Time Resolved Fluorescence

Time-resolved fluorescence (TRF) measurement is similar to fluorescence intensity (FI) measurement. The only difference is the timing of the excitation/measurement process. When measuring FI, the excitation and emission processes are simultaneous: the light emitted by the sample is measured while excitation is taking place. Even though emission systems are very efficient at removing excitation light before it reaches the detector, the amount of excitation light compared to emission light is such that FI measurements always exhibit elevated background signals. TRF offers a solution to this issue as it relies on the use of specific fluorescent molecules, called lanthanides, that have the unusual property of emitting over long periods of time (measured in milliseconds) after excitation. Lanthanides emit longer compared to most standard fluorescent dyes (e.g. fluorescein) that emit within a few nanoseconds of being excited. It is possible to excite lanthanides using a pulsed light source such as a 337 nm laser or a xenon flash lamp and measure after the excitation pulse. This results in lower measurement backgrounds than in standard FI assays. The drawbacks are that the instrumentation and reagents are typically more expensive, and that the applications must be compatible with the use of these very specific lanthanide dyes.

 
In a typical TRF assay, samples emit at one wavelength (here in red) before they react

Figure 1. In a typical TRF assay, samples emit at one wavelength (here in red) before they react.

When a reaction occurs, the sample’s emission shifts (here to orange)

Figure 2. When a reaction occurs, the sample’s emission shifts (here to orange).

 

 

The main use of TRF is found in drug screening applications, under a form called TR-FRET (time-resolved fluorescence energy transfer). Learn more about these assays on the TR-FRET page.

 

337 nm laser for Synergy Neo2 for TRF, TR-FRET assays

Figure 3. 337 nm laser for Agilent BioTek Synergy Neo2 for TRF, TR-FRET assays.

 

Time Resolved Fluorescence is available in:

 
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