Investigation of multiphoton-induced fluorescence from solutions of 5-hydroxytryptophan.

It is reported (J. B. S. Shear, C. Xu and W. W. Webb, Photochem. Photobiol. 1997, 65, 931) that multiphoton near infrared excitation of 5-hydroxytryptophan results in a transient product with green fluorescence. Visible fluorescence from multiphoton excitation enables detection of 5-hydroxytryptophan with extremely high sensitivity and also has potential applications in imaging of biological systems and investigation of protein dynamics. The characteristic fluorescence at 500 nm has now also been observed in a two laser experiment whereby 308 nm photolysis of the solution is followed by an excitation step at 430 nm. Fluorescence was observed in aerated and deaerated solutions and in the presence of ascorbate. Enhancement of fluorescence was observed on addition of ethanol. Transient absorption experiments with 308 nm photolysis showed the formation of three transient species. In the presence of ascorbate the radical formed by photoionisation was quenched, revealing a long-lived species (tau > 1 ms) with a similar absorption spectrum, which is ascribed to the fluorescing species. Fluorescence induced by multiphoton excitation had a lifetime of 910 +/- 10 ps and was also unaffected by ascorbate. In the presence of organic solvents there was an increase in fluorescence lifetime, but a decrease in overall fluorescence intensity. The fluorescence intensity and fluorescence lifetime both decreased in acidic solution (pH < 3). The results indicate that the fluorescence does not originate from the 5-indoxyl radical as previously suggested but from one or more other transient products which require further characterisation.