title: Three-dimensional reconstruction of skeletal muscle extracellular matrix ultrastructure.
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.
authors.affiliation: 1Department of Bioengineering,University of California San Diego,9500 Gilman Drive,La Jolla,CA,92093-0863,USA.
authors.forename: Allison R
authors.initials: AR
authors.lastname: Gillies
authors.affiliation: 2National Center for Microscopy and Imaging Research,University of California San Diego,9500 Gilman Drive,La Jolla,CA,92093-0608,USA.
authors.forename: Eric A
authors.initials: EA
authors.lastname: Bushong
authors.affiliation: 2National Center for Microscopy and Imaging Research,University of California San Diego,9500 Gilman Drive,La Jolla,CA,92093-0608,USA.
authors.forename: Thomas J
authors.initials: TJ
authors.lastname: Deerinck
authors.affiliation: 2National Center for Microscopy and Imaging Research,University of California San Diego,9500 Gilman Drive,La Jolla,CA,92093-0608,USA.
authors.forename: Mark H
authors.initials: MH
authors.lastname: Ellisman
authors.affiliation: 1Department of Bioengineering,University of California San Diego,9500 Gilman Drive,La Jolla,CA,92093-0863,USA.
authors.forename: Richard L
authors.initials: RL
authors.lastname: Lieber
mesh_terms: D000818:Animals; D003094:Collagen / Q000648:ultrastructure*; D005109:Extracellular Matrix / Q000648:ultrastructure*; D007091:Image Processing, Computer-Assisted; D051379:Mice; D008855:Microscopy, Electron, Scanning / Q000379:methods*; D018482:Muscle, Skeletal / Q000648:ultrastructure*
chemical_list: D003094:Collagen