Singlet Oxygen Luminescence Detection Research Articles

The Role of Singlet Oxygen in Photoreactivity and Phototoxicity of Curcumin.

Curcumin is a plant-derived yellow-orange compound widely used as a spice, dye and food additive. It is also believed to have therapeutic effects against different disorders. On the other hand, there are data showing its phototoxicity against bacteria, fungi and various mammalian cells. Since the mechanism of its phototoxic action is not fully understood, we investigated here the phototoxic potential of curcumin in liposomal model membranes and in HaCaT cells. First, detection of singlet oxygen (1 O2 ) luminescence proved that curcumin generates 1 O2 upon blue light irradiation in organic solvent and in liposomes. Then, HPLC-EC(Hg) measurements revealed that liposomal and cellular cholesterol is oxidized by 1 O2 photogenerated by curcumin. Enrichment of liposome membranes with curcumin significantly increased the oxygen photo-consumption rate compared to the control liposomes as determined by EPR oximetry. Cytotoxicity measurements, mitochondrial membrane potential analyses and protein hydroperoxides detection confirmed strong phototoxic effects of curcumin in irradiated HaCaT cells. These data show that since curcumin is advertised as a valuable dietary supplement, or a component of cosmetics for topical use, caution should be recommended especially when skin is exposed to light.

Detection of singlet oxygen luminescence for experimental corneal rose bengal photodynamic antimicrobial therapy.

Rose bengal photodynamic antimicrobial therapy (RB-PDAT) treats corneal infection by activating rose bengal (RB) with green light to produce singlet oxygen (1O2). Singlet oxygen dosimetry can help optimize treatment parameters. We present a 1O2 dosimeter for detection of 1O2 generated during experimental RB-PDAT. The system uses a 520 nm laser and an InGaAs photoreceiver with bandpass filters to detect 1O2 luminescence during irradiation. The system was validated in RB solutions and ex vivo in human donor eyes. The results demonstrate the feasibility of 1O2 dosimetry in an experimental model of RB-PDAT in the cornea.

Photooxidation Reactions of Cyclometalated Palladium(II) and Platinum(II) Complexes.

New C,N,S-cyclometalated palladium(II) and platinum(II) complexes have been synthesized and their structural, electrochemical, and photochemical properties examined. The blue color of these complexes in solution changed to yellow under visible-light irradiation. By measurement of the absorption spectra for quantifying changes in color, isosbestic points for each complex clearly indicated the presence of only two species responsible for the change of color. X-ray analysis revealed that the visible-light-induced yellow species were S-oxygenated sulfinato complexes. Photosensitized generation of singlet oxygen (1O2) was confirmed by the direct detection of singlet oxygen luminescence at 1275 nm. The present cyclometalated palladium(II) and platinum(II) complexes are efficient photosensitizers of singlet oxygen, which rapidly reacts with coordinating sulfur atoms.

Enhanced Optics for Time-Resolved Singlet Oxygen Luminescence Detection

Singlet oxygen luminescence dosimetry (SOLD) is a highly promising direct monitoring method for photodynamic therapy (PDT) in the treatment of cancer. Early SOLD systems have been hampered by inefficient excitation, poor optical collection and immature infrared single photon detection technology. We report carefully engineered improvements addressing all of these deficiencies. We use a supercontinuum source with a tunable filter to precisely target the peak absorption wavelength of the chosen photosensitizer; we have designed a compact and versatile optical package for precise alignment; we have successfully employed state-of-the-art superconducting photon counting technologies. Through these improvements, we can achieve histogram acquisition from a photosensitizer in solution test sample. This setup opens the pathway to physiological SOLD studies for PDT dosimetry.

Singlet oxygen luminescence kinetics under PDI relevant conditions of pathogenic dermatophytes and molds

A treatment of onychomycosis using the photodynamic effect would be a favorable alternative to currently used antimycotic drugs. This study should be considered as a first step towards development and control of an efficient photodynamic inactivation of onychomycosis causative pathogens. Here, we evaluate the usage of time-resolved 2D singlet oxygen luminescence detection in combination with 2D fluorescence scanning as a tool to understand the behavior of the photosensitizer when applied to fungi on Petri dishes. To investigate the interaction of photosensitizer with fungi in various concentrations and in different stages of live, a photodynamic inactivation was avoided by keeping the samples in darkness. Scans of singlet oxygen luminescence and photosensitizer fluorescence were performed over a period of 24days. Two different photosensitizer, a cationic porphyrin and cationic corrole and two fungi strains, the dermatophyte Trichophyton rubrum and the mold Scopulariopsis brevicaulis, were investigated in this study. The two-dimensional correlation of photosensitizer fluorescence and singlet oxygen luminescence revealed differences in the diffusion of both photosensitizer. Even though the singlet oxygen luminescence was quenched with increasing growth of fungi, it was found that the kinetics of singlet oxygen luminescence could be detected on Petri dishes for both photosensitizers and both fungi strains for up to seven days.

