title: Automated High-Throughput Fluorescence Lifetime Imaging Microscopy to Detect Protein-Protein Interactions.
abstract: Fluorescence resonance energy transfer (FRET) is widely used to study conformational changes of macromolecules and protein-protein, protein-nucleic acid, and protein-small molecule interactions. FRET biosensors can serve as valuable secondary assays in drug discovery and for target validation in mammalian cells. Fluorescence lifetime imaging microscopy (FLIM) allows precise quantification of the FRET efficiency in intact cells, as FLIM is independent of fluorophore concentration, detection efficiency, and fluorescence intensity. We have developed an automated FLIM system using a commercial frequency domain FLIM attachment (Lambert Instruments) for wide-field imaging. Our automated FLIM system is capable of imaging and analyzing up to 50 different positions of a slide in less than 4 min, or the inner 60 wells of a 96-well plate in less than 20 min. Automation is achieved using a motorized stage and controller (Prior Scientific) coupled with a Zeiss Axio Observer body and full integration into the Lambert Instruments FLIM acquisition software. As an application example, we analyze the interaction of the oncoprotein Ras and its effector Raf after drug treatment. In conclusion, our automated FLIM imaging system requires only commercial components and may therefore allow for a broader use of this technique in chemogenomics projects.
authors.affiliation: Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland.
authors.forename: Camilo
authors.initials: C
authors.lastname: Guzmán
authors.affiliation: Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland.
authors.forename: Christina
authors.initials: C
authors.lastname: Oetken-Lindholm
authors.affiliation: Turku Centre for Biotechnology, Åbo Akademi University, Turku, Finland daniel.abankwa@btk.fi.
authors.forename: Daniel
authors.initials: D
authors.lastname: Abankwa
mesh_terms: D057205:Automation, Laboratory / Q000379:methods*; D031541:Fluorescence Resonance Energy Transfer; D057166:High-Throughput Screening Assays; D008856:Microscopy, Fluorescence / Q000379:methods*; D061848:Optical Imaging / Q000379:methods*; D025941:Protein Interaction Mapping / Q000379:methods*; D048490:raf Kinases / Q000378:metabolism; D018631:ras Proteins / Q000378:metabolism
chemical_list: D048490:raf Kinases; D018631:ras Proteins
keywords: FLIM; FRET; automation; chemogenomic screening; high-throughput imaging