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We show that fluorescence lifetime imaging using two\u2010photon excitation of SYTO13 allows differential and simultaneous imaging of DNA and RNA in living cells, as well as sequential and repetitive assessment of staining patterns.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Two\u2010photon imaging of SYTO13 is combined with lifetime contrast, using time\u2010gated detection. We focus on distinguishing DNA and RNA in healthy and apoptotic Chinese hamster ovary cells.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>In healthy cells, SYTO13 has a fluorescence lifetime of 3.4 \u00b1 0.2 ns when associated with nuclear DNA. Bound to RNA, its lifetime is 4.1 \u00b1 0.1 ns. After induction of apoptosis, clusters of SYTO13 with fluorescence lifetime of 3.4 \u00b1 0.2 ns become apparent in the cytoplasm. They are identified as mitochondrial DNA on the basis of colocalization experiments with the DNA\u2010specific dye, DRAQ5, and the mitochondrial\u2010specific dye, CMXRos. Upon progression of apoptosis, the lifetime of SYTO13 attached to DNA shortens significantly, which is indicative of changes in the molecular environment of the dye.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>We have characterized SYTO13 as a vital lifetime probe, allowing repetitive and differential imaging of DNA and RNA. Cytometry 47:226\u2013235, 2002. \u00a9 2002 Wiley\u2010Liss, Inc.<\/jats:p><\/jats:sec>","DOI":"10.1002\/cyto.10076","type":"journal-article","created":{"date-parts":[[2002,8,25]],"date-time":"2002-08-25T23:15:07Z","timestamp":1030317307000},"page":"226-235","source":"Crossref","is-referenced-by-count":83,"title":["Discrimination of DNA and RNA in cells by a vital fluorescent probe: Lifetime imaging of SYTO13 in healthy and apoptotic cells"],"prefix":"10.1002","volume":"47","author":[{"given":"Marc A.M.J.","family":"van Zandvoort","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Cees J.","family":"de Grauw","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Hans C.","family":"Gerritsen","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Jos L.V.","family":"Broers","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Mirjam G.A.","family":"oude Egbrink","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Frans C.S.","family":"Ramaekers","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Dick W.","family":"Slaaf","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2002,3,21]]},"reference":[{"key":"e_1_2_6_2_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0955-0674(98)80007-0"},{"key":"e_1_2_6_3_2","doi-asserted-by":"publisher","DOI":"10.1006\/excr.2000.5027"},{"key":"e_1_2_6_4_2","doi-asserted-by":"publisher","DOI":"10.1615\/CritRevEukarGeneExpr.v9.i3-4.190"},{"key":"e_1_2_6_5_2","doi-asserted-by":"publisher","DOI":"10.1083\/jcb.143.6.1415"},{"key":"e_1_2_6_6_2","doi-asserted-by":"publisher","DOI":"10.1101\/gad.842600"},{"key":"e_1_2_6_7_2","doi-asserted-by":"publisher","DOI":"10.1016\/0968-0004(86)90245-8"},{"key":"e_1_2_6_8_2","article-title":"A high sensitivity spectrograph for use in fluorescence microscopy","author":"Frederix P","journal-title":"Appl Spectrosc"},{"key":"e_1_2_6_9_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0006-3495(00)76759-2"},{"key":"e_1_2_6_10_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2818.2000.00651.x"},{"key":"e_1_2_6_11_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0091-679X(08)60431-8"},{"key":"e_1_2_6_12_2","doi-asserted-by":"publisher","DOI":"10.1016\/0003-2697(92)90112-K"},{"key":"e_1_2_6_13_2","doi-asserted-by":"publisher","DOI":"10.1002\/1361-6374(199506)3:2<49::AID-BIO1>3.0.CO;2-E"},{"key":"e_1_2_6_14_2","first-page":"152","article-title":"Fluorescence lifetime imaging using a confocal laser scanning microscope","volume":"9","author":"Buurman E","year":"1992","journal-title":"Scanning"},{"key":"e_1_2_6_15_2","article-title":"A multiple time\u2010gated module for fluorescence lifetime imaging","author":"de Grauw C","journal-title":"Appl Spectrosc"},{"key":"e_1_2_6_16_2","first-page":"143","volume-title":"Handbook of fluorescent probes and research chemicals","author":"Haugland R","year":"1996"},{"key":"e_1_2_6_17_2","unstructured":"US Patents Nos. 5 436 34; 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