Time-Resolved Fluorescence (TRF)
Time-Resolved Fluorescence (TRF) uses fluorescence lifetime to gain information about a molecule or its molecular environment, as fluorescence lifetime is characteristic for each fluorescent molecule and is also influenced by the chemical composition of its environment; it can thus be used to characterize a sample.
The use of the term Time-Resolved Fluorescence in the field of Life Sciences can be sometimes confusing, as it can be used with two different meanings:
- The fluorescence of a sample monitored as a function of time after excitation by a pulse of light.
- The use of fluorophores with a distinctive fluorescence lifetime to avoid the interference caused by molecules with a very different fluorescence lifetime or other factors (most importantly, excitation light).
When discussing about microplate readers, TRF usually refers to the second meaning. The fluorophores of choice are usually chelates of lanthanides (most commonly Europium, Terbium and Samarium), because of the following properties:
- Its fluorescence lifetime is in the range of microseconds (μs), while in most fluorophores the lifetime of its fluorescence is in the range of nanoseconds (ns). That allows the measurement to be performed much after both the light of the excitation and the fluorescence of any other fluorophore in the sample have completely decayed, greatly reducing background and thus increasing the signal-to-noise ratio.
- The large difference between its excitation and emission wavelengths (Stokes' shift) and the narrow emission peaks contribute to further increasing signal-to-noise ratio.
The large signal-to-noise ratio provides a very high sensitivity. As a consequence, many assays using the Time-Resolved Fluorescence properties of lanthanide chelates have been developed. The most popular assay type using TRF are TR-FRET assays. The distinctive fluorescence lifetime and spectral properties of the different lanthanide quelates allows for multiplex assays to be developed.
One critical feature needed by a microplate reader to be able to measure TRF-based assays is that the source of excitation light has to be a flash lamp. In flash lamps, light is provided in very short pulses (around 2 μs) that repeat every few ms. This timing leaves enough time between flashes for the measurement in an appropriate time window for all commonly used lanthanide chelates.
Additional, it is important to keep in mind that filters provide the best sensitivity for TRF-based assays. If the instrument has the possibility to measure with either filters or monochromator, please select filters.