Fluorescence lifetime can be measured in either the frequency domain or the time domain. The time domain method involves the illumination of a sample (a cuvette, cells, or tissue) with a short pulse of light, followed by measuring the emission intensity against time.

Second, TCSPC reaches an extremely high time resolution. The resolution is not limited by the single-photon response uf the detector, as it is the case for analog-recording techniques. Instead, the resolution is given by the transit-time jitter of the electrons in the detector.

The second type, time-resolved fluorescence (TRF), is monitored as a function of time upon excitation. In contrast to steady-state fluorescence intensity, time-resolved fluorescence is based on the detection of intensity decays and/or on the delayed detection of the emission signal upon excitation.

TCSPC stands for time-correlated single photon counting. It is a method of using the timing of a pulsed excitation source, like a laser or LED, with the timing of the arrival of single photons on a detector to reconstruct the lifetime decay over many events (repetition of pulses and photons detected).

The distinct advantage of TCSPC is that this technique does not use the analogue detector response to produce the instrumental response function (IRF). The jitter in the rising edge of the detector analogue response dictates the width of the IRF.

where Ai and τi are the amplitudes and decay times of the M exponential components of the fluorescence decay. The mean decay time is given by 〈τe〉 = ΣAiτi.

The second type, time-resolved fluorescence (TRF), is monitored as a function of time upon excitation. In contrast to steady-state fluorescence intensity, time-resolved fluorescence is based on the detection of intensity decays and/or on the delayed detection of the emission signal upon excitation.