Hammersmith Hospital View Institution's Website 1 article published in JoVE Biology Open Source High Content Analysis Utilizing Automated Fluorescence Lifetime Imaging Microscopy Frederik Görlitz*1, Douglas J. Kelly*1, Sean C. Warren1, Dominic Alibhai2, Lucien West3, Sunil Kumar1, Yuriy Alexandrov1, Ian Munro1, Edwin Garcia1, James McGinty1, Clifford Talbot1, Remigiusz A. Serwa4, Emmanuelle Thinon4, Vincenzo da Paola3, Edward J. Murray5, Frank Stuhmeier6, Mark A. A. Neil1, Edward W. Tate4, Christopher Dunsby1,7, Paul M. W. French1 1Photonics Group, Department of Physics, Imperial College London, 2Institute for Chemical Biology, Department of Chemistry, Imperial College London, 3MRC Clinical Sciences Centre, Hammersmith Hospital, 4Chemical Biology Section, Department of Chemistry, Imperial College London, 5Retroscreen Virology Ltd, 6Pfizer Global Research and Development, Pfizer Limited, Sandwich, Kent, UK, 7Centre for Histopathology, Imperial College London We present an open source high content analysis (HCA) instrument utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy transfer (FRET) based readouts. Data acquisition for this openFLIM-HCA instrument is controlled by software written in µManager and data analysis is undertaken in FLIMfit.