Microscopic time-resolved imaging of singlet oxygen by delayed fluorescence in living cells.

Singlet oxygen is a highly reactive species which is involved in a number of processes, including photodynamic therapy of cancer. Its very weak near-infrared emission makes imaging of singlet oxygen in biological systems a long-term challenge. We address this challenge by introducing Singlet Oxygen Feedback Delayed Fluorescence (SOFDF) as a novel modality for semi-direct microscopic time-resolved wide-field imaging of singlet oxygen in biological systems. SOFDF has been investigated in individual fibroblast cells incubated with a well-known photosensitizer aluminium phthalocyanine tetrasulfonate. The SOFDF emission from the cells is several orders of magnitude stronger and much more readily detectable than the very weak near-infrared phosphorescence of singlet oxygen. Moreover, the analysis of SOFDF kinetics enables us to estimate the lifetimes of the involved excited states. Real-time SOFDF images with micrometer spatial resolution and submicrosecond temporal-resolution have been recorded. Interestingly, a steep decrease in the SOFDF intensity after the photodynamically induced release of a photosensitizer from lysosomes has been demonstrated. This effect could be potentially employed as a valuable diagnostic tool for monitoring and dosimetry in photodynamic therapy.

Singlet oxygen feedback delayed fluorescence of protoporphyrin IX in organic solutions.

Delayed fluorescence (DF) of protoporphyrin IX (PpIX) has been recently proposed as a tool for monitoring of mitochondrial oxygen tension in vivo as well as for observation of the effectiveness of photodynamic therapy (PDT) [E. G. Mik, Anesth. Analg., 2013, 117, 834-346; F. Piffaretti et al., J. Biomed. Opt., 2012, 17, 115007]. However, the efficiency of the mechanism of thermal activation (E-type DF), which was considered in the papers, is limited due to a large energy gap between the first excited singlet and the first triplet state of PpIX at room or body temperatures. Moreover, the energy gap is roughly equal to other porphyrinoid photosensitizers that generate DF mostly through the Singlet Oxygen Feedback-Induced mechanism (SOFDF) under certain conditions [M. Scholz and R. Dedic, Singlet Oxygen: Applications in Biosciences and Nanosciences, 2016, vol. 2, pp. 63-81]. The mechanisms of delayed fluorescence of PpIX dissolved either in dimethylformamide (DMF) or in the mixture of DMF with ethylene glycol (EG) were investigated at atmospheric partial pressure of oxygen by means of a simultaneous time-resolved detection of 1O2 phosphorescence and PpIX DF which makes a direct comparison of the kinetics and lifetimes of both the luminescence channels possible. Samples of PpIX (100 µM) exhibit concave DF kinetics, which is a typical footprint of the SOFDF mechanism. The dramatic decrease in the DF intensity after adding a selective 1O2 quencher sodium azide (NaN3, 10 mM) proves that >90% of DF is indeed generated through SOFDF. Moreover, the analysis of the DF kinetics in the presence of NaN3 implies that the second significant mechanism of DF generation is the triplet-triplet annihilation (P-type DF). The bimolecular mechanism of DF was further confirmed by the decrease of the DF intensity in the more viscous mixture DMF/EG and by the increase of the ratio of DF to the prompt fluorescence (PF) intensity with the increasing excitation intensity. These results show the significant role of the SOFDF mechanism in the DF of PpIX at high concentrations and at atmospheric partial pressure of oxygen and should be considered when developing diagnostic tools for clinical applications.

The singlet-oxygen-sensitized delayed fluorescence in mammalian cells: a time-resolved microscopy approach.

The present work provides a proof-of-concept that the singlet oxygen-sensitized delayed fluorescence (SOSDF) can be detected from individual living mammalian cells in a time-resolved microscopy experiment. To this end, 3T3 mouse fibroblasts incubated with 100 µM TPPS4 or TMPyP were used and the microsecond kinetics of the delayed fluorescence (DF) were recorded. The analysis revealed that SOSDF is the major component of the overall DF signal. The microscopy approach enables precise control of experimental conditions - the DF kinetics are clearly influenced by the presence of the (1)O2 quencher (sodium azide), H2O/D2O exchange, and the oxygen concentration. Analysis of SOSDF kinetics, which was reconstructed as a difference DF kinetics between the unquenched and the NaN3-quenched samples, provides a cellular (1)O2 lifetime of τΔ = 1-2 µs and a TPPS4 triplet lifetime of τT = 22 ± 5 µs in agreement with previously published values. The short SOSDF acquisition times, typically in the range of tens of seconds, enable us to study the dynamic cellular processes. It is shown that SOSDF lifetimes increase during PDT-like treatment, which may provide valuable information about changes of the intracellular microenvironment. SOSDF is proposed and evaluated as an alternative tool for (1)O2 detection in biological systems.

