Singlet Oxygen Phosphorescence Research Articles

The (un)known issue with using rose bengal as a standard of singlet oxygen photoproduction.

Rose bengal (RB) is a widely used photosensitizer for determining quantum yields of singlet oxygen generation. While it is known to aggregate in polar environments at concentrations above 2 μM, the relationship between RB concentration and singlet oxygen photogeneration remains unclear. This study investigates the shift from monomeric to dimeric RB with increasing concentration and its impact on singlet oxygen generation in D2O-based solutions and DMPC liposomes. Absorbance maxima for RB were observed at 514 nm (dimer) and 549 nm (monomer), with ionic environments influencing aggregation rates. Singlet oxygen phosphorescence showed non-linear dependency above 2 μM, indicating the effects of aggregation. Results suggest that RB concentrations should be kept at 1 μM or lower in photochemical studies to avoid aggregation-related discrepancies in singlet oxygen yield determination. These findings highlight the importance of considering RB aggregation in photochemical research and medical applications.

Monomeric and Dimeric Boron (III) Subphthalocyanines Functionalized with 4-Hydroxy-Benzoic Acid as Potential Photosensitizers and Photocatalysts in Sulfoxidation.

Axial modification of boron (III) subphthalocyanine bromides with 4-hydroxy-benzoic acid successfully led to the formation of the macrocycles with anchored 4-carboxyphenoxy group [RsPcPHBA] (R=tBu, H) in the axial position and to a new dimer [sPcPHBAsPc] as minor product. Tri-tert-butyl and unsubstituted subphthalocyanines bearing benzoate ([tBusPcBA], [sPcBA]), phenoxy-group ([tBusPcOPh], [sPcOPh])) in the axial position, have been also investigated as well as control sPcs. All compounds were characterized by NMR, IR, UV-Vis and mass spectrometry. The electrochemical properties were studied using cyclic voltammetry (CV) and square wave voltammetry (SWV). Singlet oxygen generation was systematically measured for all synthesized [RsPcX] by kinetic method of chemical trap decomposition (DPBF) and by determination of phosphorescence of singlet oxygen (at 1270 nm). Axially modified subphthalocyanines exhibit high quantum yields of singlet oxygen (1O2) generation (0.47-0.62). The observed exceptional photostability in oxygen-saturated ethanol or toluene solutions and high 1O2 quantum yields allows to use [tBusPcPHBA] as photocatalysts of selective oxidative transformations of organic sulfides to sulfoxides. Loading the catalyst to 9.7 ⋅ 10-2 mol % made it possible to achieve complete conversion of the substrate (TON up to 1700).

PH modulates efficiency of singlet oxygen production by flavin cofactors.

Flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) are frequently used interchangeably in the catalysis of various reactions as part of flavoenzymes because they have the same functional component, the isoalloxazine ring. However, they differ significantly in their conformational properties. The inclusion of two planar rings in the structure of FAD greatly increases the range of possible conformations compared to FMN. An exemplary instance of this is the remarkable disparity in singlet oxygen efficiency production, Φ Δ, between FMN and FAD. Under neutral pH conditions, FAD has low photosensitizing activity with Φ Δ ∼ 0.07 while FMN demonstrates high photosensitizing activity with Φ Δ ∼ 0.6. Both adenine rings and isoalloxazine in FAD contain pH titratable groups. Through comprehensive analysis of the kinetics of the transient absorbance of the triplet state and the phosphorescence of singlet oxygen from FAD and FMN, we determined the correlation between different conformational states and the pH-dependent generation of singlet oxygen. Based on our findings, we may deduce that within the pH range of pH 2 to pH 13, only two out of the five potential structural states of FAD are capable of efficiently producing singlet oxygen. There are two open conformations: (i) an acidic FAD conformation with a protonated adenine ring, which is around 10 times more populated than the neutral open FAD conformation, and (ii) a neutral pH FAD conformation, which is significantly less populated. The FAD conformer with a protonated adenine ring at acidic pH generates singlet oxygen with approximately 50% efficiency compared to the constantly open FMN at neutral pH. This may have implications for singlet oxygen synthesis in acidic environments.

Fertility after photodynamic inactivation of bacteria in extended boar semen.

