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

Abstract

Solutions of tryptophan and N-hydroxypyridine-2-thione (mercaptopyridine-N-oxide, MPNO) were irradiated at 335nm. Formation of 5-hydroxytryptophan was inferred from increased fluorescence at 334nm on excitation at 315nm, conditions chosen for selective detection of 5-hydroxytryptophan. Such experiments are complicated by overlapping absorption spectra in the region of 300-350nm. Similar solutions were exposed to multiphoton excitation at 750nm using 180fs pulses from a titanium:sapphire laser. In solutions containing both tryptophan and MPNO strong emission at 500nm 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 750nm 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.

Fig. 1

Schematic of the apparatus used for the experiments. The Ti:S laser produces 180 fs pulses at 75 MHz with a total average power of up to ∼100 mW at the sample. The power is adjusted by a variable neutral density filter (m1) before entering the epifluorescence microscope (Nikon TE2000). Emission is detected via a movable mirror (m2) either spectrally using a spectrograph–CCD combination or in time-resolved mode using an interference filter and microchannel plate photomultiplier (MC-PMT) linked to a time-resolved single photon counting card in the PC.

Fig. 2

Absorption spectra of HPNO (a) and MPNO (b) (right-hand scale) shown with those of Trp (c) and 5-OHTrp (d) (left-hand scale). All spectra were measured in phosphate buffered saline (pH 7.3).

Fig. 3

Detection of 5-hydroxytryptophan fluorescence on excitation of solutions of MPNO and Trp at 315 nm. The solutions were irradiated at 335 nm (slits 30 nm bandwidth) in a stirred 1 cm quartz cuvette in the sample compartment of a Spex Fluoromax fluorimeter for 2 min periods. Following each irradiation period, fluorescence was measured at 334 nm on excitation at 315 nm (5 nm slits). Solutions were air-saturated in phosphate buffer (0.05 mol dm⁻³, pH 7.3). Line A—MPNO (200 μmol⁻³) only; line B—trp (250 μmol dm⁻³) and MPNO (200 μmol⁻³); line C—trp (500 μmol dm⁻³) and MPNO (200 μmol dm⁻³); line D—trp (250 μmol dm⁻³) only.

Fig. 4

Emission spectra measured using a CCD array on multiphoton illumination (750 nm, 180 fs pulses at 75 MHz, average power 35 mW) of solutions of Trp and MPNO (2 mmol dm⁻³) in phosphate buffer (pH 7.3, 50 mmol dm⁻³). Each figure shows spectra from MPNO alone (curve a)), Trp alone (curve b)) and Trp and MPNO together (curve c)) at Trp concentrations of 0.45 mmol dm⁻³ (A), 1.8 mmol dm⁻³ (B) and 3.6 mmol dm⁻³ (C). Accumulation time 200 s.

Fig. 5

Nanosecond time-resolved fluorescence decay decays recorded from multiphoton excitation at 750 nm of solutions of (a) tryptophan (2 mmol dm⁻³) plus MPNO (2 mmol dm⁻³) and (b) MPNO (2 mmol dm⁻³). Emission was detected through a 500 nm interference filter.

Fig. 6

Log–log plot showing the effect of average laser power at 750 nm on emission intensity at:- 510 nm in solutions containing MPNO (2 mmol dm⁻³) only (♦, solid line slope of 5.9); at 500 nm (■, solid line slope of 5.1) and at 380 nm (□, dotted line slope of 3) in solutions of Trp (4 mmol dm⁻³) and MPNO (2 mmol dm⁻³); and at 500 nm in solutions of Trp (2.5 mmol dm⁻³) only (◇, dashed line slope of 8).

Fig. 7

Effect of Trp concentration on the intensities of fluorescence at 380 nm (□) and 500 nm (■) measured on 750 nm multiphoton excitation of solutions containing MPNO (2 mmol dm⁻³) in phosphate buffer. The curve shown for the 500 nm emission is that obtained by non-linear fitting of the data to Eq. (4).

Fig. 8

Effect of ethanol addition on the relative intensity (S) of hyperluminescence at 500 nm from solutions of tryptophan (4 mmol dm⁻³) and MPNO (2 mmol dm⁻³). The solid line shows the data fitted to Eq. (5). Inset. Luminescence spectra from solutions of Trp and MPNO containing ethanol at concentrations of 0 (curve 1), 12.9 (curve 2), 34.3 (curve 3), 103.0 (curve 4) and 687 mmol dm⁻³ (curve 5). Also shown are spectra from solutions containing only Trp (curve 6) and MPNO (curve 7). Apparent structure in these curves is due to transmission characteristics of the interference filters as noted in Materials and methods.