Photodynamic inactivation of Escherichia coli – Correlation of singlet oxygen kinetics and phototoxicity

Photodynamic inactivation (PDI) of bacteria may play a major role in facing the challenge of the ever expanding antibiotic resistances. Here we report about the direct correlation of singlet oxygen luminescence kinetics and phototoxicity in E. coli cell suspension under PDI using the widely applied cationic photosensitizer TMPyP. Through direct access to the microenvironment, the time resolved investigation of singlet oxygen luminescence plays a key role in understanding the photosensitization mechanism and inactivation pathway. Using the homemade set-up for highly sensitive time resolved singlet oxygen luminescence detection, we show that the cationic TMPyP is localized predominantly outside the bacterial cells but in their immediate vicinity prior to photodynamic inactivation. Throughout following light exposure, a clear change in singlet oxygen kinetics indicates a redistribution of photosensitizer molecules to at least one additional microenvironment. We found the signal kinetics mirrored in cell viability measurements of equally treated samples from same overnight cultures conducted in parallel: A significant drop in cell viability of the illuminated samples and stationary viability of dark controls. Thus, for the system investigated in this work – a Gram-negative model bacteria and a well-known PS for its PDI – singlet oxygen kinetics correlates with phototoxicity. This finding suggests that it is well possible to evaluate PDI efficiency directly via time resolved singlet oxygen detection.

A miniaturized 4 K platform for superconducting infrared photon counting detectors

We report on a miniaturized platform for superconducting infrared photon counting detectors. We have implemented a fibre-coupled superconducting nanowire single photon detector in a Stirling/Joule–Thomson platform with a base temperature of 4.2 K. We have verified a cooling power of 4 mW at 4.7 K. We report 20% system detection efficiency at 1310 nm wavelength at a dark count rate of 1 kHz. We have carried out compelling application demonstrations in single photon depth metrology and singlet oxygen luminescence detection.

A compact fiber-optic probe-based singlet oxygen luminescence detection system.

This paper presents a novel compact fiberoptic based singlet oxygen near‐infrared luminescence probe coupled to an InGaAs/InP single photon avalanche diode (SPAD) detector. Patterned time gating of the single‐photon detector is used to limit unwanted dark counts and eliminate the strong photosensitizer luminescence background. Singlet oxygen luminescence detection at 1270 nm is confirmed through spectral filtering and lifetime fitting for Rose Bengal in water, and Photofrin in methanol as model photosensitizers. The overall performance, measured by the signal‐to‐noise ratio, improves by a factor of 50 over a previous system that used a fiberoptic‐coupled superconducting nanowire single‐photon detector. The effect of adding light scattering to the photosensitizer is also examined as a first step towards applications in tissue in vivo. figureWiley-VCH Verlag & Co.KGaA

Prospects of in vivo singlet oxygen luminescence monitoring: Kinetics at different locations on living mice

BackgroundSinglet oxygen observation is considered a valuable tool to assess and optimize PDT treatment. In complex systems, such as tumors in vivo, only the direct, time-resolved singlet oxygen luminescence detection can give reliable information about generation and interaction of singlet oxygen. Up to now, evaluation of kinetics was not possible due to insufficient signal-to-noise ratio. Here we present high signal-to-noise ratio singlet oxygen luminescence kinetics obtained in mouse tumor model under PDT relevant conditions. MethodsA highly optimized system based on a custom made laser diode excitation source and a high aperture multi-furcated fiber, utilizing a photomultiplier tube with a multi photon counting device was used. ResultsLuminescence kinetics with unsurpassed signal-to-noise ratio were gained from tumor bearing nude mice in vivo upon topic application, subcutaneous injection as well as intravenous injection of different photosensitizers (chlorin e6 and dendrimer formulations of chlorin e6). Singlet oxygen kinetics in appropriate model systems are discussed to facilitate the interpretation of complex kinetics obtained from in vivo tumor tissue. ConclusionsThis is the first study addressing the complexity of singlet oxygen luminescence kinetics in tumor tissue. At present, further investigations are needed to fully explain the processes involved. Nevertheless, the high signal-to-noise ratio proves the applicability of direct time-resolved singlet oxygen luminescence detection as a prospective tool for monitoring photodynamic therapy.

Time-resolved singlet-oxygen luminescence detection with an efficient and practical semiconductor single-photon detector.