Antimicrobial resistance is an increasing challenge in semen preservation of breeding animals, especially in the porcine species. Bacteria are a natural component of semen, and their growth should be inhibited to protect sperm fertilizing capacity and the female's health. In pig breeding, where semen is routinely stored at 17°C in the liquid state, alternatives to conventional antibiotics are urgently needed. Photodynamic inactivation (PDI) of bacteria is a well-established tool in medicine and the food industry but this technology has not been widely adopted in semen preservation. The specific challenge in this setting is to selectively inactivate bacteria while maintaining sperm integrity and functionality. The aim of this study was to test the principle of PDI in liquid stored boar semen using the photosensitizer 5,10,15,20-tetrakis(N-methyl-4-pyridyl)-21H,23H-porphine (TMPyP) and a white light LED-setup. In the first step, photophysical experiments comprising singlet oxygen phosphorescence kinetics of TMPyP and determination of the photosensitizer triplet time revealed a sufficiently high production of reactive singlet oxygen in the Androstar Premium semen extender, whereas seminal plasma acted as strong quencher. In vitro experiments with extended boar semen showed that the established PDI protocol preserves sperm motility, membrane integrity, DNA integrity, and mitochondrial activity while efficiently reducing the bacteria below the detection limit. A proof-of-concept insemination study confirmed the in vivo fertility of semen after photodynamic treatment. In conclusion, using the PDI approach, an innovative tool was established that efficiently controls bacteria growth in extended boar and maintains sperm fertility. This could be a promising contribution to the One Health concept with the potential to reduce antimicrobial resistance in animal husbandry.

Plasmon-enhanced sensitization of singlet oxygen on silver island films

The influence of the plasmonic effect of silver island films on the absorption, fluorescence and long-term luminescence of rose bengal in polyvinyl butyral films has been studied. Under the plasmon effect, the optical density of the dye increased by 3.3 times, the fluorescence intensity by 8.5 times, and the long-lived fluorescence and phosphorescence by 7.08 times and 10.21, respectively. It is shown that in the plasmon field of metal nanoparticles, both an increase in the excitation rate and an increase in the rates of radiative decay of excited singlet and triplet states occur. Phosphorescence of singlet oxygen with a lifetime of 86.46 μs was observed when excited in the absorption band of rose bengal. Based on the calculation of the quenching constants of rose bengal phosphorescence by molecular oxygen molecules, it is shown that the plasmonic effect enhances the energy transfer from dye triplets to oxygen molecules in collision complexes [ТPS…3  g –] as a result of heteroannihilation. Subsequently, the triplet pair decomposes to form a singlet oxygen molecule and a photosensitizer molecule in the ground state. The plasmonic effect of silver nanoparticles leads to an increase in the generation of singlet oxygen by 4.8 times.

Photophysics of singlet oxygen generation in chitosan films with viburnum fruit extract (Viburnum opulus L.) under the influence of plasmons on a modified titanium surface

Subject of study. This study investigates the luminescence spectral and kinetic features of singlet oxygen generation in chitosan microfilms (10 µm thick) embedded with an extract of viburnum fruit (Viburnum opulus L.). These films were deposited on both glass and gold film (80 µm thick), with the gold film electrochemically deposited on a rough, modified titanium surface. Aim of study. The aim was to determine the primary photophysical patterns of singlet oxygen dynamics in chitosan films containing viburnum fruit extract, following pulsed monochromatic photoexcitation by a structured titanium surface coated with electrochemically deposited gold. Method. The films were investigated in the visible and near-infrared regions using fluorescence techniques. The luminescence lifetime of Viburnum opulus L. flavonoids in the nanosecond range was measured using multichannel photon counting with a picosecond diode. The lifetime of the flavonoid triplet states and singlet oxygen phosphorescence in the studied extract in the microsecond range under excitation by a pulsed xenon lamp were also recorded. Main results. Long-lived delayed annihilation fluorescence of Viburnum opulus L. flavonoid molecules in the 590 nm spectral region, along with luminescence at 1272 nm attributable to singlet molecular oxygen, were observed. A comprehensive kinetic analysis was performed, considering the rate constants of photophysical processes involving singlet oxygen in films with viburnum fruit extract on rough gold surfaces under conditions of sample saturation with molecular oxygen. Stable generation of singlet oxygen was achieved in the chitosan microfilms containing viburnum fruit extract. Practical significance. The findings of this study can be applied to biological methods for cancer treatment.

