FLIM FRET Technology for Drug Discovery: Automated Multiwell-Plate High-Content Analysis, Multiplexed Readouts and Application in Situ

• Published in: Chemphyschem Vol. 12; no. 3; pp. 609 - 626
• Main Authors: Kumar, Sunil, Alibhai, Dominic, Margineanu, Anca, Laine, Romain, Kennedy, Gordon, McGinty, James, Warren, Sean, Kelly, Douglas, Alexandrov, Yuriy, Munro, Ian, Talbot, Clifford, Stuckey, Daniel W., Kimberly, Christopher, Viellerobe, Bertrand, Lacombe, Francois, Lam, Eric W.-F., Taylor, Harriet, Dallman, Margaret J., Stamp, Gordon, Murray, Edward J., Stuhmeier, Frank, Sardini, Alessandro, Katan, Matilda, Elson, Daniel S., Neil, Mark A. A., Dunsby, Chris, French, Paul M. W.
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 25.02.2011; WILEY‐VCH Verlag; Wiley
• Subjects: Biological and medical sciences; Cell Line; Chemistry; drug discovery; Drug Evaluation, Preclinical; Exact sciences and technology; fluorescence lifetime imaging; Fluorescence Resonance Energy Transfer - methods; Fluorescent Dyes - chemistry; FRET; Fundamental and applied biological sciences. Psychology; gag Gene Products, Human Immunodeficiency Virus - analysis; General and physical chemistry; Green Fluorescent Proteins - chemistry; high-throughput screening; Humans; Medical sciences; Microscopy, Fluorescence; Pharmacology. Drug treatments; Protein Binding; proteins; Proteins - analysis; Rhodamines - chemistry; High throughput screening; Förster resonance energy transfer; In situ; Fluorescence; Molecular interaction; Potential; Aggregation; Assay; drug discovery; Efficiency; Technology; Pisces; Imaging; proteins; FRET; Drug; Automation; high-throughput screening; Method; Protein; Plate; Vertebrata; Brachydanio rerio; Lifetime; Mammalia; fluorescence lifetime imaging; Microscopy; Content; End; Application
• ISSN: 1439-4235; 1439-7641
• DOI: 10.1002/cphc.201000874

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. Chance of a lifetime: A fluorescence lifetime imaging (FLIM) technology platform to read out changes in Förster resonance energy transfer (FRET) efficiency is used for the study of protein interactions across the drug‐discovery pipeline. An automated FLIM multiwell‐plate reader acquires lifetime images of fixed and live cells labelled with fluorescent proteins in a few seconds per field of view (see picture) and can read a 96‐well plate in less than 15 min.