Near infrared multiphoton-induced generation and detection of hydroxyl radicals in a biochemical system

Solutions of tryptophan and N-hydroxypyridine-2-thione (mercaptopyridine-N-oxide, MPNO) were irradiated at 335 nm. Formation of 5-hydroxytryptophan was inferred from increased fluorescence at 334 nm on excitation at 315 nm, conditions chosen for selective detection of 5-hydroxytryptophan. Such experiments are complicated by overlapping absorption spectra in the region of 300–350 nm. Similar solutions were exposed to multiphoton excitation at 750 nm using 180 fs pulses from a titanium:sapphire laser. In solutions containing both tryptophan and MPNO strong emission at 500 nm was observed that was absent in solutions containing either MPNO or tryptophan only. This emission is ascribed to the characteristic fluorescence (‘hyperluminescence’) from 5-hydroxyindoles resulting from multiphoton photochemistry. The conclusion that MPNO generates hydroxyl radicals by 2-photon activation at 750 nm is confirmed by the scavenging effects of ethanol and kinetic analysis of the results. This method has potential applications in intracellular induction of oxidative stress using multiphoton near-infrared illumination, a technology that is gaining momentum as a research tool.