singlet oxygen
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Reactive oxygen species (ROS) are presently thought to play important role in an increasing number of the physiological and pathological processes in living organisms. Various chemiluminescent (CL) compounds have been studied in order to find suitable and specific probes for the detection of particular ROS species. The CL of luminol is known to be non-specific and can be induced by various oxidants. Two Cypridina luciferin analogues, CLA and MCLA, have been used for the detection of ROS in vivo. CLAs are thought to emit light only when reacting with superoxide and singlet oxygen. It is possible to distinguish the particular ROS by using a specific quencher or scavenger, e.g. superoxide dismutase (SOD) or sodium azide (NaN(3)). The CL reactions of luminol (3-aminophthalhydrazide), CLA [2-methyl-6-phenyl-3,7-dihydroimidazo(1,2α) pyrazin-3-one] and MCLA [2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo(1,2α) pyrazin-3-one] were studied in three hydrogen peroxide decomposition systems (H(2)O(2)-HRP; H(2)O(2)-CuSO(4); and H(2)O(2)-NaOCl). The measurements were carried out in phosphate buffer, pH 7.4, at 25°C, using a luminometer (Fluoroskan Ascent FL and Sirius C). NaN(3) was used as the specific quencher of singlet oxygen. The results demonstrate that the proclaimed specificity of the CL of Cypridina luciferin analogues towards singlet oxygen has to be discussed.
Formation of singlet oxygen (1O2) was reported to accompany light stress in plants, contributing to cell signaling or oxidative damage. So far, Singlet Oxygen Sensor Green (SOSG) has been the only commercialized fluorescent probe for 1O2 imaging though it suffers from several limitations (unequal penetration and photosensitization) that need to be carefully considered to avoid misinterpretation of the analysed data. Herein, we present results of a comprehensive study focused on the appropriateness of SOSG for 1O2 imaging in three model photosynthetic organisms, unicellular cyanobacteria Synechocystis sp. PCC 6803, unicellular green alga Chlamydomonas reinhardtii and higher plant Arabidopsis thaliana. Penetration of SOSG differs in both unicellular organisms; while it is rather convenient for Chlamydomonas it is restricted by the presence of mucoid sheath of Synechocystis, which penetrability might be improved by mild heating. In Arabidopsis, SOSG penetration is limited due to tissue complexity which can be increased by pressure infiltration using a shut syringe. Photosensitization of SOSG and SOSG endoperoxide formed by its interaction with 1O2 might be prevented by illumination of samples by a red light. When measured under controlled conditions given above, SOSG might serve as specific probe for detection of intracellular 1O2 formation in photosynthetic organisms.
- MeSH
- Arabidopsis metabolismus MeSH
- barva MeSH
- Chlamydomonas reinhardtii metabolismus MeSH
- fluorescenční barviva metabolismus MeSH
- fotosyntéza fyziologie MeSH
- kyslík metabolismus MeSH
- oxidace-redukce MeSH
- singletový kyslík metabolismus MeSH
- světlo MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Singlet oxygen (1O2) is formed by triplet-triplet energy transfer from triplet chlorophyll to O2 via Type II photosensitization reaction in photosystem II (PSII). Formation of triplet chlorophyll is associated with the change in spin state of the excited electron and recombination of triplet radical pair in the PSII antenna complex and reaction center, respectively. Here, we have provided evidence for the formation of 1O2 by decomposition of protein hydroperoxide in PSII membranes deprived of Mn4O5Ca complex. Protein hydroperoxide is formed by protein oxidation initiated by highly oxidizing chlorophyll cation radical and hydroxyl radical formed by Type I photosensitization reaction. Under highly oxidizing conditions, protein hydroperoxide is oxidized to protein peroxyl radical which either cyclizes to dioxetane or recombines with another protein peroxyl radical to tetroxide. These highly unstable intermediates decompose to triplet carbonyls which transfer energy to O2 forming 1O2. Data presented in this study show for the first time that 1O2 is formed by decomposition of protein hydroperoxide in PSII membranes deprived of Mn4O5Ca complex.
