Roman Dědic

Phosphorescence of singlet oxygen and meso-tetra(4-sulfonatophenyl)porphin: time and spectral resolved study

Highly sensitive spectroscopic experimental setup was built to detect infrared luminescence with both time and spectral resolution. In this contribution, phosphorescence of meso-tetra(4-sulphonatophenyl)porphin, which is used as a photosensitizer for photodynamic therapy of cancer, and emission of singlet oxygen were studied. Two-dimensional matrices of data (counts as a function of time and wavelength) were obtained. From these matrices, 1.7 μs lifetime of the photosensitizer triplet state and the same rise-time of the singlet oxygen emission were resolved. Further, 3.7 μs lifetime of singlet oxygen phosphorescence was determined.

Luminescence Study of Singlet Oxygen Production by Meso-Tetraphenylporphine

The research in the field of the photodynamic therapy of cancer (PDT) is focused on a development of photosensitizers exhibiting high quantum yield of singlet oxygen production. Direct time-resolved spectroscopic observation of singlet oxygen phosphorescence can provide time constants of its population and depopulation as well as photosensitizer phosphorescence lifetime and relative quantum yields. In our contribution, a study of time and spectral resolved phosphorescence of singlet oxygen photosensitized by meso-tetraphenylporphine in acetone together with the photosensitizer phosphorescence is presented. Time constants of singlet oxygen population and depopulation were determined at wide range of photosensitizer concentrations. The time constant of singlet oxygen generation (0.28 ± 0.01) μs is slightly shorter then the lifetime of photosensitizers triplet state (0.32 ± 0.01) μs. It is caused by lower ability of TPP aggregates to transfer excitation energy to oxygen. The lifetime of singlet oxygen (≈50 μs) decreases with increasing photosensitizer concentration. Therefore, the photosensitizer acts also as a quencher of oxygen singlet state, similarly to the effects observed in [A. A. Krasnovsky, P. Cheng, R. E. Blankenship, T. A. Moore, and D. Gust (1993). Photochem. Photobiol.57, 324–330; H. Küpper, R. Dědic, A. Svoboda, J. Hála, and P. M. H. Kroneck (2002). Biochim. Biophys. Acta Gen. Subj.1572, 107–113]. Moreover, the increasing concentraion of the photosensitizer causes a slight hypsochromic shift of the singlet oxygen luminescence maximum.

Singlet oxygen-sensitized delayed fluorescence of common water-soluble photosensitizers

Six common water-soluble singlet oxygen ((1)O2) photosensitizers - 5,10,15,20-tetrakis(1-methyl-4-pyridinio) porphine (TMPyP), meso-tetrakis(4-sulfonathophenyl)porphine (TPPS4), Al(III) phthalocyanine chloride tetrasulfonic acid (AlPcS4), eosin Y, rose bengal, and methylene blue - were investigated in terms of their ability to produce delayed fluorescence (DF) in solutions at room temperature. All the photosensitizers dissolved in air-saturated phosphate buffered saline (PBS, pH 7.4) exhibit easily detectable DF, which can be nearly completely quenched by 10 mM NaN3, a specific (1)O2 quencher. The DF kinetics has a biexponential rise-decay character in a microsecond time domain. Therefore, we propose that singlet oxygen-sensitized delayed fluorescence (SOSDF), where the triplet state of a photosensitizer reacts with (1)O2 giving rise to an excited singlet state of the photosensitizer, is the prevailing mechanism. It was confirmed by additional evidence, such as a monoexponential decay of triplet-triplet transient absorption kinetics, dependence of SOSDF kinetics on oxygen concentration, absence of SOSDF in a nitrogen-saturated sample, or the effect of isotopic exchange H2O-D2O. Eosin Y and AlPcS4 show the largest SOSDF quantum yield among the selected photosensitizers, whereas rose bengal possesses the highest ratio of SOSDF intensity to prompt fluorescence intensity. The rate constant for the reaction of triplet state with (1)O2 giving rise to the excited singlet state of photosensitizer was estimated to be ~/>1 × 10(9) M(-1) s(-1). SOSDF kinetics contains information about both triplet and (1)O2 lifetimes and concentrations, which makes it a very useful alternative tool for monitoring photosensitizing and (1)O2 quenching processes, allowing its detection in the visible spectral region, utilizing the photosensitizer itself as a (1)O2 probe. Under our experimental conditions, SOSDF was up to three orders of magnitude more intense than the infrared (1)O2 phosphorescence and by far the most important pathway of DF. SOSDF was also detected in a suspension of 3T3 mouse fibroblast cells, which underlines the importance of SOSDF and its relevance for biological systems.

