How is fluorescence lifetime measured?

Fluorescence lifetime is measured using either time-domain or frequency-domain techniques.

Time-Domain Measurement

The time-domain method directly measures the decay of fluorescence intensity after excitation with a short pulse of light. The process involves:

1. Excitation: A sample (cuvette, cells, or tissue) is illuminated with a short pulse of light (e.g., a laser pulse). This pulse excites the fluorescent molecules within the sample.

2. Emission Detection: After the excitation pulse, the emitted fluorescence is measured over time. This measurement typically uses a detector like a photomultiplier tube (PMT) or a time-correlated single-photon counting (TCSPC) system.

3. Decay Curve Analysis: The detected fluorescence intensity is plotted against time, creating a decay curve. This curve represents the exponential decay of the fluorescence signal.

4. Lifetime Calculation: The fluorescence lifetime (τ) is determined by fitting the decay curve to an exponential function. For a single exponential decay, the fluorescence intensity I(t) at time t is described by:

   I(t) = I₀ exp(-t/τ)*

   where I₀ is the initial fluorescence intensity. For more complex systems, multi-exponential decays might be observed.

Frequency-Domain Measurement

The frequency-domain method involves modulating the excitation light source at a specific frequency and measuring the phase shift and demodulation of the emitted fluorescence.

1. Modulated Excitation: The sample is excited with light that is sinusoidally modulated at a specific frequency (f).

2. Emission Detection: The emitted fluorescence will also be modulated at the same frequency, but it will be phase-shifted and demodulated (reduced in amplitude) relative to the excitation light.

3. Phase Shift and Demodulation Measurement: A detector measures the phase shift (φ) and the demodulation (m) of the fluorescence signal compared to the excitation light.

4. Lifetime Calculation: The fluorescence lifetime can be calculated from the phase shift and demodulation using the following equations:

   • τ_p = (1/ω) * tan(φ) (Lifetime from phase shift)
   • τ_m = (1/ω) * sqrt((1/m²) - 1) (Lifetime from demodulation)

   where ω = 2πf is the angular frequency. Ideally, τ_p and τ_m should be equal for a single exponential decay. Differences indicate more complex decay kinetics.

Comparison of Time-Domain and Frequency-Domain Methods

Feature	Time-Domain	Frequency-Domain
Excitation	Short pulse of light	Sinusoidally modulated light
Measurement	Fluorescence intensity decay over time	Phase shift and demodulation of fluorescence signal
Data Analysis	Fitting decay curve to exponential function	Calculation from phase shift and demodulation
Complexity	Relatively straightforward	Can be more complex for multi-exponential decays
Instrumentation	TCSPC, pulsed lasers	Modulated light sources, phase-sensitive detectors
Applicability	Suitable for a wide range of lifetimes	Particularly useful for complex decays and high-throughput measurements