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ASL depletion due to deficient cystic fibrosis transmembrane conductance regulator (CFTR)-mediated anion\/fluid secretion plays an important role in the pathogenesis of mucociliary dysfunction and chronic muco-obstructive lung disease in patients with cystic fibrosis (CF). Quantitative measurement of ASL height by confocal fluorescence microscopy following addition of fluorescent dye has contributed important insight into the (dys)regulation of ASL in health and disease. Here, we present a novel method enabling studies of ASL regulation that does not require the addition of dye by using reflected light by confocal microscopy of primary airway epithelial cultures grown at air-liquid interface (ALI). After apical volume addition to primary tracheal mouse cultures, confocal reflection microscopy yielded comparable ASL height as confocal fluorescence microscopy on cultures of wild-type mice, and was sensitive to detect ASL depletion on cultures of \u03b2ENaC-Tg mice. Under unperturbed conditions, ASL determined by confocal reflection microscopy was significantly higher in wild-type and \u03b2ENaC-Tg mice compared to values obtained by confocal fluorescence microscopy. Studies in normal and CF primary human airway epithelial cultures showed that confocal reflection microscopy was sensitive to detect effects of low temperature rescue and pharmacological modulation including improvement of CFTR function by VX-809 and VX-770 in cultures from CF patients with the F508del mutation. Our results support confocal reflection microscopy as a novel sensitive technique for quantitative studies of ASL regulation and response to therapeutic intervention under near-physiological conditions that may be applicable for studies of (patho)physiology and drug screens in healthy and CF airways.<\/jats:p>","DOI":"10.1038\/s41598-025-32061-3","type":"journal-article","created":{"date-parts":[[2025,12,11]],"date-time":"2025-12-11T18:35:31Z","timestamp":1765478131000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":1,"title":["Reflection confocal microscopy for quantitative assessment of airway surface layer dysregulation and pharmacological rescue in cystic fibrosis under near-physiological conditions"],"prefix":"10.1038","volume":"15","author":[{"given":"Ay\u00e7a Seyhan","family":"Agircan","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marko","family":"Lampe","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Heike","family":"Scheuermann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tobias","family":"Albrecht","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Simon Y.","family":"Graeber","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anita","family":"Bal\u00e1zs","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ingo","family":"Baumann","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Stephan","family":"Block","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rainer","family":"Pepperkok","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marcus A.","family":"Mall","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Julia","family":"Duerr","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2025,12,11]]},"reference":[{"key":"32061_CR1","doi-asserted-by":"publisher","first-page":"571","DOI":"10.1172\/JCI0215217","volume":"109","author":"MR Knowles","year":"2002","unstructured":"Knowles, M. 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MAM reports grants or contracts from the German Research Foundation (DFG), the German Federal Ministry for Research, Technology and Space (BMFTR), Boehringer Ingelheim, Enterprise Therapeutics, and Vertex Pharmaceuticals with payments made to the institution, fees for consultancy or advisory board participation from Boehringer Ingelheim, Enterprise Therapeutics, Kither Biotech, Pari, Splisense and Vertex Pharmaceuticals, payment or honoraria for lectures from Vertex Pharmaceuticals, travel reimbursement received for participation in advisory board meetings for Boehringer Ingelheim and Vertex Pharmaceuticals; and a patent on the Scnn1b-transgenic mouse as an animal model for chronic obstructive pulmonary disease and cystic fibrosis outside the submitted work. No conflicts of interest, financial or otherwise, are declared by the other authors.","order":2,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"43659"}}