Photoreactivity of polycyclic aromatic hydrocarbons (PAHs) and their mechanisms of phototoxicity against human immortalized keratinocytes (HaCaT)

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous organic compounds in the environment. They are produced by many anthropogenic sources of different origins and are known for their toxicity, carcinogenicity, and mutagenicity. Sixteen PAHs have been identified as Priority Pollutants by the US EPA, which are often associated with particulate matter, facilitating their dispersion through air and water. When human skin is exposed to PAHs, it might occur simultaneously with solar radiation, potentially leading to phototoxic effects. Phototoxic mechanisms involve the generation of singlet oxygen and reactive oxygen species, DNA damage under specific light wavelengths, and the formation of charge transfer complexes. Despite predictions of phototoxic properties for some PAHs, there remains a paucity of experimental data. This study examined the photoreactive and phototoxic properties of the 16 PAHs enlisted in the Priority Pollutants list. Examined PAHs efficiently photogenerated singlet oxygen and superoxide anion in simple solutions. Furthermore, singlet oxygen phosphorescence was detected in PAH-loaded HaCaT cells. Phototoxicity against human keratinocytes was evaluated using various assays. At 5 nM concentration, examined PAHs significantly reduced viability and mitochondrial membrane potential of HaCaT cells following the exposure to solar simulated light. Analyzed compounds induced a substantial peroxidation of cellular proteins after light treatment. The results revealed that a majority of the examined PAHs exhibited substantial reactive oxygen species photoproduction under UVA and violet-blue light, with their phototoxicity corresponding to their photoreactive properties. These findings improve our comprehension of the interactions between PAHs and human skin cells under environmental conditions, particularly when exposed to solar radiation.

Measuring the total photon economy of molecular species through fluorescent optical cycling.

The total photon economy of a chromophore molecular species represents a study of how absorbed photons partition among various electronic states and ultimately dissipate their excited energy into the environment. A complete accounting of these rates and pathways would allow one to optimize chromophores and their environments for applications. We describe a technique, fluorescent optical cycling (FOC), which allows for simultaneous observation of prompt and delayed emission during and after multiple pulsed excitation, ultimately granting access to multi-state photophysical rates. We exercise control over the excitation pulse train, which allows us to "optically shelve" long-lived intermediate states without the use of diode or flashlamp excitation. By recording all photon arrival times in the visible and shortwave infrared, we can simultaneously resolve fluorescence, phosphorescence, and singlet oxygen sensitization in a single experiment. We use FOC to examine the photophysics of dual emitting bis(di-R-phosphino)alkanethiophene-pyridine-platinum ([Pt(thpy)(dppm)]+) under different solvation conditions, revealing changes in intersystem crossing and phosphorescent rates induced by the external heavy atom effect. Coupling FOC with decay associated Fourier spectroscopy (DAFS), we demonstrate simultaneous correlated spectral and lifetime data in this dual emitting complex. Finally, FOC combined with superconducting nanowire single photon detectors (SNSPDs) allows us to observe the shortwave infrared region (SWIR) phosphorescence of singlet oxygen sensitized by Rose Bengal. Overall, FOC provides a powerful tool to simultaneously study multiple photophysics across timescales, even in weakly populated electronic states.

Free base porphyrin-cyanine dye conjugate: synthesis and optical properties.

The covalent combination of a cyanine dye (IR-783) with a tetraphenyl porphyrin unit through an ether linkage results in a photoactive system capable of producing singlet oxygen. The synthesis, characterization and photophysical properties of the resulting novel free base porphyrin-cyanine conjugate named TPPO-IR-783 (TOI) is reported. Excited state properties were studied in various solvents with differing polarity. The fluorescence is strongly solvent dependent, however this is not the case for singlet oxygen phosphorescence, which is only observed in tetrahydrofuran (THF), when comparing 8 different polar, non-polar and medium-polarity solvents. This novel type of porphyrin-cyanine photosensitizer has the ability to produce singlet oxygen and absorbs light at NIR wavelengths.

Activation of molecular oxygen by triplet states of S,N-doped carbon dots

S,N–doped carbon dots were synthesized and they were considered as activators of molecular oxygen, as evidenced by the observed phosphorescence of singlet oxygen upon excitation of solutions in the absorption band of carbon dots. The process of singlet–triplet annihilation develops when O2(3Σg-) molecules were added to the solution, the efficiency of which depends on the ratio of concentrations of triplet carbon dots and O2(1Δg) singlet molecules. In the presence of plasmonic nanoparticles of Au the phosphorescence of singlet oxygen was increased.

