fasmoduleringsflourometri

Fasmoduleringsflourometri, or phase-modulated fluorometry, is a technique in fluorescence spectroscopy used to determine fluorescence lifetimes by applying a sinusoidally modulated excitation source and analyzing the phase and amplitude of the emitted light with a phase-sensitive detector. It is a form of frequency-domain fluorometry.

Principle: The excitation is modulated at a radiofrequency f, and the emitted fluorescence is phase-shifted and demodulated due to the finite excited-state lifetime τ. In a simple single-exponential system, the phase shift φ and the modulation factor M relate to τ by tan φ = ωτ and M = 1/√(1+(ωτ)²), where ω = 2πf. In samples with multiple lifetimes, the measured phase and modulation reflect a distribution of lifetimes, which can be analyzed to extract τ values or lifetimes distributions. The method is insensitive to steady background and can be implemented across a range of frequencies to probe different dynamic regimes.

Instrumentation and data interpretation: Instruments use a modulated light source (such as an LED or laser) and a phase-sensitive detector or lock-in amplifier to measure φ and M. These parameters are converted to lifetime information and can be used for steady-state analysis, time-domain extrapolation, or lifetime imaging. Frequency-domain data can also support quantitative assessments of quenching, environmental effects, or binding interactions.

Applications: Fasmoduleringsflourometri is employed to measure fluorophore lifetimes in solutions, living cells, and tissues; monitor dynamic processes such as quenching, energy transfer, and binding events; and support fluorescence lifetime imaging and high-throughput screening.

Advantages and limitations: The approach provides rapid measurements and good discrimination of lifetime changes with relatively simple instrumentation, though it can be challenged by complex, multi-exponential decays and the need for accurate calibration of modulation depth and phase.