Real-time luminescence microspectroscopy monitoring of singlet oxygen in individual cells.

A new setup for direct microspectroscopic monitoring of singlet oxygen ((1)O2) has been developed in our laboratory using a novel near-infrared sensitive InGaAs 2D-array detector. An imaging spectrograph has been inserted in front of the 2D-array detector, which allows us to acquire spectral images where one dimension is spatial and the other is spectral. The work presents a detailed examination of sensitivity and noise characteristics of the setup and its ability to detect (1)O2. The (1)O2 phosphorescence-based images and near-infrared luminescence spectral images recorded from single TMPyP-containing fibroblast cells reflecting spectral changes during irradiation are demonstrated. The introduction of spectral images addresses the issue of a potential spectral overlap of (1)O2 phosphorescence with near-infrared-extended luminescence of the photosensitizer and provides a powerful tool for distinguishing and separating them, which can be applied to any photosensitizer manifesting near-infrared luminescence.

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.

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.

Time-resolved luminescence and singlet oxygen formation after illumination of the hypericin-low-density lipoprotein complex.

Time-resolved fluorescence and phosphorescence study of hypericin (Hyp) in complex with low-density lipoproteins (LDL) as well as the evolution of singlet oxygen formation and annihilation after illumination of Hyp/LDL complexes at room temperature are presented in this work. The observed shortening of the fluorescence lifetime of Hyp at high Hyp/LDL molar ratios (>25:1) proves the self-quenching of the excited singlet state of monomeric Hyp at these concentration ratios. The very short lifetime ( approximately 0.5 ns) of Hyp fluorescence at very high Hyp/LDL ratios (>150:1) suggests that at high local Hyp concentration inside LDL molecules fast and ultrafast nonradiative decay processes from excited singlet state of Hyp become more important. Contrary to the lifetime of the singlet excited state, the lifetime (its shorter component) of Hyp phosphorescence is not dependent on Hyp/LDL ratio in the studied concentration range. The amount of singlet oxygen produced as well as the integral intensity of Hyp phosphorescence after illumination of Hyp/LDL complexes resemble the dependence of the concentration of molecules of Hyp in monomeric state on Hyp/LDL until a concentration ratio of 60:1. This fact confirms that only monomeric Hyp is able to produce the excited triplet state of Hyp, which in aerobic conditions leads to singlet oxygen production. The value of singlet oxygen lifetime ( approximately 8 micros) after its formation from the excited triplet state of Hyp in LDL proves that molecules of singlet oxygen remain for a certain period of time inside LDL particles and are not immediately released to the aqueous surrounding. That Hyp exists in the complex with LDL in the monodeprotonated state is also demonstrated.

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) micros is slightly shorter then the lifetime of photosensitizer's triplet state (0.32 +/- 0.01) micros. It is caused by lower ability of TPP aggregates to transfer excitation energy to oxygen. The lifetime of singlet oxygen (approximately 50 micros) 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. Dedic, A. Svoboda, J. Hála, and P. M. H. Kroneck (2002). Biochim. Biophys. Acta Gen. Subj. 1572, 107-113]. Moreover, the increasing concentration of the photosensitizer causes a slight hypsochromic shift of the singlet oxygen luminescence maximum.

Effect of carotenoids and monogalactosyl diglyceride on bacteriochlorophyll c aggregates in aqueous buffer: implications for the self-assembly of chlorosomes.

