FLIMFRET

FLIMFRET, short for fluorescence lifetime imaging microscopy-based Förster resonance energy transfer, is a method used to study molecular interactions at nanometer distances in cells and other samples. It detects energy transfer from a donor fluorophore to an acceptor by observing changes in the donor’s fluorescence lifetime, rather than just intensity.

The technique combines the distance dependence of FRET with the lifetime information provided by FLIM. When donor and acceptor are closely spaced, energy transfer occurs and the donor’s excited-state lifetime decreases. The FRET efficiency can be inferred from the change in donor lifetime, with E ≈ 1 − (τDA/τD), where τD is the donor lifetime without the acceptor and τDA is the lifetime with the acceptor.

Measurement is typically performed by exciting the donor with a pulsed light source and recording the decay of donor fluorescence across image pixels to produce a lifetime map. Time-domain implementations use time-correlated single-photon counting (TCSPC), while frequency-domain FLIM uses phase and modulation measurements. Data analysis involves fitting decay curves to extract lifetimes or using model-free approaches such as phasor plots to visualize lifetime changes and infer FRET.

Applications of FLIMFRET include probing protein–protein interactions, monitoring conformational changes, detecting receptor activation, and mapping signaling events in live cells. Advantages include quantitative readouts that are independent of fluorophore concentration and illumination intensity, and suitability for dynamic processes. Limitations involve the need for careful spectral separation, appropriate controls for donor-only lifetimes, potential bleed-through, and the requirement for specialized instrumentation and data analysis.