Time-resolved investigations of singlet oxygen luminescence in vitro and in vivo

Singlet oxygen 

Singlet oxygen () is molecular oxygen in the lowest lying excited electronic state. The energy difference between ground state triplet oxygen () and singlet oxygen is 0.976 eV. A direct and precise method to detect singlet oxygen is the measurement of its luminescence in the near infrared spectral range at 1270 nm.
Singlet oxygen can be generated in a photosensitized process by energy transfer from dye molecules or by chemical processes.

Photosensitized generation of singlet oxygen

The energy level diagram for the excitation of singlet oxygen by energy transfer from the triplet T1 state of the photosensitizer and the deactivation of singlet oxygen and the T1 state is given by the following figure.

It contains the singlet ground state S0 of the photosensitizer, its first excited singlet state S1 and its first excited triplet state T1. The scheme contains also the ground state of oxygen 3O2 and the first excited singlet state of oxygen, (1O2). Additionally, the figure shows the ground state of the solvent and the ground state of a possible quencher. Vibronic states are not included. The relaxation rates kj and the rate constants kij describe the frequency of the occurrence of the relaxation. 
The photosensitizer absorbs the energy of the laser pulse leading to population of S1 state and a part of molecules will fill the T1 state via intersystem crossing (ISC). In comparison to the other rates, this process is considered instantaneous and it could be assumed that at t = 0 µs (start of the laser pulse) the T1 state population is completed.
In case of low laser energy, the ground state concentration [S0] of the photosensitizer is approximately equal to the complete concentration of photosensitizer [P] in solvent for all times. The concentration of oxygen molecules in the ground state [3O2] is similar to the oxygen concentration in solvent [O2] if the concentration of the triplet state photosensitizers [T1] is smaller then the concentration of the oxygen molecules in solvent.
In order to describe mathematically the energy level diagram the time dependence of the population densities [T1] of the photosensitizer triplet state and [1O2] of the oxygen singlet state must be investigated: 

The system of the differential equations can be considered a harmonic oscillator and the solutions of the system are given by

for the time dependence of the population of singlet oxygen and

for the time dependence of the population of the triplet T1 state of the photosensitizer. Here [T1]t=0 is the initial population density of the triplet state T1 of the photosensitizer. The rates beta1 and beta2 are defined by

The experimental luminescence signal at 1270 nm can be described by Equation [1O2](t). The rates beta1 and beta2 correspond the rise rate and the decay rate of the luminescence signal, respectively. 

Luminescence of singlet oxygen

In the Figure above the time independence luminescence of singlet oxygen was measured at different wavelengths from 1170 nm to 1370 nm.

The singlet oxygen was generated by 50 µM Riboflavin in H2O. The irradiation wavelength was 355 nm and solvent was air saturated. All measurements were added to a three-dimensional plot in Figure A. The typical measured logarithmic signal at 1270 nm is shown in Figure B. At 0 µs the laser pulse is shown and the solid line represent the theoretical fit according [1O2](t). The signals at 1270 nm were taken to determine the raise (beta1) and decay rate (beta2). In this case the signal raises with 3.3±0.5 µs and decays with 3.2±0.5 µs. In Figure C the wavelength scan by summing up the luminescence signals at each wavelength is shown. The maximum at 1270 nm which represent photon energy of 0.976eV is a unique evidence for the luminescence of singlet oxygen. 


Theoretical and experimental analysis of the luminescence signal of singlet oxygen for different photosensitizers
J. Baier, T. Fuß, C. Pöllmann, C. Wiesmann, K. Pindl, R. Engl, D. Baumer, M. Maier, M. Landthaler, and W. Bäumler
J. Photochem. Photobiol. B, Biol. in press 2007

The role of singlet oxygen and oxygen concentration in photodynamic inactivation of bacteria
T. Maisch, J. Baier, B. Franz, M. Maier, M. Landthaler, R.-M. Szeimies, and W. Bäumler
Proc. Natl. Acad. Sci. U.S.A. (PNAS) 104 (17): 7223. (2007) [download, abstract

Direct Detection of Singlet Oxygen Generated by UVA Irradiation in Human Cells and Skin
J. Baier, T. Maisch,  M. Maier, E. Engl, M. Landthaler, and W. Bäumler
J. Invest. Dermatol. online publication 2007 [download, abstract

Singlet oxygen generation by UVA light exposure of endogenous photosensitizers
J. Baier, T. Maisch, M. Maier, E. Engl, M. Landthaler, and W. Bäumler
Biophys. J. 2006 91: 1452-1459 [download, abstract]

Time-Resolved Investigations of Singlet Oxygen Luminescence in Water, in Phosphatidylcholine, and in Aqueous Suspensions of Phosphatidylcholine or HT29 Cells
J. Baier, M. Maier, R. Engl, M. Landthaler, and W. Bäumler
J. Phys. Chem. B 2005, 109 (7), 3041-3046 [download, abstract]

Singlet oxygen generation by 9-acetoxy-2,7,12,17-tetrakis-(beta-methoxyethyl)-porphycene (ATMPn) in solution
D. Baumer, M. Maier, R. Engl, R.-M. Szeimies and W. Bäumler
Chem. Phys. 285 (2002) 309-318

Singlet Oxygen Generation by 8-Methoxypsoralen in Deuterium Oxide: Relaxation Rate Constants and Dependence of the Generation Efficacy on the Oxygen Partial Pressure
R. Engl, R. Kilger, M. Maier, K. Scherer, C. Abels, and W. Bäumler
J. Phys. Chem. B 2002, 106 (22), 5776-5781

Bidirectional energy transfer between the triplet T1 state of photofrin and singlet oxygen in deuterium oxide
R. Kilger, M. Maier, R.-M. Szeimies and W. Bäumler
Chem. Phys. Lett. 2001, 343, (5-6), 543-548