title: Fluorescence lifetime plate reader: resolution and precision meet high-throughput.
abstract: We describe a nanosecond time-resolved fluorescence spectrometer that acquires fluorescence decay waveforms from each well of a 384-well microplate in 3 min with signal-to-noise exceeding 400 using direct waveform recording. The instrument combines high-energy pulsed laser sources (5-10 kHz repetition rate) with a photomultiplier and high-speed digitizer (1 GHz) to record a fluorescence decay waveform after each pulse. Waveforms acquired from rhodamine or 5-((2-aminoethyl)amino) naphthalene-1-sulfonic acid dyes in a 384-well plate gave lifetime measurements 5- to 25-fold more precise than the simultaneous intensity measurements. Lifetimes as short as 0.04 ns were acquired by interleaving with an effective sample rate of 5 GHz. Lifetime measurements resolved mixtures of single-exponential dyes with better than 1% accuracy. The fluorescence lifetime plate reader enables multiple-well fluorescence lifetime measurements with an acquisition time of 0.5 s per well, suitable for high-throughput fluorescence lifetime screening applications.
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Petersen
authors.affiliation: Fluorescence Innovations, Inc., Minneapolis, Minnesota 55455, USA.
authors.forename: Kurt C
authors.initials: KC
authors.lastname: Peterson
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Muretta
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Sutton E
authors.initials: SE
authors.lastname: Higgins
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Gillispie
authors.affiliation: Department of Biochemistry, Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455, USA.
authors.forename: David D
authors.initials: DD
authors.lastname: Thomas
mesh_terms: D005453:Fluorescence; D005456:Fluorescent Dyes / Q000737:chemistry*; D008956:Models, Chemical; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods
chemical_list: D005456:Fluorescent Dyes