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Micro\u2010CT imaging of contrast\u2010stained nerves enables the visualization of tissue microstructures, such as the fascicular perineurium and vasculature. In this work, we evaluated phosphotungstic acid (PTA) as a contrast agent and assessed its compatibility with downstream histology.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Human vagus nerve samples were collected from three embalmed cadavers and subjected to three different staining methods, followed by micro\u2010CT imaging: Lugol's iodine, osmium tetroxide, and PTA. Contrast ratios of adjacent tissue microstructures (perineurium, interfascicular epineurium, and fascicle) were quantified for each stain and compared. We further developed a pipeline to optimize micro\u2010CT scan acquisition parameters based on objective metrics for sharpness, noise, and pixel saturation. The PTA\u2010stained samples underwent subsequent histological processing and staining with hematoxylin and eosin, Masson's trichrome, and immunohistochemistry and were assessed for tissue degradation.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>PTA enhanced the visualization of perineurium, providing high contrast ratios compared to iodine and osmium tetroxide. Optimized scanning parameters for PTA\u2010stained nerves (55\u00a0kV and 109\u00a0\u00b5A) effectively balanced noise and sharpness. While we found that PTA is generally nondestructive for downstream histology, higher concentrations and longer exposure could alter the optical density of nuclei and affect stain differentiation in special stains.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>PTA serves as a valuable micro\u2010CT contrast agent for nerve imaging, effective in visualizing the perineurium with minimal impact on histological integrity.<\/jats:p><\/jats:sec>","DOI":"10.1111\/jon.70040","type":"journal-article","created":{"date-parts":[[2025,4,10]],"date-time":"2025-04-10T11:48:34Z","timestamp":1744285714000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Phosphotungstic Acid Staining to Visualize the Vagus Nerve Perineurium Using Micro\u2010CT"],"prefix":"10.1111","volume":"35","author":[{"given":"Aniruddha R.","family":"Upadhye","sequence":"first","affiliation":[{"name":"Department of Biomedical Engineering Case Western Reserve University  Cleveland Ohio USA"},{"name":"APT Center Louis Stokes Cleveland VA Medical Center  Cleveland Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Eleana","family":"Cintron","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Case Western Reserve University  Cleveland Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jichu","family":"Zhang","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Case Western Reserve University  Cleveland Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jennifer","family":"Coleman","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Case Western Reserve University  Cleveland Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Chaitanya","family":"Kolluru","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Case Western Reserve University  Cleveland Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael W.","family":"Jenkins","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Case Western Reserve University  Cleveland Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David","family":"Wilson","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Case Western Reserve University  Cleveland Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Nicole A.","family":"Pelot","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Duke University  Durham North Carolina USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0881-2180","authenticated-orcid":false,"given":"Andrew J.","family":"Shoffstall","sequence":"additional","affiliation":[{"name":"Department of Biomedical Engineering Case Western Reserve University  Cleveland Ohio USA"},{"name":"APT Center Louis Stokes Cleveland VA Medical Center  Cleveland Ohio USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,4,10]]},"reference":[{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1088\/1741-2552\/ab9db8"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.4103\/2152-7806.91610"},{"key":"e_1_2_6_4_1","first-page":"54","article-title":"Vagus Nerve Stimulation (VNS) and Treatment of Depression: To the Brainstem and Beyond","volume":"3","author":"O'Reardon J. 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