title: Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy.
abstract: We present an open source high content analysis instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts of fixed or live cells in multiwell plates. This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of protein interactions such as oligomerization or heterodimerization, which can be used to identify binding partners. We describe here the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein oligomerization and a time course of a FRET biosensor in live cells. A detailed description of the practical implementation is then provided with reference to a list of hardware components and a description of the open source data acquisition software written in µManager. The application of FLIMfit, an open source MATLAB-based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented. This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.
authors.affiliation: Photonics Group, Department of Physics, Imperial College London; f.gorlitz14@imperial.ac.uk.
authors.forename: Frederik
authors.initials: F
authors.lastname: Görlitz
authors.affiliation: Photonics Group, Department of Physics, Imperial College London.
authors.forename: Douglas J
authors.initials: DJ
authors.lastname: Kelly
authors.affiliation: Photonics Group, Department of Physics, Imperial College London.
authors.forename: Sean C
authors.initials: SC
authors.lastname: Warren
authors.affiliation: Institute for Chemical Biology, Department of Chemistry, Imperial College London.
authors.forename: Dominic
authors.initials: D
authors.lastname: Alibhai
authors.affiliation: MRC Clinical Sciences Centre, Hammersmith Hospital.
authors.forename: Lucien
authors.initials: L
authors.lastname: West
authors.affiliation: Photonics Group, Department of Physics, Imperial College London.
authors.forename: Sunil
authors.initials: S
authors.lastname: Kumar
authors.affiliation: Photonics Group, Department of Physics, Imperial College London.
authors.forename: Yuriy
authors.initials: Y
authors.lastname: Alexandrov
authors.affiliation: Photonics Group, Department of Physics, Imperial College London.
authors.forename: Ian
authors.initials: I
authors.lastname: Munro
authors.affiliation: Photonics Group, Department of Physics, Imperial College London.
authors.forename: Edwin
authors.initials: E
authors.lastname: Garcia
authors.affiliation: Photonics Group, Department of Physics, Imperial College London.
authors.forename: James
authors.initials: J
authors.lastname: McGinty
authors.affiliation: Photonics Group, Department of Physics, Imperial College London.
authors.forename: Clifford
authors.initials: C
authors.lastname: Talbot
authors.affiliation: Chemical Biology Section, Department of Chemistry, Imperial College London.
authors.forename: Remigiusz A
authors.initials: RA
authors.lastname: Serwa
authors.affiliation: Chemical Biology Section, Department of Chemistry, Imperial College London.
authors.forename: Emmanuelle
authors.initials: E
authors.lastname: Thinon
authors.affiliation: MRC Clinical Sciences Centre, Hammersmith Hospital.
authors.forename: Vincenzo
authors.initials: V
authors.lastname: da Paola
authors.affiliation: Retroscreen Virology Ltd.
authors.forename: Edward J
authors.initials: EJ
authors.lastname: Murray
authors.affiliation: Pfizer Global Research and Development, Pfizer Limited, Sandwich, Kent, UK.
authors.forename: Frank
authors.initials: F
authors.lastname: Stuhmeier
authors.affiliation: Photonics Group, Department of Physics, Imperial College London.
authors.forename: Mark A A
authors.initials: MA
authors.lastname: Neil
authors.affiliation: Chemical Biology Section, Department of Chemistry, Imperial College London.
authors.forename: Edward W
authors.initials: EW
authors.lastname: Tate
authors.affiliation: Photonics Group, Department of Physics, Imperial College London; Centre for Histopathology, Imperial College London.
authors.forename: Christopher
authors.initials: C
authors.lastname: Dunsby
authors.affiliation: Photonics Group, Department of Physics, Imperial College London.
authors.forename: Paul M W
authors.initials: PM
authors.lastname: French
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D019556:COS Cells; D002522:Chlorocebus aethiops; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods*; D061848:Optical Imaging; D012984:Software; D054301:gag Gene Products, Human Immunodeficiency Virus / Q000737:chemistry
chemical_list: D054301:gag Gene Products, Human Immunodeficiency Virus