Fluorescence lifetime measurements can provide quantitative
readouts of local fluorophore environment and
can be applied to biomolecular interactions via Fo¨ rster
resonant energy transfer (FRET). Fluorescence lifetime
imaging (FLIM) can therefore provide a high content
analysis (HCA) modality to map protein-protein interactions
(PPIs) with applications in drug discovery, systems
biology and basic research. We present here an automated
multiwell plate reader able to perform rapid unsupervised
optically sectioned FLIM of fixed and live
biological samples and illustrate its potential to assay
PPIs through application to Gag protein aggregation
during the HIV life cycle. We demonstrate both heteroFRET
and homo-FRET readouts of protein aggregation
and report the first quantitative evaluation of a FLIM
HCA assay by generating dose response curves through
addition of an inhibitor of Gag myristoylation. Z0 factors
exceeding 0.6 are realised for this FLIM FRET assay.
Fluorescence lifetime plate map with representative
images of high and low FRET cells and corresponding
dose response plot.