- MeSH
- chlorofyl metabolismus MeSH
- elektronová paramagnetická rezonance metody MeSH
- fotosystém II (proteinový komplex) metabolismus MeSH
- kyslík metabolismus MeSH
- oxidace-redukce MeSH
- peroxid vodíku metabolismus MeSH
- peroxidy metabolismus MeSH
- přenos energie fyziologie MeSH
- singletový kyslík metabolismus MeSH
- světlo MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
In the current study, singlet oxygen formation by lipid peroxidation induced by heat stress (40 °C) was studied in vivo in unicellular green alga Chlamydomonas reinhardtii. Primary and secondary oxidation products of lipid peroxidation, hydroperoxide and malondialdehyde, were generated under heat stress as detected using swallow-tailed perylene derivative fluorescence monitored by confocal laser scanning microscopy and high performance liquid chromatography, respectively. Lipid peroxidation was initiated by enzymatic reaction as inhibition of lipoxygenase by catechol and caffeic acid prevented hydroperoxide formation. Ultra-weak photon emission showed formation of electronically excited species such as triplet excited carbonyl, which, upon transfer of excitation energy, leads to the formation of either singlet excited chlorophyll or singlet oxygen. Alternatively, singlet oxygen is formed by direct decomposition of hydroperoxide via Russell mechanisms. Formation of singlet oxygen was evidenced by the nitroxyl radical 2,2,6,6-tetramethylpiperidine-1-oxyl detected by electron paramagnetic resonance spin-trapping spectroscopy and the imaging of green fluorescence of singlet oxygen sensor green detected by confocal laser scanning microscopy. Suppression of singlet oxygen formation by lipoxygenase inhibitors indicates that singlet oxygen may be formed via enzymatic lipid peroxidation initiated by lipoxygenase.
- MeSH
- Chlamydomonas reinhardtii metabolismus MeSH
- lipoxygenasa metabolismus MeSH
- malondialdehyd metabolismus MeSH
- peroxidace lipidů fyziologie MeSH
- reakce na tepelný šok fyziologie MeSH
- rostlinné proteiny metabolismus MeSH
- singletový kyslík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- analýza jednotlivých buněk přístrojové vybavení metody MeSH
- azid sodný chemie MeSH
- buňky 3T3 MeSH
- design vybavení MeSH
- fibroblasty chemie MeSH
- fluorescence * MeSH
- kinetika MeSH
- kyslík chemie MeSH
- mikroskopie přístrojové vybavení metody MeSH
- myši MeSH
- oxid deuteria chemie MeSH
- singletový kyslík chemie MeSH
- voda chemie MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- analýza jednotlivých buněk přístrojové vybavení metody MeSH
- buňky 3T3 MeSH
- fibroblasty metabolismus MeSH
- fotochemické procesy MeSH
- fotosenzibilizující látky MeSH
- luminiscence MeSH
- mikrospektrofotometrie přístrojové vybavení metody MeSH
- myši MeSH
- počítačové systémy MeSH
- porfyriny MeSH
- singletový kyslík metabolismus MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- buňky 3T3 MeSH
- časové faktory MeSH
- fibroblasty chemie cytologie MeSH
- fluorescence * MeSH
- fluorescenční mikroskopie MeSH
- fotochemoterapie MeSH
- fotosenzibilizující látky chemie MeSH
- indoly chemie MeSH
- kultivované buňky MeSH
- myši MeSH
- optické zobrazování * MeSH
- organokovové sloučeniny chemie MeSH
- singletový kyslík analýza chemie MeSH
- viabilita buněk MeSH
- zvířata MeSH
- Check Tag
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
Wounding, one of the most intensive stresses influencing plants ontogeny and lifespan, can be induced by herbivory as well as by physical factors. Reactive oxygen species play indispensable role both in the local and systemic defense reactions which enable "reprogramming" of metabolic pathways to set new boundaries and physiological equilibrium suitable for survival. In our current study, we provide experimental evidence on the formation of singlet oxygen (1O2) after wounding of Arabidopsis leaves. It is shown that 1O2 is formed by triplet-triplet energy transfer from triplet carbonyls to molecular oxygen. Using lipoxygenase inhibitor catechol, it is demonstrated that lipid peroxidation is initiated by lipoxygenase. Suppression of 1O2 formation in lox2 mutant which lacks chloroplast lipoxygenase indicates that lipoxygenase localized in chloroplast is predominantly responsible for 1O2 formation. Interestingly, 1O2 formation is solely restricted to chloroplasts localized at the wounding site. Data presented in this study might provide novel insight into wound-induced signaling in the local defense reaction.
- MeSH
- Arabidopsis MeSH
- fenotyp MeSH
- fluorescenční protilátková technika MeSH
- konfokální mikroskopie MeSH
- lipoxygenasa metabolismus MeSH
- lipoxygenasy genetika MeSH
- mastné kyseliny metabolismus MeSH
- molekulární zobrazování MeSH
- mutace MeSH
- proteiny huseníčku genetika MeSH
- rány a poranění metabolismus MeSH
- singletový kyslík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
