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Apart from regulating the exchange of solutes, uptake of nutrients, and excretion of waste products, their major function is to prevent uncontrolled access of foreign material to immune\u2010competent compartments. Progress in understanding this barrier function toward larger solutes and its possible defects, as can be seen in a variety of diseases, is largely hampered by a lack of methods to spatiotemporally resolve transepithelial passage of macromolecules. Using different cell culture epithelia, we applied biotinylated dextran tracers carrying an acceptor fluorophore. These bind to cell\u2010adherent avidin carrying donor fluorophore at the basolateral membranes of single\u2010layered epithelial sheets. Confocal fluorescence microscopy was applied to living epithelia in order to image apical\u2010to\u2010basolateral tracer passage as a F\u00f6rster resonance energy transfer signal of the fluorescent dextran\u2013avidin pair over time. Stimulated macromolecule passage using barrier\u2010perturbing agents proved its effectiveness for the leak imaging method presented herein. Over hours of imaging, spontaneous leaks were rare, occurring transiently on the scale of minutes and for the most part associated with rearranging cell junctions. The discussed approach to leak imaging is expected to promote the understanding of epithelial barriers, particularly, the nature and dynamics of the epithelial cell leak pathway.<\/jats:p>","DOI":"10.1111\/nyas.14847","type":"journal-article","created":{"date-parts":[[2022,6,29]],"date-time":"2022-06-29T11:11:36Z","timestamp":1656501096000},"page":"151-161","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Probing the leak pathway: Live\u2010cell imaging of macromolecule passage through epithelia"],"prefix":"10.1111","volume":"1516","author":[{"given":"Jan F.","family":"Richter","sequence":"first","affiliation":[{"name":"Jena University Hospital, Institute of Anatomy II Friedrich Schiller University Jena  Jena Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Markus","family":"Hildner","sequence":"additional","affiliation":[{"name":"Jena University Hospital, Institute of Anatomy II Friedrich 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