Aggregation of bacteriochlorophyll (BChl) c from chlorosomes, the main light-harvesting complex of green bacteria, has been studied in aqueous buffer. Unlike other chlorophyll-like molecules, BChl c is rather soluble in aqueous buffer, forming dimers. When BChl c is mixed with carotenoids (Car), the BChl c Qy transition is further redshifted, in respect to that of monomers and dimers. The results suggest that Car are incorporated in the aggregates and induce further aggregation of BChl c. The redshift of the BChl c Qy band is proportional to the Car concentration. In contrast, the mixture of bacteriochlorophyllide (BChlide) c, which lacks the nonpolar esterifying alcohol, does not form aggregates with Car in aqueous buffer or nonpolar solvents. Instead, the position of the BChlide c Qy transition remains unshifted in respect to that of the monomeric molecule, and Car precipitates with the course of time in aqueous buffer. Similar effects on both BChl c and BChlide c are also observed when monogalactosyl diglyceride (MGDG), which forms the monolayer envelope of chlorosomes, is used instead of (or together with) Car. The results show that the hydrophobic interactions of the BChl c esterifying alcohols with themselves and the nonpolar carbon skeleton of Car, or the fatty acid tails of MGDG, are essential driving forces for BChl aggregation in chlorosomes.

Unique spectroscopic properties of synthetic 15-cis beta-carotene, an important compound in photosynthesis, and a medicine for photoprotective function.

The (1(1)B(u)+) energy of synthetic 15-cis beta-carotene exhibits a linear dependence on (n(2)-1)/(n(2)+2) in non-polar and polar solvents; in this it is similar to (that of) all-trans beta-carotene. The point of intersection is at (n(2)-1)/(n(2)+2) = 0.3 for both isomers. The microenvironment of 15-cis beta-carotene in the Photosystem II reaction center was established as having a mean refractive index 1.473. Persistent spectral hole burning with a very broad (approximately 30 nm) hole observed around 500 nm (corresponding to an extremely short excited lifetime tau approximately 9 fs) indicates that 15-cis beta-carotene has/displays very efficient photoprotective quenching.

Excitation energy transfer dynamics and excited-state structure in chlorosomes of Chlorobium phaeobacteroides.

The excited-state relaxation within bacteriochlorophyll (BChl) e and a in chlorosomes of Chlorobium phaeobacteroides has been studied by femtosecond transient absorption spectroscopy at room temperature. Singlet-singlet annihilation was observed to strongly influence both the isotropic and anisotropic decays. Pump intensities in the order of 10(11) photons x pulse(-1) x cm(-2) were required to obtain annihilation-free conditions. The most important consequence of applied very low excitation doses is an observation of a subpicosecond process within the BChl e manifold (approximately 200-500 fs), manifesting itself as a rise in the red part of the Q(y) absorption band of the BChl e aggregates. The subsequent decay of the kinetics measured in the BChl e region and the corresponding rise in the baseplate BChl a is not single-exponential, and at least two components are necessary to fit the data, corresponding to several BChl e-->BChl a transfer steps. Under annihilation-free conditions, the anisotropic kinetics show a generally slow decay within the BChl e band (10-20 ps) whereas it decays more rapidly in the BChl a region ( approximately 1 ps). Analysis of the experimental data gives a detailed picture of the overall time evolution of the energy relaxation and energy transfer processes within the chlorosome. The results are interpreted within an exciton model based on the proposed structure.

Kinetics and efficiency of excitation energy transfer from chlorophylls, their heavy metal-substituted derivatives, and pheophytins to singlet oxygen.

Time-resolved measurements of the singlet oxygen infrared (1269 nm) luminescence were used to follow the kinetics and efficiency of excitation energy transfer (EET) between chlorophyll (Chl) derivatives and oxygen in acetone. The studied pigments were Mg-Chl a and b and their heavy metal (Cu(2+) and Zn(2+))-substituted analogues, as well as pheophytin (Pheo) a and b. The efficiency of EET from chlorophyll to oxygen was highly dependent on the central ion in the pigment. Cu-Chl a and Cu-Chl b had the lowest efficiencies of singlet oxygen production, while Pheo a had a higher one, and Zn-Chl a had a similar one compared to Mg-Chl a. Also the side chain (position C-7, i.e. Chl a vs. Chl b) influenced the efficiency of singlet oxygen formation. In the case of square-planar complexes like Cu-Chl and Pheo, EET was more efficient in the Chl a derivatives than in those of Chl b; the opposite effect was observed in the case of the five- or six-coordinated Mg-Chl and Zn-Chl. As for the lifetime of the Chl triplet state, the most striking difference to Mg-Chl again was found in the case of Cu-Chls, which had much shorter lifetimes. Furthermore, the central ion in Chl affected the physical quenching of singlet oxygen: its efficiency was decreasing from Mg-Chl through Zn-Chl over Cu-Chl to Pheo. The results are discussed in the context of the oxidative stress accompanying heavy metal-induced stress in plants.