Absolute fluorescence quantum yields are reported for the rhodamine 6G cation and the fluorescein dianion dyes in nine solvents. This information is combined with previously reported fluorescence lifetimes to deduce radiative and nonradiative decay rates. Along the alcohol series from methanol to octanol, rhodamine 6G displays an increasing radiative rate, in parallel with the square of the refractive index increase, and a slightly decreasing nonradiative rate. Fluorescein is different: the apparent radiative rate actually decreases, suggesting that the emissive species is perturbed in some fashion. For both dyes, fluorescence yields are enhanced in D₂O, rising to 0.98, in parallel with a corresponding increase in lifetimes. Protonated solvents invariably give shorter lifetimes and lower quantum yields, contrary to some previous speculation. From this work and an analysis of existing literature values, more precise values have been obtained for two previously proposed absolute quantum yield standards. The yield of fluorescein in 0.1 N NaOH(aq) is 0.925 ± 0.015, and for rhodamine 6G in ethanol, it is 0.950 ± 0.015. In both cases, the solutions are assumed to be in the limit of low concentration, excited close to their long-wave absorption band and at room temperature but may be either air-saturated or free of oxygen.

Fluorescence excitation efficiency is of great importance for photodynamic diagnosis. Because usually a difference in the interstitial pH between normal and tumor tissue occurs, it is necessary to assess the impact of pH on the fluorescence emission intensity of the photosensitizer meta-tetrahydroxyphenylchlorin (mTHPC) in this context. The results obtained by in vitro fluorescence measurements clearly indicate that pH values below 6 lead to a significant decrease in the fluorescence intensity. In the physiological range of pH 6.5–7.2, however, no pH dependence was found. Besides the decrease in the fluorescence intensity of mTHPC for pH < 6, changes in the spectral shape of the absorption were found. These changes can be utilized for “dual-wavelength ratio imaging,” using mTHPC as a pH-sensitive indicator with the excitation pair 405 nm/436 nm in the range of pH 3.5–6.

The absorption and fluorescence spectra of 3-aminobenzo-1,2,4-triazine di-N-oxide (tirapazamine) have been recorded and exhibit a dependence on solvent that correlates with the Dimroth E_T30 parameter. Time-dependent density functional theory calculations reveal that the transition of tirapazamine in the visible region is π → π* in nature. The fluorescence lifetime is 98 ± 2 ps in water. The fluorescence quantum yield is ∼0.002 in water. The fluorescence of tirapazamine is efficiently quenched by electron donors via an electron-transfer process. Linear Stern–Volmer fluorescence quenching plots are observed with sodium azide, potassium thiocyanate, guanosine monophosphate and tryptophan (Trp) methyl ester hydrochloride. Guanosine monophosphate, tyrosine (Tyr) methyl ester hydrochloride and Trp methyl ester hydrochloride appear to quench the fluorescence at a rate greater than diffusion control implying that these substrates complex with tirapazamine in its ground state. This complexation was detected by absorption spectroscopy.

Photochemical pathways leading to the phototoxicity of the aloe vera constituent aloe emodin were studied. The results indicate a photochemical mechanism involving singlet oxygen to be the most likely pathway responsible for the observed phototoxicity. Aloe emodin was found to efficiently generate singlet oxygen when irradiated with UV light (Φ_Δ = 0.56 in acetonitrile). The survival of human skin fibroblast cells in the presence of aloe emodin was found to decrease upon irradiation with UV light. A further decrease in cell survival was observed in D₂O compared with H₂O, suggesting the involvement of singlet oxygen as the primary pathway. Laser flash photolysis experiments were also carried out on aloe emodin alone and in the presence of various biological substrates. Aloe emodin proved to be relatively photostable (Φ = 1 × 10⁻⁴) and a poor photo-oxidant (E^*_red = 1.02 V). Only absorption bands caused by the triplet state of aloe emodin (λ_max = 480 nm) and the aloe emodin conjugate base (λ_max = 520 nm) were observed in the transient spectra.

The influence of the medium heterogeneity upon the bimolecular rate constants for the physical quenching, k_q, and chemical quenching, k_r, of singlet molecular oxygen O₂(¹Δ_g) by seven natural and three synthetic carotenoids (CAR) with different substituent patterns was studied in a reverse micelle system of sodium bis(2-ethylhexyl)sulfosuccinate, hexane and water. Because O₂(¹Δ_g) was generated inside the water pools of the reverse micelles by photosensitization of the water-soluble dye rose bengal and the CAR are mainly located in the external hexane pseudophase, the quenching process was interpreted using a pseudophase model for the partition of O₂(¹Δ_g) between the water pools and the organic pseudophases. The k_q values were mainly dependent on the extent of the double-bond conjugation of the CAR, as demonstrated by a good empirical relationship between log(k_q) and the energy E(S) of the longest wavelength transition π→π* of the CAR. In contrast, the k_r values were almost independent of the extent of the double-bond–conjugated system and about four orders of magnitude lower than k_q. However, in all cases, CAR photobleaching was observed with the formation of various oxidation products, depending on the photosensitization time. Chromatographic and spectroscopic product analysis for the reaction products of β-carotene with O₂(¹Δ_g) indicated the formation of the β-carotene-5,8-endoperoxide as the primary oxidation product.