In clinical applications, such as PhotoDynamic Therapy, direct singlet-oxygen detection through its luminescence in the near-infrared range (1270 nm) has been a challenging task due to its low emission probability and the lack of suitable single-photon detectors. Here, we propose a practical setup based on a negative-feedback avalanche diode detector that is a viable alternative to the current state-of-the art for different clinical scenarios, especially where geometric collection efficiency is limited (e.g. fiber-based systems, confocal microscopy, scanning systems etc.). The proposed setup is characterized with Rose Bengal as a standard photosensitizer and it is used to measure the singlet-oxygen quantum yield of a new set of photosensitizers for site-selective photodynamic therapy.

Singlet oxygen luminescence kinetics in a heterogeneous environment - identification of the photosensitizer localization in small unilamellar vesicles.

In vivo measurement of singlet oxygen luminescence kinetics is affected by the heterogeneity of biological samples. Even though singlet oxygen luminescence detection is technically getting easier, the analysis of signals from biological samples is still far from quantitative real time surveillance as it is aspired by the community. In this paper small unilamellar vesicles (SUVs) are used for modelling the general behaviour of heterogeneous samples. The geometry of the SUVs can be determined independently using dynamic light scattering. Therefore an accurate theoretical description of the generation, deactivation and diffusion of the singlet oxygen is possible. The theoretical model developed here perfectly fits the experimental results. Thus the location of the singlet oxygen generating a photosensitizer molecule can be exactly determined from the kinetics of the singlet oxygen luminescence. The application of the used theoretical approach thus allows for accurate quantitative measurements in SUVs.

Correlation of in vivo tumor response and singlet oxygen luminescence detection in mTHPC-mediated photodynamic therapy

Excited-state singlet oxygen (1 O 2), generated during photodynamic therapy (PDT), is believed to be the primary cytotoxic agent with a number of clinically approved photosensitizers. Its relative concentration in cells or tissues can be measured directly through its near-infrared (NIR) luminescence emission, which has correlated well with in vitro cell and in vivo normal skin treatment responses. Here, its correlation with the response of tumor tissue in vivo is examined, using the photosensitizer meso-tetrahydroxyphenylchlorin (mTHPC) in an animal model comprising luciferase- and green fluorescent protein (GFP)-transduced gliosarcoma grown in a dorsal window chamber. The change in the bioluminescence signal, imaged pretreatment and at 2, 5 and 9 d post treatment, was used as a quantitative measure of the tumor response, which was classified in individual tumors as "non", "moderate" and "strong" in order to reduce the variance in the data. Plotting the bioluminescence-based response vs the 1 O 2 counts demonstrated clear correlation, indicating that 1 O 2 luminescence provides a valid dosimetric technique for PDT in tumor tissue.

Luminescence investigation of photosensitizer distribution in skin: correlation of singlet oxygen kinetics with the microarchitecture of the epidermis

This is the first study showing that singlet oxygen kinetics of topically applied photosensitizers coincides with the microarchitecture of skin, e.g., fissures and hair follicles. The kinetics indicate a chemical interaction of singlet oxygen with the skin, which allows differentiating between residual crème, e.g., in the follicular orifice, and photosensitizer penetrated into the skin. We show the feasibility of an easy-to-use fiber optic application providing the opportunity for in situ investigation, as well as a setup with focused optics for high-resolution two-dimensional scanning of singlet oxygen luminescence kinetics in skin samples. The results show that time-resolved singlet oxygen luminescence detection in tissue is a desirable tool for medical therapy, diagnostics, and evaluation of singlet oxygen interaction with biological environments.

Highly sensitive time resolved singlet oxygen luminescence detection using LEDs as the excitation source

For the first time singlet oxygen luminescence kinetics in living cells were detected at high precision using LED light for excitation. As LED technology evolves, the light intensity emitted by standard LEDs allows photosensitized singlet oxygen luminescence detection in solution and cell suspensions. We present measurements superior to those of most actual laser powered setups regarding precision of singlet oxygen kinetics in solutions and cell suspensions. Data presented here show that LED based setups allow the determination of the photosensitizer triplet and singlet oxygen decay times in vitro with an accuracy of 0.1 μs. This enables monitoring of the photosensitizer efficiency and interaction with the cellular components using illumination doses small enough not to cause cell death.

