Contrast-enhanced tissue processing of fibrillin-rich elastic fibres for 3D visualization by volume scanning electron microscopy

Lewis, Philip N. ORCID: https://orcid.org/0000-0002-3353-0708, Young, Robert D. ORCID: https://orcid.org/0000-0002-8300-8002, Souza, R. B., Quantock, Andrew J. ORCID: https://orcid.org/0000-0002-2484-3120 and Meek, Keith M. ORCID: https://orcid.org/0000-0002-9948-7538 2021. Contrast-enhanced tissue processing of fibrillin-rich elastic fibres for 3D visualization by volume scanning electron microscopy. Methods and Protocols 4 (3) , 56. 10.3390/mps4030056

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Abstract

Elastic fibres constitute an important component of the extracellular matrix and currently are the subject of intensive study in order to elucidate their assembly, function and involvement in cell–matrix interactions and disease. However, few studies to date have investigated the 3D architecture of the elastic fibre system in bulk tissue. We describe a protocol for preparation of tissue samples, including primary fixation and backscatter electron contrast-enhancement steps, through dehydration into stable resin-embedded blocks for volume electron microscopy. The use of low molecular weight tannic acid and alcoholic lead staining are critical stages in this procedure. Block preparation by ultramicrotomy and evaporative metal coating prior to microscopical examination are also described. We present images acquired from serial block face scanning electron microscopy of cornea and aorta showing target structures clearly differentiated from cells and other matrix components. The processing method imparts high contrast to fibrillin-containing elastic fibres, thus facilitating their segmentation and rendering into 3D reconstructions by image analysis software from large serial image datasets.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Optometry and Vision Sciences
Additional Information:	This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/).
Publisher:	MDPI
ISSN:	2409-9279
Funders:	MRC, BBSRC
Date of First Compliant Deposit:	20 August 2021
Date of Acceptance:	11 August 2021
Last Modified:	26 May 2023 16:56
URI:	https://orca.cardiff.ac.uk/id/eprint/143430

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