We present an open source high content analysis instrument
utilizing automated fluorescence lifetime imaging (FLIM) for assaying protein interactions using Förster resonance energy
transfer (FRET) based readouts of fixed or live cells in multiwell plates.
This provides a means to screen for cell signaling processes read out using intramolecular FRET biosensors or intermolecular FRET of
protein interactions such as oligomerization or heterodimerization,
which can be used to identify binding partners. We describe here
the functionality of this automated multiwell plate FLIM instrumentation and present exemplar data from our studies of HIV Gag protein
oligomerization and a time course of a FRET biosensor in live cells.
A detailed description of the practical implementation is then
provided with reference to a list of hardware components and
a description of the open source data acquisition software written
in μ Manager. The application of FLIMfit, an open source MATLAB-
based client for the OMERO platform, to analyze arrays of multiwell plate FLIM data is also presented. The protocols for
imaging fixed and live cells are outlined and a demonstration of an automated multiwell plate FLIM experiment using cells expressing fluorescent protein-based FRET constructs is presented.
This is complemented by a walk-through of the data analysis for this specific FLIM FRET data set.