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The Application of Fluorescence Lifetime Readouts in High-Throughput Screening
1 School of Physics, University of Exeter, Exeter, UK.
* To whom correspondence should be addressed. E-mail: J.Moger{at}Exeter.ac.uk.
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 102, but the same accuracy with a 2-component decay requires a peak photon count of 103. When a 3rd component is introduced, the minimum peak count increases to 104. 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. Key Words: high-throughput screening, fluorescence lifetime, FIDA, fluorescence polarization
First published on August 30, 2006, doi:10.1177/1087057106291541 This article has been cited by other articles:
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