Hole burning study of CP 34 pigment protein of iron-deprived cyanobacterium Synechococcus elongatus

Abstract Persistent hole burning results obtained with the isolated chlorophyll-binding protein CP 34 induced under Fe stress in the cyanobacterium Synechococcus elongatus are presented. Excited state lifetimes were determined from zero phonon hole widths while chlorophyll–protein coupling was characterised from phonon side-band holes and inhomogeneously broadened function. Comparison of these values to those obtained previously with other chlorophyll–protein complexes from the same cyanobacterium supports an antenna role as well as chlorophyll-storage function of CP 34 in Fe limited cyanobacteria.

Parallel fluorescence and phosphorescence monitoring of singlet oxygen photosensitization in rats

The time- and spectral-resolved set-up for measurements of weak infrared luminescence of photosensitizers (PSs) and singlet oxygen using optical lightguides was used on skin of laboratory animals in vivo. Wistar rats with abdominal incisions treated by methylis aminolevulinitis (MAL) were used as a model. A control group of animals with abdominal incisions was also tested. Spectrally resolved fluorescence of the PS was acquired during the treatment from the same spot. The intensity and spectral profile of the fluorescence signal from the skin can be used to guide the detection setup to the investigated spots in the lesion. The rate of bleaching of Protoporphyrin IX band and appearance of a band of its photoproducts during the treatment can be characterized by the exposition ED, under which the latter becomes dominant feature in fluorescence spectrum. ED value differs statistically significantly between the normal skin and the lesion treated by MAL. No direct proportionality was found between the fluorescence signal and singlet oxygen production. Nevertheless, the strong fluorescence signal is necessary but not a sufficient condition for higher singlet oxygen production in vivo. ED value correlates rather well with production of singlet oxygen, but differently in lesion and normal skin. Lifetimes of singlet oxygen differ between spots outside and in the lesion. PS triplet state lifetimes exhibit weak difference between spots treated and untreated by MAL.

Low-temperature spectroscopy of bacteriochlorophyll c aggregates

Chlorosomes from green photosynthetic bacteria belong to the most effective light-harvesting antennas found in nature. Quinones incorporated in bacterichlorophyll (BChl) c aggregates inside chlorosomes play an important redox-dependent photo-protection role against oxidative damage of bacterial reaction centers. Artificial BChl c aggregates with and without quinones were prepared. We applied hole-burning spectroscopy and steady-state absorption and emission techniques at 1.9 K and two different redox potentials to investigate the role of quinones and redox potential on BChl c aggregates at low temperatures. We show that quinones quench the excitation energy in a similar manner as at room temperature, yet the quenching process is not as efficient as for chlorosomes. Interestingly, our data suggest that excitation quenching partially proceeds from higher excitonic states competing with ultrafast exciton relaxation. Moreover, we obtained structure-related parameters such as reorganization energies and inhomogeneous broadening of the lowest excited state, providing experimental ground for theoretical studies aiming at designing plausible large-scale model for BChl c aggregates including disorder.

Chapter 28:Singlet Oxygen-Sensitized Delayed Fluorescence

The chapter provides a review on singlet oxygen-sensitized delayed fluorescence (SOSDF). The molecular mechanism, kinetics properties, experimental instrumentation, and specific applications of SOSDF are overviewed. Concurrent types of delayed fluorescence that may overlap with SOSDF are also discussed. It provides a framework for distinguishing among different types of delayed fluorescence, which is necessary for correct analysis of the experimental data. SOSDF of a photosensitizer offers an alternative method for singlet oxygen (1O2) detection and monitoring employing the photosensitizer itself as a probe. SOSDF allows to determine the rate constants of 1O2 formation and deactivation in a time-resolved experiment similarly to direct 1O2 near-infrared phosphorescence. However, SOSDF can be orders of magnitude more intense and manifests itself in the visible spectrum, which makes the detection less experimentally demanding. SOSDF has been observed in a wide range of systems, including living cells and tissues incubated with photosensitizers. These properties make SOSDF a promising method for online monitoring during photodynamic therapy treatment.

Laser-induced hole filling in reaction center of photosystem II

Abstract Laser-induced hole-filling of a primary hole (at λ pri ) after burning of a secondary hole (at λ sec ) was measured in Q y absorption spectra of the photosystem II reaction center at 4.2 K. The areas of the primary burnt holes were filled more for λ pri > λ sec than for λ pri λ sec . The observed laser-induced hole filling kinetics followed biexponential decays for λ pri > λ sec , while for λ pri λ sec they were only monoexponential. Two different laser-induced hole filling mechanisms were connected with two different decay parameters. The first laser-induced hole filling mechanism was interpreted as photoinduced relaxation of the protein matrix, while the second one was due to excited energy transfer in the reaction center of photosystem II.