Three-photon excitation of a tryptophan derivative using a fs-Ti: Sapphire laser

We found that N-acetyl-L-tryptophanamide (NATA) could be excited at 840 nm using a mode-locked Ti : sapphire laser. The emission spectra of NATA were the same for one-photon (1 hv) excitation at 280 nm as for excitation at 840 nm. The emission intensity of NATA was found to depend on the cube of the laser power at 840 nm, consistent with simultaneous absorption of three 840-nm photons. The intensity-decay times were the same for 280- and 840-nm excitation, suggesting the same excited state is reached for each mode of excitation. However, the anisotropies were different for 280- and 840-nm excitation. At 280 nm the time-zero anisotropy was 0.130, whereas at 840 nm the time-zero anisotropy was negative (-0.06), indicating different orientations of the transition moments for one- and three-photon excitation. These results suggest that the intrinsic fluorescence of proteins may be excited using the fundamental output of a Ti : sapphire laser.