Förster resonance energy transfer (FRET)-based picosecond lifetime reference for instrument response evaluation

Förster resonance energy transfer (FRET) can be utilized to achieve ultrashort fluorescence responses in time-domain fluorometry. In a poly(vinyl) alcohol matrix, the presence of 60 mM Rhodamine 800 acceptor shortens the fluorescence lifetime of a pyridine 1 donor to about 20 ps. Such a fast fluorescence response is very similar to the instrument response function (IRF) obtained using scattered excitation light. A solid fluorescent sample (e.g a film) with picosecond lifetime is ideal for IRF measurements and particularly useful for time-resolved microscopy. Avalanche photodiode detectors, commonly used in this field, feature color- dependent-timing responses. We demonstrate that recording the fluorescence decay of the proposed FRET-based reference sample yields a better IRF approximation than the conventional light-scattering method and therefore avoids systematic errors in decay curve analysis.