Automated multiwell fluorescence lifetime imaging for Förster resonance energy transfer assays and high content analysis

2015 ◽  
Vol 7 (10) ◽  
pp. 4071-4089 ◽  
Author(s):  
Douglas J. Kelly ◽  
Sean C. Warren ◽  
Dominic Alibhai ◽  
Sunil Kumar ◽  
Yuriy Alexandrov ◽  
...  
Keyword(s):  
Content Analysis ◽  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Förster Resonance Energy Transfer ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
High Content Analysis ◽  
Förster Resonance

An HCA-FLIM instrument is presented alongside exemplar oligomerisation, intermolecular and intramolecular FRET assays that require robust measurement of small lifetime changes.

scholarly journals Screening for protein-protein interactions using Förster resonance energy transfer (FRET) and fluorescence lifetime imaging microscopy (FLIM)

2016 ◽  
Vol 6 (1) ◽  
Author(s):  
Anca Margineanu ◽  
Jia Jia Chan ◽  
Douglas J. Kelly ◽  
Sean C. Warren ◽  
Delphine Flatters ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Fluorescence Lifetime ◽  
Protein Interactions ◽  
Resonance Energy Transfer ◽  
Resonance Energy ◽  
Forster Resonance Energy Transfer ◽  
Förster Resonance Energy Transfer ◽  
Fluorescence Lifetime Imaging ◽  
Lifetime Imaging ◽  
Förster Resonance

Abstract We present a high content multiwell plate cell-based assay approach to quantify protein interactions directly in cells using Förster resonance energy transfer (FRET) read out by automated fluorescence lifetime imaging (FLIM). Automated FLIM is implemented using wide-field time-gated detection, typically requiring only 10 s per field of view (FOV). Averaging over biological, thermal and shot noise with 100’s to 1000’s of FOV enables unbiased quantitative analysis with high statistical power. Plotting average donor lifetime vs. acceptor/donor intensity ratio clearly identifies protein interactions and fitting to double exponential donor decay models provides estimates of interacting population fractions that, with calibrated donor and acceptor fluorescence intensities, can yield dissociation constants. We demonstrate the application to identify binding partners of MST1 kinase and estimate interaction strength among the members of the RASSF protein family, which have important roles in apoptosis via the Hippo signalling pathway. K D values broadly agree with published biochemical measurements.

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