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Three-Dimensional Reconstruction of Skeletal Muscle Extracellular Matrix Ultrastructure

Published online by Cambridge University Press:  02 October 2014

Allison R. Gillies ,
Eric A. Bushong ,
Thomas J. Deerinck ,
Mark H. Ellisman  and
Richard L. Lieber
Allison R. Gillies
Affiliation:
Department of Bioengineering, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0863, USA
Eric A. Bushong
Affiliation:
National Center for Microscopy and Imaging Research, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0608, USA
Thomas J. Deerinck
Affiliation:
National Center for Microscopy and Imaging Research, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0608, USA
Mark H. Ellisman
Affiliation:
National Center for Microscopy and Imaging Research, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0608, USA Department of Neurosciences, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0608, USA
Richard L. Lieber*
Affiliation:
Department of Bioengineering, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0863, USA Department of Orthopaedic Surgery, University of California San Diego, 9500 Gilman Drive, La Jolla, CA, 92093-0863, USA
*
*Corresponding author. [email protected]
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Abstract

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.

Keywords

skeletal muscleextracellular matrixcollagenserial block face scanning electron microscopyperimysium
Type
Biological Applications
Information
Microscopy and Microanalysis , Volume 20 , Issue 6 , December 2014 , pp. 1835 - 1840
Copyright
© Microscopy Society of America 2014 

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