A 2 GHz frequency-domain fluorometer; picosecond resolution of protein fluorescence and anisotropy decays

We developed a frequency-domain fluorometer which operates from 4 to 2000 MHz. The modulated excitation is provided by the harmonic content of a laser pulse train (7.59 MHz, 5 psec) from a syncronously pumped and cavity dumped dye laser. The phase angle and modulation of the emission are measured with a microchannel plate photomultiplier. Cross-correlation detection is performed outside the PMT. The performance was verified by measurement of known time delays and examination of standard fluorophores. The detector displayed no detectable color effect, with the 300 to 600 nm difference being less than 5 psec. The precision of the measurements is adequate to detect differences of 20 psec for decay times of 500 ps. This new instrument was used to examine tyrosine and tryptophan intensity and anisotropy decays from peptides and proteins. The data demonstrate that triply-exponential tyrosine intensity decays are easily recoverable, even if the mean decay time is less than 1 nsec. Importantly, the extended frequency range provides good resolution of rapid and/or multi-exponential tyrosine anisotropy decays. Correlation times as short as 15 psec have been recovered for indole, with an uncertainty of ± 3 psec. We recovered a doubly exponential anisotropy decay of oxytoxin (29 and 454 psec), which probably reflects torsional motions of the phenol ring and overall rotational diffusion. Also, the 2 GHz data reveal the time-dependent (Equation presented) terms found in the presence of collisional quenching.