Three-Dimensional Reconstruction of Skeletal Muscle Extracellular Matrix Ultrastructure

The skeletal muscle extracellular matrix (ECM) supports muscle’s passive mechanical function and provides a unique environment for extracellular tissues such as nerves, blood vessels, and a cadre of mononuclear cells. Within muscle ECM, collagen is thought to be the primary load-bearing protein, yet...

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Bibliographic Details
Published in:Microscopy and microanalysis 2014-12, Vol.20 (6), p.1835-1840
Main Authors: Gillies, Allison R., Bushong, Eric A., Deerinck, Thomas J., Ellisman, Mark H., Lieber, Richard L.
Format: Article
Language:English
Subjects:
Animals
Biological Applications
Blood vessels
Collagen
Collagen - ultrastructure
Electron microscopes
Extracellular matrix
Extracellular Matrix - ultrastructure
Fibroblasts
Image Processing, Computer-Assisted
Mice
Microscopy, Electron, Scanning - methods
Muscle, Skeletal - ultrastructure
Musculoskeletal system
Ruthenium
Scanning electron microscopy
Software
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Summary:The skeletal muscle extracellular matrix (ECM) supports muscle’s passive mechanical function and provides a unique environment for extracellular tissues such as nerves, blood vessels, and a cadre of mononuclear cells. Within muscle ECM, collagen is thought to be the primary load-bearing protein, yet its structure and organization with respect to muscle fibers, tendon, and mononuclear cells is unknown. Detailed examination of extracellular collagen morphology requires high-resolution electron microscopy performed over relatively long distances because multinucleated muscle cells are very long and extend from several millimeters to several centimeters. Unfortunately, there is no tool currently available for high resolution ECM analysis that extends over such distances relevant to muscle fibers. Serial block face scanning electron microscopy is reported here to examine skeletal muscle ECM ultrastructure over hundreds of microns. Ruthenium red staining was implemented to enhance contrast and utilization of variable pressure imaging reduced electron charging artifacts, allowing continuous imaging over a large ECM volume. This approach revealed previously unappreciated perimysial collagen structures that were reconstructed via both manual and semi-automated segmentation methods. Perimysial collagen structures in the ECM may provide a target for clinical therapies aimed at reducing skeletal muscle fibrosis and stiffness.
ISSN:1431-9276
1435-8115
DOI:10.1017/S1431927614013300