The term Time-Resolved Fluorescence (TRF) can have two different meanings:
- The fluorescence of a sample monitored as a function of time after excitation by a pulse of light. For disambiguation, this procedure is often called Fluorescence Lifetime measurement.
- 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 like excitation light.
When discussing about microplate readers, TRF usually refers to the second meaning.
The fluorophores of choice are usually chelates of lanthanides and are 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 after both the light of the excitation and the fluorescence of any other fluorophore in the sample have completely decayed. This greatly reduces background and increases 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.
Applications of Time-Resolved Fluorescence
The large signal-to-noise ratio of methods based on TRF provides very high sensitivity. Consequently, 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 chelates allows for multiplex assays to be developed.
Transcreener® GDP TR-FRET Red Assay with Mithras² Validation of the Mithras² Multimode Microplate Reader with the Transcreener® GDP TR-FRET Red assay
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Transcreener® GDP TR-FRET Red Assay with TriStar² S Validation of the TriStar² S Multimode Microplate Reader with the Transcreener® GDP TR-FRET Red assay
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cAMP/IP-One HTplex HTRF® assay with Mithras cAMP/IP-One HTplex cell-based experiment performed on Mithras LB 940 Multimode Microplate Reader using HTRF® technology
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Microplate readers suitable for TRF
A microplate reader capable of measuring high-sensitivity fluorescence using filters and equipped with a flash lamp is the best combination for TRF (monochromator-based instrument may also be used but with lower performance). A detector with extended spectral range (above 650 nm) may be required for some applications.