A PARALLEL MULTIHARMONIC FREQUENCY-DOMAIN FLUOROMETER FOR MEASURING EXCITED-STATE DECAY KINETICS FOLLOWING ONE-PHOTON, 2-PHOTON, OR 3-PHOTON EXCITATION

We report on the performance of a new, multiharmonic frequency-domain instrument that uses the high harmonic content of a passively mode-loc ked, pulse-picked femtosecond Ti-sapphire laser as the excitation sour ce for the determination of one-, two-, or three-photon excited time-r esolved fluorescence anisotropy and intensity decay kinetics. In opera tion, the new instrument can provide a complete frequency-domain data set at 100 modulation frequencies in less than 1 min. The new instrume nt exhibits 5-10-ps measurement precision and it can rapidly and accur ately recover complex excited-state fluorescence anisotropy and intens ity decay kinetics under one-, two-, or three-photon excitation for di lute or optically dense samples that exhibit single or multiexponentia l decay kinetics, This latter aspect of the instrument is demonstrated by successfully determining the excited-state intensity decay kinetic s for a dilute aqueous solution of rhodamine 6G dissolved in a high co ncentration of bromocresol green. This approach is extended by determi ning the excited-state fluorescence intensity decay kinetics of dilute fluorescein directly in undiluted, whole blood as a function of pH un der two-photon excitation conditions. The high-speed capabilities of t he new instrument are exploited by performing two-photon excited fluor escence anisotropy decay experiments on the fly for site-selectivity l abeled bovine serum albumin as it undergoes enzymatic digestion by try psin.