The light-dependent reaction between N-acetyl-l-tryptophanamide (NATA) and chloroform has been examined using fluorescence, NMR and reverse phase chromatography. The emission of NATA in the presence of CHCl₃ decreases at 360 nm and increases at longer wavelengths (∼480 nm) upon illumination with 280 nm light. The action spectrum for the formation of the 480 nm emitting product(s) has the same shape as the excitation spectra of the indole fluorophore in NATA. The pH of the solution decreases as the reaction proceeds. The reaction rate depends on the intensity of the illumination and is of the first order with respect to both [NATA] and [CHCl₃]. NMR and reverse phase chromatography results demonstrate that multiple products are formed. The reaction products give new peaks between 8.9 and 10.5 ppm in the ¹H-NMR that are assigned to –CHO groups, which are added to the indole ring. Some of the products react with 2,4-dinitrophenylhydrazine and thus confirm this assignment. A scheme is proposed in which the excited indole gives off a solvated electron to initiate a series of steps that yield indole derivatives in which a –CHO group has replaced a –H in the indole ring. Similar reactions are observed when 5-hydroxytryptophan, 5-fluorotryptophan or N-methylindolacetate is used instead of tryptophan or when the chloroform is replaced with other trichlorinated compounds, such as trichloroacetic acid, trichloroethanol and trichloroethane, as well as the tribrominated compound, bromoform, and the monoiodinated compound, iodoactetate.

Trees influence the amount of solar UV radiation that reaches pedestrians. A three-dimensional model was developed to predict the ultraviolet-B (UV-B) irradiance fields in open-tree canopies where the spacing between trees is equal to or greater than the width of individual tree crowns. The model predicted the relative irradiance (fraction of above-canopy irradiance) under both sunlit and shaded conditions under clear skies with a mean bias error of less than 0.01 and a root mean square error of 0.07. Both model and measurements showed that the locations people typically perceive as shady, low-irradiance locations in the environment can actually have significant UV-B exposure (40–60% of that under direct sunlight). The relationship of tree cover in residential neighborhoods to erythemal UV-B exposure for children and adults was modeled for the 4 h around noon in June and July. Results showed that human exposures (on the horizontal) in cities located at 15 and 30° latitudes are nearly identical. For latitudes between 15 and 60°, ultraviolet protection factors (UPF) were less than 2 for less than 50% tree cover. A UPF of 10 was possible at all latitudes for tree cover of 90%.

The relationship between the structural and functional changes of the photosynthetic apparatus in the flower bud of Lilium longiflorum during chloroplast–chromoplast transition was examined. Compared with green petals, there was a five-fold increase of the carotenoid content in yellow petals, whereas the chlorophyll content decreased five-fold. Absorption and emission fluorescence spectra of chromoplasts indicated that newly synthesized carotenoids were not associated with photosystem II (PSII) photochemistry. The maximum quantum yield in the remaining PSII reaction centers remained constant during the chromoplast formation, whereas the photosynthetic electron transport beyond PSII became inhibited, as indicated by a marked decrease of the O₂ evolution capacity, of the photochemical quenching of chlorophyll-a fluorescence and of the operational quantum yield of photosynthetic electron transport. Deconvoluted fluorescence emission spectra indicated a more rapid degradation of photosystem I (PSI) complexes than of PSII during chromoplast formation. Compared with green petals, the spillover between PSII and PSI decreased by approximately 40% in yellow petals. Our results indicate that during chloroplast–chromoplast transition in the flower bud of L. longiflorum, PSII integrity was preserved longer than the rest of the photosynthetic apparatus.

