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Current imaging modalities and biochemical assays require many specific markers that should be detected to identify the hypoxic response, especially at a level of single cells. This study presents a single-cell molecular imaging approach utilizing Fourier-Transform Infrared and Raman spectroscopy. Those methods enable the simultaneous detection of proteins, lipids, and nucleic acids encoded in their unique vibrational fingerprints. By establishing ratiometric estimators, we measured upregulated lipid metabolism, structural changes of proteins and asses DNA:RNA ratio at the single-cell level induced by oxygen depletion. Moreover, this approach allows for analyzing changes within specific cellular compartments, including nuclei, providing a comprehensive understanding of how hypoxia affects cellular functions and metabolism. Our findings pave the way for future investigations into the cellular adaptations to hypoxia in brain endothelial cells, potentially revealing novel therapeutic targets for neurodegenerative diseases.<\/jats:p>","DOI":"10.1038\/s41598-025-11000-2","type":"journal-article","created":{"date-parts":[[2025,7,18]],"date-time":"2025-07-18T14:12:31Z","timestamp":1752847951000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Marker-independent vibrational spectroscopy imaging recognizes the hypoxia effect in the human brain endothelium"],"prefix":"10.1038","volume":"15","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-3156-4194","authenticated-orcid":false,"given":"Aleksandra","family":"Pragn\u0105ca","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8008-8684","authenticated-orcid":false,"given":"Anna","family":"Antolak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9913-5479","authenticated-orcid":false,"given":"Zuzanna J.","family":"Krysiak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0340-4054","authenticated-orcid":false,"given":"Monika","family":"Le\u015bniak","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3273-4610","authenticated-orcid":false,"given":"Agata","family":"Borkowska","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0455-1072","authenticated-orcid":false,"given":"Robert","family":"Zdanowski","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0582-2743","authenticated-orcid":false,"given":"Kamilla","family":"Malek","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,7,18]]},"reference":[{"key":"11000_CR1","doi-asserted-by":"publisher","first-page":"490","DOI":"10.1016\/j.redox.2018.01.008","volume":"15","author":"JN Cobley","year":"2018","unstructured":"Cobley, J. 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