{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2025,10,24]],"date-time":"2025-10-24T07:51:53Z","timestamp":1761292313818},"reference-count":29,"publisher":"Wiley","issue":"4","license":[{"start":{"date-parts":[[2006,2,23]],"date-time":"2006-02-23T00:00:00Z","timestamp":1140652800000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Cytometry Pt A"],"published-print":{"date-parts":[[2006,4]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background:<\/jats:title><jats:p>Fluorescence lifetime microscopy (FLIM) is currently one of the best techniques to perform accurate measurements of interactions in living cells. It is independent of the fluorophore concentration, thus avoiding several common artifacts found in F\u00f6rster Resonance Energy Transfer (FRET) imaging. However, for FLIM to achieve high performance, a rigorous instrumental setup and characterization is needed.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods:<\/jats:title><jats:p>We use known fluorophores to perform characterization experiments in our instrumental setup. This allows us to verify the accuracy of the fluorescence lifetime determination, and test the linearity of the instrument by fluorescence quenching.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results:<\/jats:title><jats:p>We develop and validate here a protocol for rigorous characterization of time\u2010domain FLIM instruments. Following this protocol, we show that our system provides accurate and reproducible measurements. We also used HeLa cells expressing proteins fused to Green Fluorescent Proteins variants (CFP and YFP) to confirm its ability to detect interactions in living cells by FRET.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions:<\/jats:title><jats:p>We report a well\u2010designed protocol in which precise and reproducible lifetime measurements can be performed. It is usable for all confocal\u2010based FLIM instruments and is a useful tool for anyone who wants to perform quantitative lifetime measurements, especially when studying interactions in living cells using FRET. \u00a9 2006 Wiley\u2010Liss, Inc.<\/jats:p><\/jats:sec>","DOI":"10.1002\/cyto.a.20240","type":"journal-article","created":{"date-parts":[[2006,2,24]],"date-time":"2006-02-24T00:16:50Z","timestamp":1140740210000},"page":"299-306","source":"Crossref","is-referenced-by-count":30,"title":["Setup and characterization of a multiphoton FLIM instrument for protein\u2013protein interaction measurements in living cells"],"prefix":"10.1002","volume":"69A","author":[{"given":"Fran\u00e7ois","family":"Waharte","sequence":"first","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Corentin","family":"Spriet","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"Laurent","family":"H\u00e9liot","sequence":"additional","affiliation":[],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"311","published-online":{"date-parts":[[2006,2,23]]},"reference":[{"key":"e_1_2_6_2_2","doi-asserted-by":"publisher","DOI":"10.1002\/andp.19484370105"},{"key":"e_1_2_6_3_2","doi-asserted-by":"publisher","DOI":"10.1016\/S0091-679X(08)61962-7"},{"key":"e_1_2_6_4_2","doi-asserted-by":"publisher","DOI":"10.1038\/nbt896"},{"key":"e_1_2_6_5_2","doi-asserted-by":"publisher","DOI":"10.1046\/j.1365-2818.2003.01100.x"},{"key":"e_1_2_6_6_2","doi-asserted-by":"publisher","DOI":"10.1002\/cyto.a.20053"},{"key":"e_1_2_6_7_2","doi-asserted-by":"publisher","DOI":"10.1177\/27.1.438507"},{"key":"e_1_2_6_8_2","doi-asserted-by":"publisher","DOI":"10.1039\/b412924p"},{"key":"e_1_2_6_9_2","doi-asserted-by":"crossref","unstructured":"KoenigK 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