Singlet oxygen generation under optical excitation of polytetrafluoroethylene

Singlet oxygen together with radical reactions plays an important role in biochemistry of living organisms, and is one of the main active substances in photodynamic treatment of organs and body fluids. Along with traditional singlet oxygen photosensitizers based on biocompatible dyes, the singlet oxygen photogeneration ability of matrix materials such as organic solvents and metal oxide surfaces has recently been investigated. Despite a good compatibility with bio substances, polytetrafluoroethylene (also known under the trademark as Teflon®) remains unaddressed in these studies. Here we present our photoluminescence studies of singlet oxygen generation in polytetrafluoroethylene samples under ultraviolet and visible irradiation. We found a good singlet oxygen photogeneration with an efficiency no less and possibly exceeding it in carbon tetrachloride, a known singlet‑oxygen-retentive medium. Based on the results obtained, it is assumed that the phosphorescence of singlet oxygen is observed from the volume of polytetrafluoroethylene with direct optical excitation of oxygen adsorbed or dissolved in the structure of polytetrafluoroethylene by analogy with direct optical excitation of oxygen in the volume of various solvents. Cooling a PTFE sample down to −20 °C allows to enhance the singlet oxygen concentration in the polymer indicating the enthalpy of oxygen sorption of ΔHsorp = −1.51 kcal/mol. Our results can heighten medical interest to fluoropolymers and, in particular, to polytetrafluoroethylene making it a promising singlet oxygen generator for photodynamic uses.

Photophysical properties of Radachlorin photosensitizer in solutions of different pH, viscosity and polarity

We present a thorough experimental investigation of fluorescence properties of Radachlorin photosensitizer in solutions of different acidity, viscosity and polarity. Experiments were performed using time-resolved fluorescence lifetime imaging and time-resolved analysis of polarized fluorescence. Variations of solution acidity resulted in considerable changes of Radachlorin fluorescence quantum yield and lifetime in the pH range from 4 to 7, but did not affect the rotational diffusion time, and almost did not change the quantum yield and characteristic times of singlet oxygen phosphorescence. Variations of solution polarity and viscosity were achieved by changing ethanol or methanol fraction in aqueous solution. The decrease of solution polarity resulted in nonlinear rise of Radachlorin fluorescence quantum yield and lifetime up to alcohol concentration of 50%–65%, as well as in considerable rise of singlet oxygen quantum yield and significant changes in characteristic times of its phosphorescence. Variations of solution viscosity resulted in changes of rotational diffusion time of Radachlorin molecules, which appeared to be in perfect correlation with methanol solution viscosity. Good correspondence with ethanol solution viscosity was observed only up to 50% alcohol fraction. Deviations of rotational diffusion time of Radachlorin molecules from direct proportionality with solution viscosity at higher ethanol concentrations were suggested to be due to different solvation conditions. The data obtained can give important reference points for analysis of microenvironment of Radachlorin molecules, their intracellular localization and performance in singlet oxygen generation.

Singlet oxygen lifetime changes in dying glioblastoma cells.

Time-resolved phosphorescence detection was employed to determine the lifetime of singlet oxygen in live cells. Using hypericin as a photosensitizer, singlet oxygen was generated in U87MG glioblastoma cells. The phosphorescence of singlet oxygen was detected in aqueous cell suspensions following pulsed laser excitation. Our goal was to eliminate or reduce the problems associated with lifetime measurements in water-based cell suspensions. The apparatus enabled simultaneous singlet oxygen phosphorescence and transient absorption measurements, reducing uncertainty in lifetime estimation. The changes in singlet oxygen lifetime were observed during early and late apoptosis induced by photodynamic action. Our findings show that the effective lifetime of singlet oxygen in the intracellular space of the studied glioblastoma cells is 0.4 μs and increases to 1.5 μs as apoptosis progresses. Another group of hypericin, presumably located in the membrane blebs and the plasma membrane of apoptotic cells, generates singlet oxygen with a lifetime of 1.9 μs.

Can l-ascorbic acid and trans-resveratrol protect HaCaT cells from fine particulate matter toxicity?

