Spectral unmixing plate reader: High-throughput, high-precision FRET Assays in living cells

Tory M. Schaaf, Kurt C. Peterson, Benjamin D. Grant, David D. Thomas, Gregory D. Gillispie

Research output: Contribution to journalArticle

7 Scopus citations

Abstract

We have developed a microplate reader that records a complete high-quality fluorescence emission spectrum on a wellby-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.

Original languageEnglish (US)
Pages (from-to)250-261
Number of pages12
JournalSLAS Discovery
Volume22
Issue number3
DOIs
StatePublished - Jan 1 2017

Keywords

  • Biosensor
  • Drug screening
  • Fluorescence lifetime
  • SERCA
  • Top-read

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