Time-resolved fluorescence

Although conventional fluorophores are extremely popular, they have several limitations that can be overcome using time-resolved fluorescence methods.

Firstly, the simultaneous excitation/emission process from conventional fluorophores can result in high background signal. Secondly, the Stokes shift (the difference between the maximum absorbance and emission wavelengths) of many commercially available fluorophores is relatively small, meaning that these reagents can suffer from self-quenching due to the overlap between their absorption and emission spectra. Thirdly, biological matrices, such as serum or tissue samples, often contain autofluorescent substances, which are excited and emit light simultaneously in the same way as fluorophores and are a source of background signals. Similarly, in high-throughput screening formats, false positives can occur due to the fluorescent nature of certain types of test compounds.

Time-resolved fluorescence (TRF) can help overcome some of these problems. TRF is very similar to standard fluorescence detection, apart from the timing of the excitation and emission processes.

During standard fluorometric detection, excitation and emission are simultaneous with the light emitted. By contrast, TRF relies on using specific fluorescent molecules, called lanthanide chelate labels, which have a long fluorescence lifetime and allow detection of the emitted light after excitation has occurred. The most commonly used lanthanide chelate label is the europium ion (Eu³⁺).

Find out more about Abcam's time-resolved fluorescence conjugation kits on our website