Continuous exposure of human skin to air pollution can result in a range of undesirable skin conditions. In our recent study, UV and visible light were found to increase cytotoxicity of fine particulate matter (PM2.5 ) against human keratinocytes. Since it is impossible to avoid exposure of human skin to PM2.5 , effective strategies are needed to reduce their damaging effects. l-ascorbic acid and resveratrol were tested as potential topical agents against pollution-related skin impairment. Although these agents were previously found to ameliorate PM-dependent damage, the effect of light and seasonal variation of particles were not previously studied. EPR spin-trapping, DPPH assay, and singlet oxygen phosphorescence were used to determine the scavenging activities of the antioxidants. MTT, JC-10 and iodometric assays were used to analyze the effect on PM2.5 -induced cytotoxicity, mitochondrial damage and oxidation of lipids. Live-cell imaging was employed to examine wound-healing properties of cells. Light-induced, PM2.5 -mediated oxidative damage was examined by immunofluorescent staining. Both antioxidants effectively scavenged free radicals and singlet oxygen produced by PM2.5 , reduced cell death and prevented oxidative damage to HaCaT cells. l-ascorbic acid and resveratrol, especially when applied in combination, can protect HaCaT cells against the dark and light induced toxicity of PM2.5 .

Protective action of graphene oxide against singlet oxygen generation by cosmetic dye methylene blue

Nanohybrid methylene blue/graphene oxide (MB/GO) was prepared via non-covalent functionalization with the focus on spectroscopic characterization of MB molecules adsorbed on GO. Fluorescence was not detected for the MB/GO hybrid which indicates that other competitive fast singlet excited state deactivation process must take place. It was demonstrated by measuring singlet oxygen (1O2) phosphorescence at 1270 nm that MB functionalized with GO does not generate 1O2. Nanosecond transient absorption spectroscopy experiments pointed to a lack of population of triplet state by MB/GO hybrid material. Femtosecond pump–probe experiments revealed much faster dynamics of the singlet excited state of MB/GO in comparison to free MB, which was attributed to the photoinduced electron transfer from MB to GO. Suppressing of the singlet oxygen quantum efficiency by MB upon functionalization to GO might be useful for designing graphene-based dye additives to color cosmetics with a reduced risk of the 1O2 generation on the skin.

Photoreactivity and phototoxicity of experimentally photodegraded hair melanosomes from individuals of different skin phototypes.

Even though melanin is commonly viewed as natural photoprotectant, the pigment demonstrates residual photoreactivity, which under certain conditions could contribute to UVA-dependent melanomagenesis. Skin melanin is constantly exposed to external stressors, including solar radiation, which could induce photodegradation of the pigment. Although photodegradation of melanin pigments was studied in synthetic models and RPE melanosomes, photochemical and photobiological effects of experimental photodegradation of human skin melanin of different chemical composition remain unknown. In this work, melanosomes isolated from hair of individuals of different skin phototypes (I-III, V) were exposed to high-intensity violet light and its impact on physical and chemical properties of the pigments were analyzed using electron paramagnetic resonance (EPR), spectrophotometry and dynamic light scattering (DLS). Photoreactivity of photodegraded melanins was examined by EPR oximetry, EPR spin-trapping and time-resolved singlet oxygen phosphorescence. Antioxidant potential of the pigments was measured using the EPR DPPH assay. Cellular effect of the exposure of melanosome-loaded HaCaT cells to UV-Vis light was determined by MTT assay, JC-10 assay, and iodometric assay. The data revealed that experimental photodegradation increased photoreactivity of natural melanins, while decreasing their antioxidant capacity. Photodegraded melanin was responsible for higher cell death, a decrease in mitochondrial membrane potential and elevated levels of lipid hydroperoxides.

Singlet Oxygen In Vivo: It Is All about Intensity-Part 2.

Recently, we reported induced anoxia as a limiting factor for photodynamic tumor therapy (PDT). This effect occurs in vivo if the amount of generated singlet oxygen that undergoes chemical reactions with cellular components exceeds the local oxygen supply. The amount of generated singlet oxygen depends mainly on photosensitizer (PS) accumulation, efficiency, and illumination intensity. With illumination intensities above a certain threshold, singlet oxygen is limited to the blood vessel and the nearest vicinity; lower intensities allow singlet oxygen generation also in tissue which is a few cell layers away from the vessels. While all experiments so far were limited to light intensities above this threshold, we report experimental results for intensities at both sides of the threshold for the first time, giving proof for the described model. Using time-resolved optical detection in NIR, we demonstrate characteristic, illumination intensity-dependent changes in signal kinetics of singlet oxygen and photosensitizer phosphorescence in vivo. The described analysis allows for better optimization and coordination of PDT drugs and treatment, as well as new diagnostic methods based on gated PS phosphorescence, for which we report a first in vivo feasibility test.

