Luminescence and fluorescence-based assay methods prevail in modern HTS. Although invaluable as technologies, the problem of compound-related assay interferences such as quenching, auto-fluorescence, and light scattering becomes important. The degree of interference experienced may depend on variables such as assay reagents, composition of the chemical library, and reader technology.
Where the amount of interference is unacceptably high, the only option is to delete the data and thus lose all information pertaining to compound activity. False positives and negatives occur when such interferences go undetected. Fluorescence lifetime is increasingly being viewed as an alternative reporter since it largely mitigates such issues.
As a result of significant improvements in assay reagents and the availability of HTS-compatible readers, fluorescence lifetime (FLT) has recently gained new appeal as a screening technology. FLT (also referred to as tau or τ) is the average time between excitation of a fluorophore and its return to ground energy state.
In simple systems, fluorescence intensity decays exponentially, defining FLT as the time in which the fluorescence intensity decays to 1/e (~37%) of that immediately following excitation. FLT provides an attractive reporter modality as it is largely unaffected by auto-fluorescence, light scattering, and inner filter effects providing more robust assays. Furthermore FLT, being an intrinsic parameter of the fluorophore, is independent of probe concentration, which facilitates miniaturization into high-density microplate formats, making FLT a highly attractive and enabling reporting technology for HTS and profiling applications.
There are two approaches currently employed for the measurement of fluorescence lifetimes in microplates: time-correlated single photon counting (TCSPC) and real-time decay curve analysis (RT-DCA). To enable such measurements, the optics in readers comprise a pulsed laser as an excitation source, emission filters, and sensitive photomultipliers tube (PMT) detectors. The approaches differ in the method used to quantify fluorescence emissions in the time domain.
TCSPC involves measuring the time for the first emitted photon to reach the PMT detector following a brief excitation pulse. Repeating this measurement produces a histogram of detected “first photons.” Commonly, TCSPC experiments are set up to record 3,000–10,000 counts in the peak channel resulting in plate read times (384-well) of circa 5–20 minutes. Shorter acquisition times can be utilized for certain applications but may compromise data quality.
In real-time decay curve analysis (RT-DCA), the entire emission curve is recorded after a single excitation pulse, enabling high-throughput time-resolved data to be acquired. The Ameon™ microplate reader (TTP Labtech) contains a digitizer that is capable of detecting thousands of photons per laser pulse enabling plate-read times (1,536-well) of ~2 mins. This makes it possible to implement FLT measurements in studies which were previously challenging, including HTS.