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Existing solutions for various segmentation tasks are optimized for either: high\u2010resolution three\u2010dimensional imaging, relying on well\u2010defined object boundaries (e.g., whole neuron segmentation in volumetric electron microscopy datasets); or low\u2010resolution two\u2010dimensional imaging, boundary\u2010invariant but poorly suited to large 3D objects (e.g., whole\u2010cell segmentation of light microscopy images). However, there is a middle ground that challenges current segmentation tools: large 3D objects with ambiguous boundaries, such as mitochondria in whole\u2010cell 3D electron microscopy datasets. To address this, we developed\n                    <jats:bold>Sk<\/jats:bold>\n                    eleton\u2010\n                    <jats:bold>O<\/jats:bold>\n                    riented\n                    <jats:bold>O<\/jats:bold>\n                    bjec\n                    <jats:bold>t S<\/jats:bold>\n                    egmentation (SKOOTS)\u2014a novel, general\u2010purpose 3D segmentation framework for efficiently segmenting densely packed, morphologically complex objects. SKOOTS is fast, accurate, and memory\u2010efficient, and can be applied to segment mitochondria and other structures in both 3D light and electron microscopy datasets. By combining skeleton\u2010based instance segmentation with a scalable embedding approach, SKOOTS bridges a key gap in existing segmentation strategies and enables biologically meaningful, large\u2010scale analysis of 3D biomedical imaging data. We demonstrate this by segmenting &gt;15\u00a0000 mitochondria from cochlear hair cells and supporting cells across experimental conditions in under 2 h on a consumer\u2010grade PC, enabling downstream morphological analysis that revealed subtle structural changes following aminoglycoside exposure. SKOOTS is fully open\u2010source, easy to retrain, and designed to support diverse datasets, making it broadly accessible to the research community.\n                  <\/jats:p>","DOI":"10.1002\/advs.202517738","type":"journal-article","created":{"date-parts":[[2026,3,31]],"date-time":"2026-03-31T19:35:31Z","timestamp":1774985731000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["SKOOTS: Skeleton\u2010Oriented Object Segmentation for Mitochondria in High\u2010Resolution Cochlear EM Datasets"],"prefix":"10.1002","volume":"13","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-3618-5356","authenticated-orcid":false,"given":"Christopher J.","family":"Buswinka","sequence":"first","affiliation":[{"name":"Eaton Peabody Laboratories Mass Eye and Ear  Boston MA USA"},{"name":"Department of Otolaryngology Head and Neck Surgery Harvard Medical School  Boston MA USA"},{"name":"Speech and Hearing Biosciences and Technology graduate Program Harvard University  Cambridge MA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0347-9614","authenticated-orcid":false,"given":"Richard T.","family":"Osgood","sequence":"additional","affiliation":[{"name":"Eaton Peabody Laboratories Mass Eye and Ear  Boston MA USA"},{"name":"Department of Otolaryngology Head and Neck Surgery Harvard Medical School  Boston MA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hidetomi","family":"Nitta","sequence":"additional","affiliation":[{"name":"Eaton Peabody Laboratories Mass Eye and Ear  Boston MA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2076-6818","authenticated-orcid":false,"given":"Artur A.","family":"Indzhykulian","sequence":"additional","affiliation":[{"name":"Eaton Peabody Laboratories Mass Eye and Ear  Boston MA USA"},{"name":"Department of Otolaryngology Head and Neck Surgery Harvard Medical School  Boston MA USA"},{"name":"Speech and Hearing Biosciences and Technology graduate Program Harvard University  Cambridge MA USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2026,3,31]]},"reference":[{"key":"e_1_2_8_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/Nrm2275"},{"key":"e_1_2_8_3_1","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.23\u201011\u201004499.2003"},{"key":"e_1_2_8_4_1","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.3231\u201006.2006"},{"key":"e_1_2_8_5_1","doi-asserted-by":"publisher","DOI":"10.1128\/MCB.24.18.8195\u20108209.2004"},{"key":"e_1_2_8_6_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.cell.2011.10.018"},{"key":"e_1_2_8_7_1","doi-asserted-by":"publisher","DOI":"10.1093\/cvr\/cvq237"},{"key":"e_1_2_8_8_1","doi-asserted-by":"publisher","DOI":"10.1152\/jn.00019.2010"},{"key":"e_1_2_8_9_1","doi-asserted-by":"publisher","DOI":"10.7554\/eLife.48914"},{"key":"e_1_2_8_10_1","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.0363\u201019.2019"},{"key":"e_1_2_8_11_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrn.2017.170"},{"key":"e_1_2_8_12_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bbabio.2008.12.005"},{"key":"e_1_2_8_13_1","doi-asserted-by":"publisher","DOI":"10.1111\/j.1742\u20104658.2010.07755.x"},{"key":"e_1_2_8_14_1","doi-asserted-by":"publisher","DOI":"10.1172\/JCI84939"},{"key":"e_1_2_8_15_1","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.051627098"},{"key":"e_1_2_8_16_1","doi-asserted-by":"publisher","DOI":"10.1038\/nrm1150"},{"key":"e_1_2_8_17_1","doi-asserted-by":"publisher","DOI":"10.1007\/s10495\u2010007\u20100756\u20102"},{"key":"e_1_2_8_18_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.mad.2010.04.006"},{"key":"e_1_2_8_19_1","doi-asserted-by":"publisher","DOI":"10.1002\/cne.21345"},{"key":"e_1_2_8_20_1","first-page":"66","volume-title":"MitoEM Dataset: Large\u2010scale 3D Mitochondria Instance Segmentation From EM Images","author":"Wei D.","year":"2020"},{"key":"e_1_2_8_21_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.bios.2020.112886"},{"key":"e_1_2_8_22_1","doi-asserted-by":"publisher","DOI":"10.1007\/978\u20101\u201062703\u2010137\u20100_16"},{"key":"e_1_2_8_23_1","doi-asserted-by":"publisher","DOI":"10.1523\/JNEUROSCI.2901\u201019.2020"},{"key":"e_1_2_8_24_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.conb.2018.04.030"},{"key":"e_1_2_8_25_1","doi-asserted-by":"publisher","DOI":"10.1162\/neco.2009.10\u201008\u2010881"},{"key":"e_1_2_8_26_1","unstructured":"S. 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