ζ-Carotene: Generation and Quenching of Singlet Oxygen, Comparison with Phytofluene.

It is known that C40 carotenoids with a short chain of conjugated double bonds (CDB) (5 and 7, respectively) are universal precursors in the biosynthesis of colored carotenoids in plant cells. Previously, using mainly stationary measurements of photosensitized phosphorescence of singlet oxygen (1O2), we discovered that phytofluene efficiently generates 1O2 in aerated solution and therefore, can serve as a source of the UV photodynamic stress in living cells [Ashikhmin et al., Biochemistry (Moscow), 2020, 85, 773]. In the present paper, by using novel pulsed light emitting diodes (LEDs), aerated hexafluorobenzene as a solvent and time-resolved measurements of 1O2 phosphorescence we confirmed that phytofluene efficiently photosensitizes 1O2 formation. The quantum yield of this process according to the novel experiments is about 0.4. An ability to generate 1O2 was also found in aerated solutions of ζ-carotene although the quantum yield of this process is 30-fold lower that in phytofluene solutions. Both carotenoids were found to quench 1O2 in the dark with the quenching rate constants equal to (3.6 ± 0.9)×107 and (2.1 ± 0.2)×108 M-1s-1, respectively. To our knowledge, the rate constant of 1O2 quenching by ζ-carotene has been reported in the present paper for the first time. It follows from the data obtained that the rate constants of 1O2 quenching by both carotenoids are much (by 2-3 orders of magnitude) smaller than the rate constant of the diffusion-limited biomolecular reactions. Hence, both carotenoids are weak protectors against 1O2 oxidative activity. It is more likely that they are potential promoters of photodynamic stress in living cells.

Photophysical properties of methylene blue in aqueous solution sprayed onto biological surfaces

The paper presents a set of experiments revealing photophysical processes associated with methylene blue assisted photodynamic inactivation of pathogens on different surfaces, both inorganic (glass) and organic (superficial fascial flaps of chicken and fish and mushroom cap peel). The experiments were performed in normoxic and hyperoxic conditions. The characteristic times of singlet oxygen generation and degradation on the surfaces were evaluated from the analysis of time-resolved signals of singlet oxygen phosphorescence. The photobleaching kinetics of methylene blue on the surfaces were found to be significantly different and dependant on solution oxygenation. The characteristic decay times varied from dozens of seconds for glass to dozens of minutes for fascial flaps and mushroom peel. The interpretation of the experimental results obtained was done on the basis of a set of master equations describing photosensitizer-assisted singlet oxygen generation and photosensitizer photobleaching. The results obtained on methylene blue photodynamics and conclusions made were compared with results of similar experiments performed earlier with Radachlorin photosensitizer.

Singlet Oxygen In Vivo: It Is All about Intensity.

The presented work addresses the influence of illumination intensity on the amount and locations of singlet oxygen generation in tumor tissue. We used time-resolved optical detection at the typical emission wavelength around 1270 nm and at 1200 nm where there is no singlet oxygen phosphorescence to determine the phosphorescence kinetics. The discussed data comprise in vivo measurements in tumor-laden HET-CAM and mice. The results show that illumination that is too intense is a major issue, affecting many PDT treatments and all singlet oxygen measurements in vivo so far. In such cases, photosensitization and oxygen consumption exceed oxygen supply, limiting singlet oxygen generation to the blood vessels and walls, while photosensitizers in the surrounding tissue will likely not participate. Being a limitation for the treatment, on one hand, on the other, this finding offers a new method for tumor diagnosis when using photosensitizers exploiting the EPR effect. In contrast to high-intensity PDT, some papers reported successful treatment with nanoparticular drugs using much lower illumination intensity. The question of whether, with such illumination, singlet oxygen is indeed generated in areas apart from vessels and walls, is addressed by numerical analysis. In addition, we discuss how to perform measurements at such low intensities.