Fluorescent lifetime of amino acid tryptophan in the protein xylanase

We employ fluorescence lifetime microscopy (FLIM) to quantify the emission lifetime of a model biopolymer, xylanase. We excite tryptophan using a femtosecond pulsed laser and quantify the emission lifetime by Time Correlated Single Photon Counting (TCSPC). We observe two major fluorescence decay rates associated with tryptophan. In addition to the intrinsic decay rate of tryptophan in this protein (~2.4ns), we observed a fast decay that indicates quenching between tryptophan and its neighbors. We also observed an increase in emission intensity after higher power laser exposure, as opposed to the expected decrease in the intensity due to photobleaching. We characterize the relation between the lifetime decay rates of tryptophan emission and the increase in its intensity after high power laser pulses.