SUPR: Spectral Unmixinig Plate Reader for Live-Cell FRET Biosensor Drug Screening

We have developed a high-throughput Spectral Unmixing Plate Reader (SUPR) which can acquire a fluorescence emission spectrum across 384 wells, with short (< 4 min) read time for the entire plate. The entire spectrum is recorded at once with a compact spectrometer equipped with an uncooled one-dimensional array detector. For application to FRET in living cells, FRET protein biosensors, with genetically encoded GFP (donor) and RFP (acceptor) tags, are expressed in HEK cells, and single-component spectra are acquired from water (Raman), untransfected cells (autofluorescence), cells expressing donor only, and cells expressing acceptor only. Each observed spectrum is decomposed into a linear combination of component spectra to determine FRET efficiency. The method enables unprecedented speed, precision, and accuracy. It rigorously and painlessly accounts for phenomena such as bleed-through of the donor emission into the acceptor channel, cross-talk associated with direct excitation of the acceptor, and cell autofluorescence. Excitation and detection are both from the top, so low-cost polypropylene plates typically used in qPCR work very well in this application. This novel technology has been combined with our previously developed fluorescence lifetime technology (Petersen et al, Rev Sci Inst, 2014) allowing for high-throughput quantitation of FRET efficiency from independent measurements. We have screened small-molecule libraries with an intramolecular cardiac calcium pump FRET biosensor, expressed in living cells (Gruber et al., J. Biomol Screening, 2014) using this multiplexed fluorescence detection platform and identified several previously unknown small-molecule effectors. The information obtained from both fluorescence lifetime and spectrum measurements reduced the number of false-positives and increased confidence in our hit selection methods. The development of this technology and screening assay will prove useful for drug discovery but also answering basic biological questions with fluorescence based assays.