Detecting Free Radicals (Singlet Oxygen) Using a Fluorescence Spectrometer

Detecting the singlet state of Oxygen (1O2) is important in a wide range of applications such as fine chemical synthesis, wastewater treatment and the photodynamic treatment of cancer. 1O2 has a natural bandwidth of around 18 nm and exhibits a weak emission band at approximately 1270 nm.

Direct excitation of molecular oxygen into it's first excited singlet state by the absorption of light is forbidden as its ground state is in a triplet state. Hence, 1O2 is usually produced by chemical reactions or by photosensitisation, which involves the absorption of light by photoactive dyes that transfer the energy absorbed to the molecular oxygen through their triplet state.

It has always been challenging to detect 1O2 emission as the signal at 1270 nm is very weak. Histroically, germanium and InGaAs detectors were mainly employed for steady-state applications. Due to an additional reduction in the signal amplitude when the detector’s RC constant is maintained at a low level (in order to acquire the required temporal resolution) lifetime applications were rather limited.

Modern photomultipliers (PMT) have a sensitivity of 1400 nm and above to enable steady-state and lifetime measurements of 1O2. Even while using modern detectors, a powerful excitation source and a highly efficient optical system are mandatory. For such applications, the FLS980 proves to be an ideal spectrometer.

Materials and Methods

A FLS980 Fluorescence Spectrometer, fitted with a 60 W microsecond flashlamp, a 450 W Xe lamp and single monochromators was used to measure the emission spectra. 1200 and 400 nm gratings were used respectively at the emission and excitation arms.

An integration time of 2 seconds was achieved using a two-stage TE-cooled InGaAs detector (Hamamatsu, G8605-23) with 20 repeats and 0.1 second dwell, and an NIR-PMT (Hamamatsu, R5509-72) with 4 repeats and a 0.5 second dwell. The long wave-pass filters integrated in the FLS980 spectrometer enabled filtering of higher diffraction orders. Air-saturated samples of tris(2,2’-bipyridyl)dichlororuthenium(II) hexahydrate Ru(bpy)3 (Sigma-Aldrich, 544981) dissolved in D2O (Sigma-Aldrich, 151882) and Erythrosin B (Sigma-Aldrich, 19,826-9) dissolved in anhydrous ethanol (Sigma-Aldrich, 459836) with respective optical densities (ODs) of 0.35 and 0.5 were prepared. Additional saturation of the Ru(bpy)3 sample with oxygen was performed for around 10 minutes.

Quartz cuvettes with a path length of 10 mm at right angle geometry were used for measuring the samples. The Erythrosin B sample was excited at 530 nm with emission and excitation bandwidths of 15 nm, whereas the Ru(bpy)3 samples were excited at it's 450 nm absorption peak. For direct comparison of the spectra from different detectors, noise-normalization of the data was done by applying a scaling that is equivalent to the noise ratio at 1360 to 1400 nm, i.e., f = INIR-PMT(λ)/IInGaAs(λ), offset in a vertical manner.

Steady-State Emission

The NIRPMT and InGaAs detectors were used to measure the 1O2 emission spectra depicted in Figure 1. The 1O2 emission generated by Erythrosin at about 1270 nm displays a single emission peak, whereas the 1O2 emission generated by Ru(bpy)3 is overlapped by the emission tail of the sensitizer molecule. As depicted in Figure 1b, the background emission with respect to the saturated O2 sample is minimized as a result of quenching of the triplet state, similar to that observed for Ru(bpy)3 dissolved in aqueous solutions.

Steady-State Emission

Steady-State Emission

Figure 1. Emission spectra of 1O2 in Erythrosin B and Ru(bpy)3 under λexc = 530nm and λexc = 450nm, respectively

For the 1O2 emission generated by Erythrosin B, the estimated signal to noise ratio (SNR) is 45 for the InGaAs detector and 195 for the NIR-PMT detector. This SNR difference is due to the difference in sensitivity, quantum efficiency, active area, integration and sources of noise in both the detectors:

  1. Noise: The only source of noise in the NIR-PMT is photon noise (Poisson noise) as the NIR-PMT is a single photon counting detector. The photon noise is the square root of the quantified signal (or the square root of the signal plus detector dark counts). The InGaAs detector is equipped with a lock-in amplifier that reduces noise fluctuations, and hence is noise-limited by the noise equivalent power (NEP).
  2. Sensitivity: Single photon counting and analogue detection differ in sensitivity because single photon counting is intrinsically digital and accepts only signal counts above a noise floor.
  3. Active area: A standard InGaAs chip has an active area of 3 mm, while NIR-PMT has an active area of 24 mm. This factor has no impact on small monochromator slits, but in the case of weak samples, the increase in sensitivity is restricted as the monochromator slits are opened to have a greater focus on the detector than the active area.
  4. Quantum efficiency: The quantum efficiency of the InGaAs detector is nearly 2 orders of magnitude greater than that of the NIR-PMT at 1270 nm. This efficiency is further dependent on the detectors’ operating temperature.
  5. Integration: With regard to the analogue detection, the integration time is set by the electrical circuit, while in the case of single photon counting the integration time is set directly by the duration of time the photons are counted.

The intrinsic higher source of noise in the analogue detector indicates a greater SNR in the NIR-PMT and consequently higher sensitivity at 1270 nm, although the InGaAs could have greater quantum efficiency. For direct comparison of the 1O2 signal provided by the two detectors, noise-normalization of the emission spectra of 1O2 from Erythrosin B (Figure 1) is shown in Figure 2.

Emission spectra of 1O2 in Erythrosin B

Figure 2. Emission spectra of 1O2 in Erythrosin B obtained by NIR-PMT and InGaAs detectors.

Time-Resolved Emission

InGaAs detectors can measure the emission spectra of 1O2; however, a photon counting detector is required to measure its lifetime. Therefore, the NIR-PMT was used to measure the decay of 1O2 observed at 1270 nm. The result is illustrated in Figure 3. The exponential fit led to a lifetime of 15.3µs for 1O2 generated from Erythrosin B dissolved in ethanol, and 59.47 µs for Ru(bpy)3 dissolved in D2O, which correlated well with the reported decay values.

Life time decay of Erythrosin B in Ethanol

Life time decay of Ru(bpy)3 in D2O

Figure 3. Life time decay of (a) Erythrosin B in Ethanol and (b) Ru(bpy)3 in D2O

Time-resolved emission spectra (TRES) can be acquired by performing time-resolved scans over the 1O2 emission. Thus, a differentiation between the 1O2 emission and the phosphorescence generated by the sensitizer can be made. The emission spectra generated from Ru(bpy)3 at 200-400µs with a step of 20 µs are depicted in Figure 4.

Time-resolved emission spectra (TRES) of 1O2 generated from Ru(bpy)3

Figure 4. Time-resolved emission spectra (TRES) of 1O2 generated from Ru(bpy)3

Conclusion

This article presents the time-resolved and steady-state emission spectra of 1O2 generated from Erythrosin B and Ru(bpy)3.samples Moreover, the lifetimes were fitted as single exponentials and are found to correlate with the reported lifetimes. Hence, we can conclude that both time-resolved and steady-state measurements of 1O2 spectra can be acquired by using single photon counting detectors like NIR-PMT, whereas InGaAs detectors can be employed only for making steady-state measurements.

This information has been sourced, reviewed and adapted from materials provided by Edinburgh Instruments.

For more information on this source, please visit Edinburgh Instruments.

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