Effect of oxygen concentration on singlet oxygen luminescence detection

Singlet oxygen (1O2) is a major phototoxic component in photodynamic therapy (PDT) and its generation is dependent on the availability of tissue oxygen. To examine the effect of oxygen concentration on 1O2 detection, two hydrophilic photosensitizer (PS), rose bengal (RB) and meso-metra (N-methyl-4-pyridyl) porphine tetra tosylate (TMPyP) were used as model PS. Irradiation was carried out using 523nm under hypoxic (2%, 13%), normoxic (21%) and hyperoxic (65%) conditions. The spectral and spatial resolved 1O2 luminescence was measured by near-infrared (NIR) photomultiplier tube (PMT) and camera, respectively. Upon the irradiation, the emission signal mainly consisted of background scattering light, PS fluorescence and phosphorescence, and 1O2 luminescence. The PS phosphorescence was evidently dependent on the oxygen concentration and PS type, which resulted in the change of emission profile of 1O2 luminescence. This change was further demonstrated on 1O2 luminescence image. The present study suggests that the low oxygen concentration could affect 1O2 luminescence detection.

Singlet oxygen luminescence detection with a fiber-coupled superconducting nanowire single-photon detector

Direct monitoring of singlet oxygen (¹O₂) luminescence is a particularly challenging infrared photodetection problem. ¹O₂, an excited state of the oxygen molecule, is a crucial intermediate in many biological processes. We employ a low noise superconducting nanowire single-photon detector to record ¹O₂ luminescence at 1270 nm wavelength from a model photosensitizer (Rose Bengal) in solution. Narrow band spectral filtering and chemical quenching is used to verify the ¹O₂ signal, and lifetime evolution with the addition of protein is studied. Furthermore, we demonstrate the detection of ¹O₂ luminescence through a single optical fiber, a marked advance for dose monitoring in clinical treatments such as photodynamic therapy.

Time-resolved singlet oxygen luminescence detection under photodynamic therapy relevant conditions: comparison ofex vivoapplication of two photosensitizer formulations

Singlet oxygen plays a crucial role in photo-dermatology and photodynamic therapy (PDT) of cancer. Its direct observation by measuring the phosphorescence at 1270 nm, however, is still challenging due to the very low emission probability. It is especially challenging for the time-resolved detection of singlet oxygen kinetics in vivo which is of special interest for biomedical applications. Photosensitized generation of singlet oxygen, in pig ear skin as model for human skin, is investigated here. Two photosensitizers (PS) were topically applied to the pig ear skin and examined in a comparative study, which include the amphiphilic pheophorbide-a and the highly hydrophobic perfluoroalkylated zinc phthalocyanine (F64PcZn). Fluorescence microscopy indicates the exclusive accumulation of pheophorbide-a in the stratum corneum, while F64PcZn can also accumulate in deeper layers of the epidermis of the pig ear skin. The kinetics obtained with phosphorescence measurements show the singlet oxygen interaction with the PS microenvironment. Different generation sites of singlet oxygen correlate with the luminescence kinetics. The results show that singlet oxygen luminescence detection can be used as a diagnostic tool, not only for research, but also during treatment. The detection methodology is suitable for the monitoring of chemical quenchers’ oxidation as well as saturation at singlet oxygen concentration levels relevant to PDT treatment protocols.

Combined effects of singlet oxygen and hydroxyl radical in photodynamic therapy with photostable bacteriochlorins: Evidence from intracellular fluorescence and increased photodynamic efficacy in vitro

Sulfonamides of halogenated bacteriochlorins bearing Cl or F substituents in the ortho positions of the phenyl rings have adequate properties for photodynamic therapy, including strong absorption in the near-infrared (λmax≈750nm, ε≈105M−1cm−1), controlled photodecomposition, large cellular uptake, intracellular localization in the endoplasmic reticulum, low cytotoxicity, and high phototoxicity against A549 and S91 cells. The roles of type I and type II photochemical processes are assessed by singlet oxygen luminescence and intracellular hydroxyl radical detection. Phototoxicity of halogenated sulfonamide bacteriochlorins does not correlate with singlet oxygen quantum yields and must be mediated both by electron transfer (superoxide ion, hydroxyl radicals) and by energy transfer (singlet oxygen). The photodynamic efficacy is enhanced when cellular death is induced by both singlet oxygen and hydroxyl radicals.

New aspects in singlet oxygen luminescence detection in biological relevant systems

New aspects in singlet oxygen luminescence detection in biological relevant systems

Optimal discrimination threshold for the detection of singlet oxygen luminescence

Direct detection of singlet oxygen (1O2) luminescence around 1270 nm is the golden standard of 1O2 identification. In this study, the effect of the discrimination threshold on the detection of 1O2 luminescence that generated from the photoirradiation of Rose Bengal (RB) was evaluated by using a self-developed photon-counting detection system. The obtained results show that the discrimination threshold for photon counting has a significant impact on the intensity and shape of the measured 1O2 luminescence, which resulted in the variation of 1O2 lifetimes. The optimal discrimination threshold is determined to be about −0.0412 V, and the corresponding 1O2 lifetime in air-saturated distilled water is 4.26±0.06 μs.