title: Spectral Unmixing Plate Reader: High-Throughput, High-Precision FRET Assays in Living Cells.
abstract: We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a well-by-well basis under true high-throughput screening (HTS) conditions. The read time for an entire 384-well plate is less than 3 min. This instrument is particularly well suited for assays based on fluorescence resonance energy transfer (FRET). Intramolecular protein biosensors with genetically encoded green fluorescent protein (GFP) donor and red fluorescent protein (RFP) acceptor tags at positions sensitive to structural changes were stably expressed and studied in living HEK cells. Accurate quantitation of FRET was achieved by decomposing each observed spectrum into a linear combination of four component (basis) spectra (GFP emission, RFP emission, water Raman, and cell autofluorescence). Excitation and detection are both conducted from the top, allowing for thermoelectric control of the sample temperature from below. This spectral unmixing plate reader (SUPR) delivers an unprecedented combination of speed, precision, and accuracy for studying ensemble-averaged FRET in living cells. It complements our previously reported fluorescence lifetime plate reader, which offers the feature of resolving multiple FRET populations within the ensemble. The combination of these two direct waveform-recording technologies greatly enhances the precision and information content for HTS in drug discovery.
authors.affiliation: 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.
authors.forename: Tory M
authors.initials: TM
authors.lastname: Schaaf
authors.affiliation: 2 Fluorescence Innovations, Inc., Minneapolis, MN, USA.
authors.forename: Kurt C
authors.initials: KC
authors.lastname: Peterson
authors.affiliation: 2 Fluorescence Innovations, Inc., Minneapolis, MN, USA.
authors.forename: Benjamin D
authors.initials: BD
authors.lastname: Grant
authors.affiliation: 1 Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, MN, USA.|3 Photonic Pharma LLC, Minneapolis, MN.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
authors.affiliation: 2 Fluorescence Innovations, Inc., Minneapolis, MN, USA.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Gillispie
mesh_terms: D000476:Alkanesulfonates / Q000494:pharmacology; D001391:Azo Compounds / Q000494:pharmacology; D015374:Biosensing Techniques; D055808:Drug Discovery / Q000295:instrumentation / Q000379:methods*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005453:Fluorescence; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods*; D015870:Gene Expression; D017930:Genes, Reporter; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D057809:HEK293 Cells; D057166:High-Throughput Screening Assays; D006801:Humans; D019044:Image Cytometry / Q000295:instrumentation / Q000379:methods*; D007211:Indoles / Q000494:pharmacology; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D019284:Thapsigargin / Q000494:pharmacology; D000097573:Red Fluorescent Protein
chemical_list: D000476:Alkanesulfonates; D001391:Azo Compounds; D004791:Enzyme Inhibitors; D049452:Green Fluorescent Proteins; D007211:Indoles; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D019284:Thapsigargin; D000097573:Red Fluorescent Protein; C000598590:BHQ-1 compound; C506943:ATP2A1 protein, human; C000543:cyclopiazonic acid
keywords: SERCA; biosensor; drug screening; fluorescence lifetime; top-read