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EMbedding and Backscattered Scanning Electron Microscopy: A Detailed Protocol for the Whole-Specimen, High-Resolution Analysis of Cardiovascular Tissues
- Source :
- Frontiers in Cardiovascular Medicine, Frontiers in Cardiovascular Medicine, Vol 8 (2021)
- Publication Year :
- 2021
-
Abstract
- 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.
- Subjects :
- 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
Subjects
Details
- ISSN :
- 2297055X
- Volume :
- 8
- Database :
- OpenAIRE
- Journal :
- Frontiers in cardiovascular medicine
- Accession number :
- edsair.doi.dedup.....289d786a0d50d66b50860ba793bff274