A time-resolved fluorometer for high-throughput screening

Fluorescence instrumentation for high-throughput screening (HTS) must be sensitive, accurate and reliable. An instrument must be capable of robust measurement as well. HTS assays are often complicated by interfering signals from background fluorescence, scattering, absorption and quenching. Traditional fluorescence intensity methods do not remove the effect of interferences well. Fluorescence lifetime methods, however, have the ability to retrieve the true assay signal from a signal plagued with interferences. We have developed fluorescence-lifetime methods specifically for high-throughput screening with a high-frequency time-resolved fluorometer created from the optics of a high-throughput screening instrument, a high-speed LED or laser diode light source and the phase and modulation method of fluorescence lifetime measurement. The prototype instrument is capable of measuring the lifetime of samples from 1-1000 ns, able to measure a single frequency in less than one second and able to distinguish nanomolar concentrations of fluorophores in small volumes (10 mu l). Furthermore, the often complex and troublesome fluorescence lifetime measurement is made simple and reliable with this prototype. To demonstrate robustness and reliability, we used lifetime-based methods to measure a model system with background fluorescence from biological membranes. Specifically, methods based upon long-lifetime fluorophores can significantly improve immunity to assay interferences.