* Author for Correspondence

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.


INTRODUCTION
The detection of reactive oxygen species (ROS), such as hydroxyl radicals, in biological systems is challenging due to their high reactivity and extremely short lifetimes that are typically sub-microsecond [1,2]. A frequent approach is to use a scavenger which leads to distinctive products: examples include salicylate which is hydroxylated to a characteristic ratio of isomeric products [3,4] and dimethylsulfoxide which is oxidised to methanesulfinate by hydroxyl radical [5,6]. Spin traps have been extensively used for such purposes [7,8] and this permits indirect imaging of radical species in tissues such as skin [9]. Luminescent probes have also been used to detect oxidative stress in cellular systems, and examples include 2',7'dichlorodihydrofluorescein and dihydrorhodamine which are oxidised to their fluorescent products [10], although there is some concern as to their specificity regarding the type of ROS (hydroxyl, superoxide etc ) detected. Further consequences of ROS activity within cells are important and secondary biological products stemming from radical chemistry can also be investigated using imaging techniques, for example lipid peroxidation [11,12]. At the more chemically specific level tryptophan is one of the most reactive amino acids and is subject to attack by electrophilic reagents such as the hydroxyl radical, reacting with a near diffusion controlled rate constant [13]. In this respect there have been numerous studies showing how tryptophan is degraded in proteins exposed to hydroxyl radicals and use of this is made in the 'footprinting' method for probing protein surfaces [14,15].
Tryptophan is also a target for reaction of reactive nitrogen species such as peroxynitrite [16]. Botchway  There is currently much interest in oxidative stress in the cellular environment in relation to biochemical responses such as apoptosis and DNA repair. Continuous levels of oxidative stress may be induced by peroxyl radical generators such as the azo-initiators or through Fenton reagents. However, the opportunity for spatial and temporal control of oxidative stress using photo-activated precursors is now possible.
Hydroxyl radicals may be generated by ultraviolet excitation of compounds such as pyridine-N-oxides, often referred to as "photoFenton" reagents [17][18][19]. The generation of hydroxyl radicals from such reagents has been identified by trapping with coumarin-carboxylic acid and the consequent formation of the highly fluorescent 7-hydroxycoumarin carboxylate [20]. A potential problem with one-photon excitation of photoFenton reagents such as 2-hydroxypyridine-N-oxide (HPNO) and 2-mercaptopyridine-N-oxide (MPNO) are their short wavelength (UV) absorption maxima at ~ 310 nm and ~330 nm respectively at physiological pH (see Figure 2).
Such wavelengths are absorbed by several biochemical chromphores and so have limited penetration in tissues. The use of multiphoton excitation in the near infrared (ca 700 to 900 nm) with a femtosecond titanium-sapphire (Ti:Saph) laser has the potential to circumvent these problems and also allows pseudo-confocal three dimensional microscopic imaging of cellular systems due to the femtolitre volume at the focus of the laser beam [21][22][23]. Although cross-sections for 2-and 3-photon absorption are many orders of magnitude less than for the corresponding one-photon process, the high peak power within a ca 100 femtoseconds laser pulse may be used to induce photochemistry such as uncaging [24] and generation of reactive oxygen species from the triplet states of dyes [23,25]. Focusing these extreme peak laser powers (MW to GW) may cause dielectric breakdown in the solvent and Nishimura and Kinjo [26] have reported the formation of a green emission in solutions of Botchway et al: Multiphoton generation of hydroxyl radicals tryptophan exposed to femtosecond laser pulses with peak power densities up to 1.2 x 10 12 W cm -2 . Shear et al [27,28] were the first to report an unusual green emission ('hyperluminescence') from serotonin on multiphoton near infrared excitation. This was shown to be due to the generation of a photochemical product in a 4-photon event at 830 nm that is subsequently excited by a further two photons to generate the green fluorescence. Although such hyperluminescence has been further characterised [29,30], the nature of the emitting species remains unknown although it appears to be relatively specific for 5-hydroxyindoles. Laser flash photolysis has identified a triplet state. Together with neutral and cation radicals similarly identified, these are likely to be involved as precursors of the emitting state [31]. The observation of green luminescence from tryptophan solutions exposed to high laser powers [26] suggests the formation of hydroxyl radicals from laser-induced breakdown of water and subsequent formation and excitation of 5-hydroxytryptophan. We have now explored the possibility of hydroxyl radical generation by 2-photon excitation of photoFenton agents and its detection from reaction with tryptophan.

