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Time-resolved fluorescence spectroscopy is an extension of fluorescence spectroscopy. Here, the fluorescence of a sample is monitored as a function of time after excitation by a flash of light. The time resolution can be obtained in a number of ways, depending on the required sensitivity and time resolution:
Time-resolved fluorescence energy transfer (TR-FRET) is the practical combination of time-resolved fluorometry (TRF) with Förster resonance energy transfer (FRET) that offers a powerful tool for drug discovery researchers.
In addition to the changes in the fluorescence intensity, all of them allow the recording of time-resolved fluorescent spectra [50] [51] and fluorescence anisotropies, [52] [53] which provide information about the relaxation of the excited electronic states and the type of the emitting excited states.
Fluorescence lifetimes can be determined in the time domain by using a pulsed source. When a population of fluorophores is excited by an ultrashort or delta pulse of light, the time-resolved fluorescence will decay exponentially as described above. However, if the excitation pulse or detection response is wide, the measured fluorescence, d(t ...
Thermally activated delayed fluorescence (TADF) is a process through which surrounding thermal energy changes population of excited states of molecular compounds and thus, alters light emission. The TADF process usually involves an excited molecular species in a triplet state , which commonly has a forbidden transition to the singlet ground ...
In chemistry, the method is more often referred to as fluorescence spectroscopy, but the instrumentation is the same. The relaxation processes can be studied using time-resolved fluorescence spectroscopy to find the decay lifetime of the photoluminescence.
Time-resolved fluorescence (TRF) measurement is very 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.
Suitable fluorophores (e.g. for STORM) reside in a non-fluorescent dark state for most of the time and are activated stochastically, typically with an excitation laser of low intensity. A readout laser stimulates fluorescence and bleaches or photoswitches the fluorophores back to a dark state, typically within 10–100 ms.