EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues
| Autor: | Anton G. Kutikhin, A. R. Shabaev, A. V. Frolov, Daria K. Shishkova, Rinat A. Mukhamadiyarov, Tatiana V. Glushkova, Anton A Lyapin, Alexander N Stasev, L. A. Bogdanov, Vladislav A Koshelev, Alexander E Kostyunin |
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| Rok vydání: | 2021 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
Vascular smooth muscle Scanning electron microscope Uranyl acetate Cardiovascular Medicine cardiovascular research grinding and polishing law.invention epoxy resin calcification chemistry.chemical_compound law Microscopy Methods biocompatibility testing Diseases of the circulatory (Cardiovascular) system mineral deposits electron microscopy sample preparation Chemistry Osmium tetroxide RC666-701 Ultrastructure Electron microscope Cardiology and Cardiovascular Medicine Biomedical engineering |
| Zdroj: | Frontiers in Cardiovascular Medicine Frontiers in Cardiovascular Medicine, Vol 8 (2021) |
| ISSN: | 2297-055X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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