collection PubMed
description Serial block face scanning electron microscopy (SBF‐SEM) is a relatively new technique that allows the acquisition of serially sectioned, imaged and digitally aligned ultrastructural data. There is a wealth of information that can be obtained from the resulting image stacks but this presents a new challenge for researchers – how to computationally analyse and make best use of the large datasets produced. One approach is to reconstruct structures and features of interest in 3D. However, the software programmes can appear overwhelming, time‐consuming and not intuitive for those new to image analysis. There are a limited number of published articles that provide sufficient detail on how to do this type of reconstruction. Therefore, the aim of this paper is to provide a detailed step‐by‐step protocol, accompanied by tutorial videos, for several types of analysis programmes that can be used on raw SBF‐SEM data, although there are more options available than can be covered here. To showcase the programmes, datasets of skeletal muscle from foetal and adult guinea pigs are initially used with procedures subsequently applied to guinea pig cardiac tissue and locust brain. The tissue is processed using the heavy metal protocol developed specifically for SBF‐SEM. Trimmed resin blocks are placed into a Zeiss Sigma SEM incorporating the Gatan 3View and the resulting image stacks are analysed in three different programmes, Fiji, Amira and MIB, using a range of tools available for segmentation. The results from the image analysis comparison show that the analysis tools are often more suited to a particular type of structure. For example, larger structures, such as nuclei and cells, can be segmented using interpolation, which speeds up analysis; single contrast structures, such as the nucleolus, can be segmented using the contrast‐based thresholding tools. Knowing the nature of the tissue and its specific structures (complexity, contrast, if there are distinct membranes, size) will help to determine the best method for reconstruction and thus maximize informative output from valuable tissue.
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spelling pubmed-59471722018-05-17 A guide to analysis and reconstruction of serial block face scanning electron microscopy data COCKS, E. TAGGART, M. RIND, F.C. WHITE, K. J Microsc Original Articles Serial block face scanning electron microscopy (SBF‐SEM) is a relatively new technique that allows the acquisition of serially sectioned, imaged and digitally aligned ultrastructural data. There is a wealth of information that can be obtained from the resulting image stacks but this presents a new challenge for researchers – how to computationally analyse and make best use of the large datasets produced. One approach is to reconstruct structures and features of interest in 3D. However, the software programmes can appear overwhelming, time‐consuming and not intuitive for those new to image analysis. There are a limited number of published articles that provide sufficient detail on how to do this type of reconstruction. Therefore, the aim of this paper is to provide a detailed step‐by‐step protocol, accompanied by tutorial videos, for several types of analysis programmes that can be used on raw SBF‐SEM data, although there are more options available than can be covered here. To showcase the programmes, datasets of skeletal muscle from foetal and adult guinea pigs are initially used with procedures subsequently applied to guinea pig cardiac tissue and locust brain. The tissue is processed using the heavy metal protocol developed specifically for SBF‐SEM. Trimmed resin blocks are placed into a Zeiss Sigma SEM incorporating the Gatan 3View and the resulting image stacks are analysed in three different programmes, Fiji, Amira and MIB, using a range of tools available for segmentation. The results from the image analysis comparison show that the analysis tools are often more suited to a particular type of structure. For example, larger structures, such as nuclei and cells, can be segmented using interpolation, which speeds up analysis; single contrast structures, such as the nucleolus, can be segmented using the contrast‐based thresholding tools. Knowing the nature of the tissue and its specific structures (complexity, contrast, if there are distinct membranes, size) will help to determine the best method for reconstruction and thus maximize informative output from valuable tissue. John Wiley and Sons Inc. 2018-01-15 2018-05 /pmc/articles/PMC5947172/ /pubmed/29333754 http://dx.doi.org/10.1111/jmi.12676 Text en © 2018 The Authors. Journal of Microscopy published by John Wiley & Sons Ltd on behalf of Royal Microscopical Society. This is an open access article under the terms of the http://creativecommons.org/licenses/by/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.
spellingShingle Original Articles
COCKS, E.
TAGGART, M.
RIND, F.C.
WHITE, K.
A guide to analysis and reconstruction of serial block face scanning electron microscopy data
title A guide to analysis and reconstruction of serial block face scanning electron microscopy data
title_full A guide to analysis and reconstruction of serial block face scanning electron microscopy data
title_fullStr A guide to analysis and reconstruction of serial block face scanning electron microscopy data
title_full_unstemmed A guide to analysis and reconstruction of serial block face scanning electron microscopy data
title_short A guide to analysis and reconstruction of serial block face scanning electron microscopy data
title_sort guide to analysis and reconstruction of serial block face scanning electron microscopy data
topic Original Articles
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5947172/
https://www.ncbi.nlm.nih.gov/pubmed/29333754
http://dx.doi.org/10.1111/jmi.12676
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