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EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues
Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining suffer from a relatively low resolution of light microscopy, whereas the fluorescence imaging of plaques and bioprosthetic heart valv...
Autores principales: | , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
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Frontiers Media S.A.
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8573413/ https://www.ncbi.nlm.nih.gov/pubmed/34760942 http://dx.doi.org/10.3389/fcvm.2021.739549 |
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author | Mukhamadiyarov, Rinat A. Bogdanov, Leo A. Glushkova, Tatiana V. Shishkova, Daria K. Kostyunin, Alexander E. Koshelev, Vladislav A. Shabaev, Amin R. Frolov, Alexey V. Stasev, Alexander N. Lyapin, Anton A. Kutikhin, Anton G. |
author_facet | Mukhamadiyarov, Rinat A. Bogdanov, Leo A. Glushkova, Tatiana V. Shishkova, Daria K. Kostyunin, Alexander E. Koshelev, Vladislav A. Shabaev, Amin R. Frolov, Alexey V. Stasev, Alexander N. Lyapin, Anton A. Kutikhin, Anton G. |
author_sort | Mukhamadiyarov, Rinat A. |
collection | PubMed |
description | Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining suffer from a relatively low resolution of light microscopy, whereas the fluorescence imaging of plaques and bioprosthetic heart valves yields considerable background noise from the convoluted extracellular matrix that often results in a low signal-to-noise ratio. Besides, the sectioning of calcified or stent-expanded blood vessels or mineralised heart valves leads to a critical loss of their integrity, demanding other methods to be developed. Here, we designed a conceptually novel approach that combines conventional formalin fixation, sequential incubation in heavy metal solutions (osmium tetroxide, uranyl acetate or lanthanides, and lead citrate), and the embedding of the whole specimen into epoxy resin to retain its integrity while accessing the region of interest by grinding and polishing. Upon carbon sputtering, the sample is visualised by means of backscattered scanning electron microscopy. The technique fully preserves calcified and stent-expanded tissues, permits a detailed analysis of vascular and valvular composition and architecture, enables discrimination between multiple cell types (including endothelial cells, vascular smooth muscle cells, fibroblasts, adipocytes, mast cells, foam cells, foreign-body giant cells, canonical macrophages, neutrophils, and lymphocytes) and microvascular identities (arterioles, venules, and capillaries), and gives a technical possibility for quantitating the number, area, and density of the blood vessels. Hence, we suggest that our approach is capable of providing a pathophysiological insight into cardiovascular disease development. The protocol does not require specific expertise and can be employed in virtually any laboratory that has a scanning electron microscope. |
format | Online Article Text |
id | pubmed-8573413 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Frontiers Media S.A. |
record_format | MEDLINE/PubMed |
spelling | pubmed-85734132021-11-09 EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues Mukhamadiyarov, Rinat A. Bogdanov, Leo A. Glushkova, Tatiana V. Shishkova, Daria K. Kostyunin, Alexander E. Koshelev, Vladislav A. Shabaev, Amin R. Frolov, Alexey V. Stasev, Alexander N. Lyapin, Anton A. Kutikhin, Anton G. Front Cardiovasc Med Cardiovascular Medicine Currently, an ultrastructural analysis of cardiovascular tissues is significantly complicated. Routine histopathological examinations and immunohistochemical staining suffer from a relatively low resolution of light microscopy, whereas the fluorescence imaging of plaques and bioprosthetic heart valves yields considerable background noise from the convoluted extracellular matrix that often results in a low signal-to-noise ratio. Besides, the sectioning of calcified or stent-expanded blood vessels or mineralised heart valves leads to a critical loss of their integrity, demanding other methods to be developed. Here, we designed a conceptually novel approach that combines conventional formalin fixation, sequential incubation in heavy metal solutions (osmium tetroxide, uranyl acetate or lanthanides, and lead citrate), and the embedding of the whole specimen into epoxy resin to retain its integrity while accessing the region of interest by grinding and polishing. Upon carbon sputtering, the sample is visualised by means of backscattered scanning electron microscopy. The technique fully preserves calcified and stent-expanded tissues, permits a detailed analysis of vascular and valvular composition and architecture, enables discrimination between multiple cell types (including endothelial cells, vascular smooth muscle cells, fibroblasts, adipocytes, mast cells, foam cells, foreign-body giant cells, canonical macrophages, neutrophils, and lymphocytes) and microvascular identities (arterioles, venules, and capillaries), and gives a technical possibility for quantitating the number, area, and density of the blood vessels. Hence, we suggest that our approach is capable of providing a pathophysiological insight into cardiovascular disease development. The protocol does not require specific expertise and can be employed in virtually any laboratory that has a scanning electron microscope. Frontiers Media S.A. 2021-10-25 /pmc/articles/PMC8573413/ /pubmed/34760942 http://dx.doi.org/10.3389/fcvm.2021.739549 Text en Copyright © 2021 Mukhamadiyarov, Bogdanov, Glushkova, Shishkova, Kostyunin, Koshelev, Shabaev, Frolov, Stasev, Lyapin and Kutikhin. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms. |
spellingShingle | Cardiovascular Medicine Mukhamadiyarov, Rinat A. Bogdanov, Leo A. Glushkova, Tatiana V. Shishkova, Daria K. Kostyunin, Alexander E. Koshelev, Vladislav A. Shabaev, Amin R. Frolov, Alexey V. Stasev, Alexander N. Lyapin, Anton A. Kutikhin, Anton G. EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues |
title | EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues |
title_full | EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues |
title_fullStr | EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues |
title_full_unstemmed | EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues |
title_short | EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues |
title_sort | embedding and backscattered scanning electron microscopy: a detailed protocol for the whole-specimen, high-resolution analysis of cardiovascular tissues |
topic | Cardiovascular Medicine |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8573413/ https://www.ncbi.nlm.nih.gov/pubmed/34760942 http://dx.doi.org/10.3389/fcvm.2021.739549 |
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