title: FLIM FRET technology for drug discovery: automated multiwell-plate high-content analysis, multiplexed readouts and application in situ.
abstract: A fluorescence lifetime imaging (FLIM) technology platform intended to read out changes in Förster resonance energy transfer (FRET) efficiency is presented for the study of protein interactions across the drug-discovery pipeline. FLIM provides a robust, inherently ratiometric imaging modality for drug discovery that could allow the same sensor constructs to be translated from automated cell-based assays through small transparent organisms such as zebrafish to mammals. To this end, an automated FLIM multiwell-plate reader is described for high content analysis of fixed and live cells, tomographic FLIM in zebrafish and FLIM FRET of live cells via confocal endomicroscopy. For cell-based assays, an exemplar application reading out protein aggregation using FLIM FRET is presented, and the potential for multiple simultaneous FLIM (FRET) readouts in microscopy is illustrated.
authors.affiliation: Photonics Group, Department of Physics, Imperial College London, London SW7 2AZ, UK. sunil.kumar@imperial.ac.uk
authors.forename: Sunil
authors.initials: S
authors.lastname: Kumar
authors.forename: Dominic
authors.initials: D
authors.lastname: Alibhai
authors.forename: Anca
authors.initials: A
authors.lastname: Margineanu
authors.forename: Romain
authors.initials: R
authors.lastname: Laine
authors.forename: Gordon
authors.initials: G
authors.lastname: Kennedy
authors.forename: James
authors.initials: J
authors.lastname: McGinty
authors.forename: Sean
authors.initials: S
authors.lastname: Warren
authors.forename: Douglas
authors.initials: D
authors.lastname: Kelly
authors.forename: Yuriy
authors.initials: Y
authors.lastname: Alexandrov
authors.forename: Ian
authors.initials: I
authors.lastname: Munro
authors.forename: Clifford
authors.initials: C
authors.lastname: Talbot
authors.forename: Daniel W
authors.initials: DW
authors.lastname: Stuckey
authors.forename: Christopher
authors.initials: C
authors.lastname: Kimberly
authors.forename: Bertrand
authors.initials: B
authors.lastname: Viellerobe
authors.forename: Francois
authors.initials: F
authors.lastname: Lacombe
authors.forename: Eric W-F
authors.initials: EW
authors.lastname: Lam
authors.forename: Harriet
authors.initials: H
authors.lastname: Taylor
authors.forename: Margaret J
authors.initials: MJ
authors.lastname: Dallman
authors.forename: Gordon
authors.initials: G
authors.lastname: Stamp
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Murray
authors.forename: Frank
authors.initials: F
authors.lastname: Stuhmeier
authors.forename: Alessandro
authors.initials: A
authors.lastname: Sardini
authors.forename: Matilda
authors.initials: M
authors.lastname: Katan
authors.forename: Daniel S
authors.initials: DS
authors.lastname: Elson
authors.forename: Mark A A
authors.initials: MA
authors.lastname: Neil
authors.forename: Chris
authors.initials: C
authors.lastname: Dunsby
authors.forename: Paul M W
authors.initials: PM
authors.lastname: French
mesh_terms: D002460:Cell Line; D004353:Drug Evaluation, Preclinical; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins / Q000737:chemistry; D006801:Humans; D008856:Microscopy, Fluorescence; D011485:Protein Binding; D011506:Proteins / Q000032:analysis*; D012235:Rhodamines / Q000737:chemistry; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000032:analysis
chemical_list: D005456:Fluorescent Dyes; D011506:Proteins; D012235:Rhodamines; D054301:gag Gene Products, Human Immunodeficiency Virus; C026188:rhodamine 6G; D049452:Green Fluorescent Proteins; C029773:rhodamine B