How long is long enough? Optimising time-resolved fluorescence measurements

Fluorescence emission is a time-dependent process yielding a high number of photons within nanoseconds upon excitation and then decreasing with increasing time.

Dr Mario Schneider Dr Mario Schneider (1)

A plot of the number of photons versus time after excitation is known as fluorescence lifetime decay curve (short: decay curve). 

In a conventional fluorescence intensity measurement, the incoming fluorescence signal is integrated by the detector for a fixed time interval starting at the same time as the light source flashes. However, in time-resolved fluorescence measurements, since lanthanides emit light over a longer time upon excitation (usually microseconds), the start of the integration and the overall integration time must be appropriately chosen. But how long is long enough? 

For many assays, the integration start and integration time are given by the kit manufacturers. These settings might be even further optimized or need to be established from scratch in case of new assay developments. This can be easily done using PHERAstar microplate readers thanks to their Decay Curve Monitoring function. 

Information contained therein can be used for optimizing time-resolved fluorescence (TRF), homogeneous time-resolved fluorescence (HTRF®), and time-resolved Förster resonance energy transfer (TR-FRET) assays. The latter two assays use two spectrally different dyes, one donor and one acceptor dye. In a typical TR-FRET assay the donor dye becomes excited by the light source and transfers the energy to the acceptor if in close proximity, and the acceptor itself starts emitting photons (fig. 1). This is why TR-FRET is often used to assess the interaction of two molecules labelled with either the donor or acceptor in so-called binding assays

Fig. 1: Schematic of the TR-FRET technology. Donor (green) transfers energy to the acceptor (red) that emits light. Both signals have a longer lifetime than the unspecific background autofluorescence signal (Bg, grey) and can be measured in the integration time window.

Time-resolved FRET inherits the same advantages as other time-resolved fluorescence methods, e.g. being able to suppress background by setting an appropriate integration start and integration time interval. The novel Integration Time Wizard available in the MARS software for PHERAstar readers helps to optimize these parameters. After recording the decay curves for the donor and acceptor, for instance, the Integration Time Wizard can be used to optimize the detection time window of a TR-FRET binding assay. A heat-map in the integration time wizard helps to identify the best settings of the integration start and integration time based on assay quality metrics such as Z´, assay window, etc. The combinations of integration start and integration time with the highest maximum assay quality metrics (marked in dark red) are likely the ones best suited for the corresponding assay.

Fig. 2: The novel Integration Time Wizard available in the MARS software for PHERAstar readers helps to optimize integration start and integration time interval.

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