MATERIALS AND METHODS
All chemicals were obtained from Sigma-Aldrich and used as received. Solutions were prepared in triply filtered Milli-Q purified water (total organic content <2 ppm) and buffered to pH 7.3 with phosphate. Fluorescence spectra were measured in a Spex

a) UV excitation
Both MNPO and HPNO have been shown to be "photoFenton" reagents, liberating free hydroxyl radicals on illumination with quantum yields of ca. 0.11 to 0.28 [19].

b) Multiphoton near-infrared excitation
Using multiphoton excitation with 180 fs pulses in the near-infrared for activation of this photochemistry has a number of advantages, including simultaneous photoexcitation of both photoFenton agent, generating hydroxyl radical, and 5-OHTrp, permitting generation of the specific green ('hyperluminescence') emission [27][28][29] from 5-OHTrp. In multiphoton processes there is no competing absorption by solutes as there is in the one-photon case. Consequently, the inner filter effect at 750 nm is almost completely absent. (spectrum a) has a long wavelength limit at ~350 nm, and it appears that there is insufficient cross section for 2-photon excitation at 750 nm.

c) Time-resolved fluorescence measurements
Further confirmation that the enhanced 500 nm emission is due to hyperluminescence  [29]) and confirms the green emission as arising from 5-OHTrp.  Figure 4 therefore use a laser power which is a compromise between that sufficiently high to produce a reasonable hyperluminescence signal in an overall 5-photon process but not so high as to induce significant radical formation by breakdown in the solvent.  (2) and (3) (with second order rate constants k 2 and k 3 respectively). The luminescent signal at 500 nm (S) may be related to its maximum value (S max ) by:-

e) Effect of Trp concentration
This kinetic analysis ignores bimolecular recombination of hydroxyl radicals, since their steady state concentration is expected to be at least 3 orders of magnitude less than those of Trp and MPNO. Non-linear least squares fitting to equation (4) of the curve for the 500 nm signal in Figure 7 gives k 3 /k 2 = 0.82 ± 0.23. Taking the second order rate constant for reaction of • OH with tryptophan, k 3 , as 1.4 x 10 10 dm 3 mol -1 s -1 [13] gives a value for k 2 of (1.1 ± 0.3) x 10 10 dm 3 mol -1 s -1 . Aveline et al [18] quote a value of k 2 measured by pulse radiolysis of 9 x 10 9 dm 3 mol -1 s -1 , whilst the related compound HPNO reacts with • OH with a second order rate constant of 2 x 10 10 dm 3 mol -1 s -1 [32]. Applying this kinetic model to the data therefore produces a reasonable fit to the known kinetics of the system and confirms our interpretation of the multiphoton chemistry.

f) Scavenging by ethanol
Ethanol is an effective scavenger of hydroxyl radical with a second order rate constant (k e ) of 1.9 x 10 9 dm 3 mol -1 s -1 [13], producing carbon-centred radicals which react with oxygen to form peroxyl radicals with modest reactivity. It is not anticipated that either the radicals or chemical products from ethanol will react with tryptophan to  Figure 4C) is removed. This observation is good evidence that photolysis of MPNO produces a reactive radical such hydroxyl, which is then involved in production of 5-OHTrp and hyperluminescence on multiphoton irradiation. The validity of this is supported by kinetic analysis of the data shown in The best fit to the data to equation (5) is shown by the solid curve in Figure 8 taking k e to be 3.5 x 10 9 dm 3 mol -1 s -1 , which is in reasonable agreement with the actual value [13].
Botchway et al: Multiphoton generation of hydroxyl radicals Overall the results described here show that multiphoton excitation of MPNO at 750 nm generates the hydroxyl radical and this may then be scavenged by tryptophan to yield 5-hydroxytryptophan that is subsequently excited in a 5-photon process similar to that previously described [27] to produce the characteristic green emission. It is also possible that hydroxyl radicals react with tryptophan to give another radical product (such as the 5-indoxyl radical) which might be excited to produce the fluorescence. However, the evidence points towards the formation of 5-hydroxytryptophan as this can be detected in the one-photon experiment from UVexcited fluorescence and the spectroscopic properties of the green emission (wavelength, fluorescence lifetime) are essentially identical to those determined previously for multiphoton excitation of 5-OHTrp. Furthermore, the relatively high concentration of 5-OHTrp within the confocal volume indicated by the relative intensity of the green emission suggests that 5-OHTrp accumulates between successive sub-picosecond pulses of the Ti-Sapphire laser.

CONCLUSIONS
The results show that multiphoton near-infrared excitation of the photoFenton reagent MPNO is capable of generating significant yields of hydroxyl radical within the confocal volume at the laser focus. Simultaneous detection of hydroxyl radicals was enabled by adding tryptophan as a hydroxyl radical scavenger producing 5-OHTrp which was re-excited to form a species emitting at 500 nm. This may have the potential to detect hydroxyl radical generation in more complex biochemical and biological systems. In comparison with the normal one-photon excitation in the UV, the near-infrared excitation has several advantages. These include the lack of competing absorption by the solutes due to their lower cross sections at 750 nm, the simultaneous excitation of the probe, and the potential to be applied to cellular systems for the study of hydroxyl radical induced stress. In the latter context, oxidative stress could be induced at a specific intracellular locus as is currently being explored by direct photodamage of DNA [33,34] and resulting effects imaged in the scanning confocal microscope. The results demonstrate the feasibility for developing further the use of multiphoton activated ROS for studying oxidative stress at the chemically specific level within living cells.

Acknowledgments
This work was made possible through access to the Confocal Microscopy Laboratory at the Central Laser Facility, CCLRC Rutherford Appleton Laboratory, and through the studentship to AGC funded by the CCLRC Biomed Network.     Emission was detected through a 500 nm interference filter.