Singlet oxygen (¹O₂) is believed to be the major cytotoxic agent involved in photodynamic therapy (PDT). Measurement of ¹O₂ near-infrared (NIR) luminescence at 1270 nm in biological environments is confounded by the strongly reduced ¹O₂ lifetime and probably has never been achieved. We present evidence that this is now possible, using a new NIR-sensitive photomultiplier tube. Time-resolved ¹O₂ luminescence measurements were made in various solutions of aluminum tetrasulphonated phthalocyanine (AlS₄Pc) and Photofrin. Measurements were also performed on suspensions of leukemia cells incubated with AlS₄Pc, and a true intracellular component of the ¹O₂ signal was clearly identified. Time-resolved analysis showed a strongly reduced ¹O₂ lifetime and an increased photosensitizer triplet-state lifetime in the intracellular component. In vivo measurements were performed on normal skin and liver of Wistar rats sensitized with 50 mg/kg AlS₄Pc. In each case, a small but statistically significant spectral peak was observed at 1270 nm. The ¹O₂ lifetime based on photon count rate measurements at 1270 nm was 0.03–0.18 μs, consistent with published upper limits. We believe that these are the first direct observations of PDT-generated intracellular and in vivo ¹O₂. The detector technology provides a new tool for PDT research and possibly clinical use.

We have synthesized a series of symmetrical phenothiazines in which the methyl groups of methylene blue have been substituted by longer alkyl chains. Intrinsic photosensitizing ability was not altered by increasing the chain length. However, in vitro phototoxicity after 2 h incubation of RIF-1 murine fibrosarcoma cells followed the order n-propyl > n-pentyl > n-butyl > n-hexyl > ethyl > methyl, with ethyl and n-propyl analogues being 14- and 130-fold more phototoxic than methylene blue, respectively. All analogues also had an improved ratio of phototoxicity : dark toxicity (4:1 to 27:1) compared with methylene blue (3:1). Phototoxicity did not correlate with cellular phenothiazine levels, suggesting that the site of subcellular localization may be more important. After 2 h incubation of RIF-1 cells with the phototoxicity LD₅₀ concentration, methylene blue and all analogues were observed to be localized in the lysosomes by fluorescence microscopy. On exposure to light, methylene blue relocalized to the nucleus, the ethyl analogue did not relocalize, whereas the more phototoxic n-propyl – n-hexyl analogues relocalized to the mitochondria. Relocalization to the mitochondria was associated with an octanol : buffer partition coefficient ≥ 1. Therefore, the longer-chain analogues of methylene blue show significantly improved phototoxicity in vitro and, in addition, are expected to avoid the problems of mutagenicity associated with the nuclear localization of methylene blue.

In current clinical practice, photodynamic therapy (PDT) is carried out with prescribed drug doses and light doses as well as fixed drug–light intervals and illumination fluence rates. This approach can result in undesirable treatment outcomes of either overtreatment or undertreatment because of biological variations between different lesions and patients. In this study, we explore the possibility of improving PDT dosimetry by monitoring drug photobleaching and photoproduct formation. The study involved 60 mice receiving the same drug dose of a novel verteporfin-like photosensitizer, QLT0074, at 0.3 mg/kg body weight, followed by different light doses of 5, 10, 20, 30, 40 or 50 J/cm² at 686 nm and a fluence rate of 70 mW/cm². Photobleaching and photoproduct formation were measured simultaneously, using fluorescence spectroscopy. A ratio technique for data processing was introduced to reliably detect the photoproduct formed by PDT on mouse skin in vivo. The study showed that the QLT0074 photoproduct is stable and can be reliably quantified. Three new parameters, photoproduct score (PPS), photobleaching score (PBS) and percentage photobleaching score (PBS%), were introduced and tested together with the conventional dosimetry parameter, light dose, for performance on predicting PDT-induced outcome, skin necrosis. The statistical analysis of experimental results was performed with an ordinal logistic regression model. We demonstrated that both PPS and PBS improved the prediction of skin necrosis dramatically compared to light dose. PPS was identified as the best single parameter for predicting the PDT outcome.

The photodynamic properties of meta-tetra(hydroxy-phenyl)chlorin (mTHPC), a promising second-generation photosensitizer, were investigated using a human colon adenocarcinoma cell line (Colo 201 cells). The study on photocytotoxicity using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay showed that mTHPC was an effective photosensitizer on Colo 201 cells. The photocytotoxicity of mTHPC showed both drug and light dose–dependent characteristics. To reach LD₅₀, namely, the dose at which 50% of the cells were killed, only 0.45 ± 0.15 μg/mL of mTHPC and 3 J/cm² of light dose were required. The presence of 10% fetal calf serum in culture medium significantly decreased the incorporation of mTHPC into cells and resulted in the reduction of photodynamic efficacy. Using confocal laser scanning microscopy, mTHPC was first shown to localize in lysosomes rather than in mitochondria. Furthermore, nuclear stainings demonstrated that photodynamic therapy with mTHPC induced apoptosis in Colo 201 cells.

