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To further elucidate this microbial pathogenesis and evaluate the mechanism of action of novel interventions, additional advances in TEM imaging are needed. Here we present detailed protocols for fixation, decalcification, and epoxy embedment of bone tissue for standard TEM imaging studies, as well as the application of immunoelectron microscopy to confirm <jats:italic>S. aureus<\/jats:italic> occupation within sub\u2010micron canaliculi. We also describe the first application of the novel Automated\u2010Tape\u2010UltraMicrotome system with three\u2010dimensional reconstruction and volumetric analyses to quantify <jats:italic>S. aureus<\/jats:italic> occupation within the osteocyte\u2010lacuno canalicular networks. Reconstruction of the three\u2010dimensional volume broadened our perspective of <jats:italic>S. aureus<\/jats:italic> colonization of the canalicular network and, surprisingly, revealed adjacent noninfected canaliculi. This observation has led us to hypothesize that viable osteocytes of the osteocyte\u2010lacuno canalicular networks respond and resist infection, opening future research directions to explain the paradox of adjacent uninfected canaliculi and life\u2010long deep bone infection in patients with chronic osteomyelitis.<\/jats:p>","DOI":"10.1002\/jor.24968","type":"journal-article","created":{"date-parts":[[2020,12,30]],"date-time":"2020-12-30T15:30:19Z","timestamp":1609342219000},"page":"376-388","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":12,"title":["Emerging electron microscopy and 3D methodologies to interrogate <i>Staphylococcus aureus<\/i> osteomyelitis in murine models"],"prefix":"10.1002","volume":"39","author":[{"given":"Karen L.","family":"de Mesy Bentley","sequence":"first","affiliation":[{"name":"Center for Musculoskeletal Research University of Rochester Medical Center  Rochester New York USA"},{"name":"Department 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