The application of fluorescence lifetime readouts in high-throughput screening.
Measurement of fluorescence lifetime is a well-established technique, which has recently been introduced into the portfolio of assay formats used in high-throughput screening (HTS). This investigation establishes appropriate conditions for using lifetime measurements to reduce the impact of compound interference effects during large-scale HTS of corporate screening files. Experimental data on mixtures of standard fluorophores and interfering compounds (from 5 HTS campaigns) have been combined with a theoretical model to identify the minimum data quality required, defined by the photon count in the peak channel, for discrimination of biological activity. Single-component fluorophore lifetimes can be recovered with an error of 1%, with a peak photon count of 10(2), but the same accuracy with a 2-component decay requires a peak photon count of 10(3). When a 3rd component is introduced, the minimum peak count increases to 10(4). The influence of scattered light on lifetime determination was investigated using an emulsion (diameters 25-675 nm). The measured decays of interfering compounds, identified as autofluorescent, show that the vast majority have a very short lifetime that can readily be resolved from the reporter fluorophore, using appropriate data-fitting methods.
Moger
Julian
J
School of Physics, University of Exeter, Exeter, UK. J.Moger@Exeter.ac.uk
Gribbon
Philip
P
Sewing
Andreas
A
Winlove
C Peter
CP
D004353:Drug Evaluation, Preclinical / Q000379:methods*; D005453:Fluorescence; D005454:Fluorescence Polarization; D005456:Fluorescent Dyes / Q000032:analysis* / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry; D012542:Scattering, Radiation; D012680:Sensitivity and Specificity
D005456:Fluorescent Dyes; D010455:Peptides