{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,1,24]],"date-time":"2026-01-24T02:18:03Z","timestamp":1769221083468,"version":"3.49.0"},"reference-count":36,"publisher":"American Society for Cell Biology (ASCB)","issue":"11","content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["MBoC"],"published-print":{"date-parts":[[2015,6]]},"abstract":"<jats:p>We report an improved variant of mKeima, a monomeric long Stokes shift red fluorescent protein, hmKeima8.5. The increased intracellular brightness and large Stokes shift (\u223c180 nm) make it an excellent partner with teal fluorescent protein (mTFP1) for multiphoton, multicolor applications. Excitation of this pair by a single multiphoton excitation wavelength (MPE, 850 nm) yields well-separable emission peaks (\u223c120-nm separation). Using this pair, we measure homo- and hetero-oligomerization interactions in living cells via multiphoton excitation fluorescence correlation spectroscopy (MPE-FCS). Using tandem dimer proteins and small-molecule inducible dimerization domains, we demonstrate robust and quantitative detection of intracellular protein\u2013protein interactions. We also use MPE-FCCS to detect drug\u2013protein interactions in the intracellular environment using a Coumarin 343 (C343)-conjugated drug and hmKeima8.5 as a fluorescence pair. The mTFP1\/hmKeima8.5 and C343\/hmKeima8.5 combinations, together with our calibration constructs, provide a practical and broadly applicable toolbox for the investigation of molecular interactions in the cytoplasm of living cells.<\/jats:p>","DOI":"10.1091\/mbc.e14-10-1473","type":"journal-article","created":{"date-parts":[[2015,4,16]],"date-time":"2015-04-16T02:31:42Z","timestamp":1429151502000},"page":"2054-2066","source":"Crossref","is-referenced-by-count":20,"title":["Live-cell multiphoton fluorescence correlation spectroscopy with an improved large Stokes shift fluorescent protein"],"prefix":"10.1091","volume":"26","author":[{"given":"Yinghua","family":"Guan","sequence":"first","affiliation":[{"name":"Department of Systems Biology, Harvard Medical School, Boston, MA 02115"},{"name":"Renal Division, Brigham and Women's Hospital, Boston, MA 02115"}]},{"given":"Matthias","family":"Meurer","sequence":"additional","affiliation":[{"name":"Zentrum f\u00fcr Molekulare Biologie der 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Portugal"}]},{"given":"Ilia","family":"Kats","sequence":"additional","affiliation":[{"name":"Zentrum f\u00fcr Molekulare Biologie der Universit\u00e4t Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH-Allianz, 69120 Heidelberg, Germany"}]},{"given":"Michael W.","family":"Davidson","sequence":"additional","affiliation":[{"name":"National High Magnetic Field Laboratory, Florida State University, Tallahassee, FL 32310"}]},{"given":"Ralph","family":"Mazitschek","sequence":"additional","affiliation":[{"name":"Center for Systems Biology, Massachusetts General Hospital, Boston, MA 02114"},{"name":"Broad Institute of Harvard and MIT, Cambridge, MA 02142"}]},{"given":"Thomas E.","family":"Hughes","sequence":"additional","affiliation":[{"name":"Department of Cell Biology and Neuroscience, Montana State University, Bozeman, MT 59717"}]},{"given":"Mikhail","family":"Drobizhev","sequence":"additional","affiliation":[{"name":"Department of Physics, Montana State University, Bozeman, MT 59717"}]},{"given":"Michael","family":"Knop","sequence":"additional","affiliation":[{"name":"Zentrum f\u00fcr Molekulare Biologie der Universit\u00e4t Heidelberg and Deutsches Krebsforschungszentrum, DKFZ-ZMBH-Allianz, 69120 Heidelberg, Germany"}]},{"given":"Jagesh V.","family":"Shah","sequence":"additional","affiliation":[{"name":"Department of Systems Biology, Harvard Medical School, Boston, MA 02115"},{"name":"Renal Division, Brigham and Women's Hospital, Boston, MA 02115"}]}],"member":"1076","reference":[{"key":"B1","doi-asserted-by":"crossref","first-page":"531","DOI":"10.1042\/BJ20060874","volume":"400","author":"Ai H-W","year":"2006","journal-title":"Biochem J"},{"key":"B2","doi-asserted-by":"crossref","first-page":"2842","DOI":"10.1038\/nprot.2007.410","volume":"2","author":"Bacia K","year":"2007","journal-title":"Nat Protocols"},{"key":"B3","doi-asserted-by":"crossref","first-page":"756","DOI":"10.15252\/msb.20145422","volume":"10","author":"Boeke 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