title: Time-resolved luminescence resonance energy transfer imaging of protein-protein interactions in living cells.
abstract: Lanthanide-based or luminescence resonance energy transfer (LRET) microscopy can be used to sensitively image interactions between reporter-labeled proteins in living mammalian cells. With LRET, luminescent lanthanide complexes are used as donors, conventional fluorophores are used as acceptors, and donor-sensitized acceptor emission occurs at time scales that reflect the long (~ms) lanthanide emission lifetime. These long-lived signals can be separated from short-lifetime (~ns) sample autofluorescence and directly excited acceptor fluorescence by using pulsed light to excite the specimen and by implementing a short delay (>100 ns) before detection, thereby increasing measurement sensitivity. As practical implementation of time-resolved LRET microscopy requires several potentially unfamiliar experimental techniques, we explicitly describe herein methods to label proteins in living mammalian cells with luminescent terbium complexes, image interactions between terbium-labeled proteins and green fluorescent protein fusions, and quantitatively analyze LRET images.
authors.affiliation: Department of Chemistry, University of Illinois at Chicago, Chicago, Illinois, USA.
authors.forename: Harsha E
authors.initials: HE
authors.lastname: Rajapakse
authors.forename: Lawrence W
authors.initials: LW
authors.lastname: Miller
mesh_terms: D000818:Animals; D058948:Cell Tracking / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins; D007091:Image Processing, Computer-Assisted / Q000379:methods; D028581:Lanthanoid Series Elements / Q000737:chemistry; D049449:Luminescence; D051379:Mice; D008856:Microscopy, Fluorescence / Q000379:methods*; D018715:Microscopy, Video / Q000379:methods; D041681:NIH 3T3 Cells; D060066:Protein Interaction Maps; D013194:Staining and Labeling; D013725:Terbium / Q000737:chemistry
chemical_list: D028581:Lanthanoid Series Elements; D013725:Terbium; D049452:Green Fluorescent Proteins