title: Wide-Field Multi-Parameter FLIM: long-term minimal invasive observation of proteins in living cells.
abstract: Time-domain Fluorescence Lifetime Imaging Microscopy (FLIM) is a remarkable tool to monitor the dynamics of fluorophore-tagged protein domains inside living cells. We propose a Wide-Field Multi-Parameter FLIM method (WFMP-FLIM) aimed to monitor continuously living cells under minimum light intensity at a given illumination energy dose. A powerful data analysis technique applied to the WFMP-FLIM data sets allows to optimize the estimation accuracy of physical parameters at very low fluorescence signal levels approaching the lower bound theoretical limit. We demonstrate the efficiency of WFMP-FLIM by presenting two independent and relevant long-term experiments in cell biology: 1) FRET analysis of simultaneously recorded donor and acceptor fluorescence in living HeLa cells and 2) tracking of mitochondrial transport combined with fluorescence lifetime analysis in neuronal processes.
authors.affiliation: Leibniz Institute for Neurobiology, Magdeburg, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Vitali
authors.forename: Fernando
authors.initials: F
authors.lastname: Picazo
authors.forename: Yury
authors.initials: Y
authors.lastname: Prokazov
authors.forename: Alessandro
authors.initials: A
authors.lastname: Duci
authors.forename: Evgeny
authors.initials: E
authors.lastname: Turbin
authors.forename: Christian
authors.initials: C
authors.lastname: Götze
authors.forename: Juan
authors.initials: J
authors.lastname: Llopis
authors.forename: Roland
authors.initials: R
authors.lastname: Hartig
authors.forename: Antonie J W G
authors.initials: AJ
authors.lastname: Visser
authors.forename: Werner
authors.initials: W
authors.lastname: Zuschratter
mesh_terms: D000465:Algorithms; D001692:Biological Transport; D002477:Cells / Q000378:metabolism / Q000648:ultrastructure*; D004526:Efficiency; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D008928:Mitochondria / Q000378:metabolism / Q000502:physiology; D019370:Observation / Q000379:methods; D038761:Photobleaching; D011506:Proteins / Q000032:analysis / Q000378:metabolism*; D059010:Single-Cell Analysis / Q000379:methods; D013997:Time Factors; D059008:Time-Lapse Imaging / Q000379:methods
chemical_list: D011506:Proteins; D049452:Green Fluorescent Proteins