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A promising treatment approach applies photodynamic therapy (PDT) with hypericin as a photosensitizer. To generate a comprehensive understanding of the photosensitizer-tumor interactions, the first part of our study is focused on investigating the distribution and penetration behavior of hypericin in glioma cell spheroids by fluorescence microscopy. In the second part, fluorescence lifetime imaging microscopy (FLIM) was used to correlate fluorescence lifetime (FLT) changes of hypericin to environmental effects inside the spheroids. In this context, 3D tumor spheroids are an excellent model system since they consider 3D cell\u2013cell interactions and the extracellular matrix\u00a0is similar to tumors in vivo. Our analytical approach considers hypericin as probe molecule for FLIM and as photosensitizer for PDT at the same time, making it possible to directly draw conclusions of the state and location of the drug in a biological system. The knowledge of both state and location of hypericin  makes a fundamental understanding of the impact of hypericin PDT in brain tumors possible. Following different incubation conditions, the hypericin distribution in peripheral and central cryosections of the spheroids were analyzed. Both fluorescence microscopy and FLIM revealed a hypericin gradient towards the spheroid core for short incubation periods or small concentrations. On the other hand, a homogeneous hypericin distribution is observed for long incubation times and high concentrations. Especially, the observed FLT change is crucial for the PDT efficiency, since the triplet yield, and hence the O\n                    <jats:sub>2<\/jats:sub>\n                    activation, is directly proportional to the FLT. Based on the FLT increase inside spheroids, an incubation time\u2009&gt;\u200930\u00a0min is required to achieve most suitable conditions for an effective PDT.\n                  <\/jats:p>\n                  <jats:p>\n                    <jats:bold>Graphical abstract<\/jats:bold>\n                  <\/jats:p>","DOI":"10.1007\/s00216-022-04107-2","type":"journal-article","created":{"date-parts":[[2022,5,10]],"date-time":"2022-05-10T18:02:22Z","timestamp":1652205742000},"page":"4849-4860","update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Accumulation and penetration behavior of hypericin in glioma tumor spheroids studied by fluorescence microscopy and confocal fluorescence lifetime imaging microscopy"],"prefix":"10.1007","volume":"414","author":[{"given":"Miriam C.","family":"Bassler","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tim","family":"Rammler","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Frank","family":"Wackenhut","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sven","family":"zur Oven-Krockhaus","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ivona","family":"Secic","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rainer","family":"Ritz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alfred J.","family":"Meixner","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marc","family":"Brecht","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2022,5,10]]},"reference":[{"issue":"4","key":"4107_CR1","doi-asserted-by":"publisher","first-page":"778","DOI":"10.1002\/ijc.33588","volume":"149","author":"J Ferlay","year":"2021","unstructured":"Ferlay J, Colombet M, Soerjomataram I, Parkin DM, Pi\u00f1eros M, Znaor A, Bray F. 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