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Abstract

Using a new type of fluorescence lifetime spectrometer, the time resolution of single shot fluorescence detection is improved by two orders of magnitude. The spectrometer is based on a streak camera and an excimer laser pumped distributed feedback dye laser (DFDL) providing UV pulses of 50 μJ and 15 ps FWHM, corresponding to a peak power of ∼ 5 · 106 W in the ultraviolet. —This spectrometer is used to investigate the fluorescence decay of peptides containing a tyrosine as the single fluorophore. Peptides with tyrosine at the N-terminus reveal a biexponential decay, while peptides with C-terminal tyrosine are mainly monoexponential. This difference can be explained by the relative orientation of the fluorescing phenol ring and the quenching peptide bond. —The fluorescence decay of a number of peptides with tyrosine in a non terminal position can be described by two lifetimes, 2.3 · 0.1 ns and 380 ± 70 ps, where the percentage of the long lifetime increases to up to 100% when the rotational flexibility of the peptide chain decreases, either due to increasing weight of the chain or due to formation of an H-bridge. —Similar processes seem to be relevant for the fluorescence of the dye coumarin 120 coupled to nucleotides. The fluorescence lifetime of such molecules is significantly different, so that the four DNA nucleotides can be identified on the basis of the fluorescence decay of the attached dye.