Photodynamic therapy (PDT) produces reactive species that alter vascular wall biology and vessel wall proteins. In this study, we examined platelet adhesion to PDT-treated (photosensitizer = Photofrin; fluence 100 J/cm²; λ = 630 nm) extracellular matrix (ECM), fibrinogen, von Willebrand factor (vWF) and collagen Types I and III, under flow conditions in a recirculating perfusion chamber. Platelet adhesion was quantified by image analysis. The effect of PDT on vWF was assessed by measuring the binding of domain-specific antibodies to treated vWF. PDT significantly decreased platelet adhesion to the ECM, fibrinogen and vWF. However, PDT of collagen resulted in significantly increased platelet adhesion, with large aggregate formation. PDT affected mostly the A1 (glycoprotein [GP]-Ib–binding site), A2 and A3 (collagen-binding site) domains of vWF but not the D'-D3 (factor VIII–binding site) and B-C1 (GP-IIb/IIIa–binding site) domains. In conclusion, PDT can alter the ECM, resulting in decreased platelet adhesion. However, vessels with high collagen content, such as veins and small arteries, may become increasingly prone to thrombosis. The results of this study may thus play a role in understanding the thrombogenic properties and mechanisms of vascular PDT.

Iontophoretic transport of the prodrug 5-aminolevulinic acid (ALA), which is used for photodynamic therapy (PDT), across human stratum corneum (SC) was studied quantitatively in vitro. The experiments were carried out in a three-compartment iontophoresis cell consisting of two electrode chambers equipped with Ag–AgCl electrodes, each separated from a central acceptor chamber by a sheet of SC, supported by a dialysis membrane, to mimic the side-by-side configuration normally used in vivo. Acceptor fluid samples were collected every hour for a period of 30 h in a fraction collector and analyzed by high-performance liquid chromatography–fluorometry after derivatization of the ALA. The iontophoretic ALA flux was studied as a function of the applied current density and the ALA concentration in the donor solution (1, 2.5 or 10% ALA). Depending on the ALA concentration in the donor cell, iontophoresis enhances the flux from close to the detection limit of 0.23 nmol cm⁻² h⁻¹ at zero current density (passive diffusion) to several hundred or thousand nanomoles per square centimeter per hour at current densities ranging from 100 to 1000 μA cm⁻². For example, interpolating our data we find that with an ALA concentration of 2% in the donor chamber, a current density of 0.255 mA cm⁻² transports 0.065 μmol cm⁻² ALA across the SC in 10 min (conditions of Rhodes et al. (1997), J. Invest. Dermatol. 108, 87–91). For passive diffusion we find that a 5 h topical application of 20% ALA results in the transport of 0.05 μmol cm⁻². Thus, the amount of ALA that passively diffuses through the SC in several hours, leading to therapeutic levels of protoporphyrin IX (PpIX) in the epidermis, can be delivered by iontophoresis in 10 min or less. However, because the formation of sufficient PpIX also requires several hours and also because the SC overlying skin lesions such as basal cell carcinoma (BCC) is not intact, the clinical benefit of topical ALA delivery by iontophoresis for PDT of BCC is yet to be established.

We present a mathematical layer model to quantitatively calculate the diffusion of 5-aminolevulinic acid (ALA) in the skin in vivo, its uptake into the cells and its conversion to protoporphyrin IX (PpIX) and subsequently to heme. The model is a modification and extension of a recently presented three-compartment model. The diffusion of ALA in the skin (epidermis, dermis) is described by the time-dependent diffusion equation, and the sink in this equation accounts for ALA uptake in the cells. As boundary conditions, we use the ALA flux across the human stratum corneum (SC) in vitro during passive or iontophoretic ALA delivery as measured in vitro. Besides the diffusion equation, the model includes three additional equations, similar in form to those of the three-compartment model but with a different interpretation. Our additional equations are supposed to describe, respectively, the conversion of ALA in the cytoplasm to some intermediate compound in the mitochondria and the conversion of the latter to PpIX and of PpIX to heme. The first conversion is a process of the Michaelis–Menten type, the other two are first-order rate processes. When fitted to the published data of PpIX fluorescence from normal human skin following iontophoresis of ALA, the model yields the tissue concentration of PpIX as a function of time after ALA application. The computed concentrations are in good agreement with the published phototoxic concentrations of PpIX in the tissues obtained from extraction. The model parameters obtained from the fit are subsequently used to compute the PpIX concentration in normal human skin after 4 h topical application of 10, 20 and 40% ALA. This again yields the PpIX concentrations in tissue, in good agreement with the published values. The saturation of the PpIX concentration as a function of applied ALA concentration is calculated and agrees with clinical observations on the effectiveness of photodynamic therapy. Photobleaching is simulated, with subsequent resynthesis of PpIX in qualitative agreement with experiment. Finally, the model predicts that only 2.5–3.5% of the ALA entering the skin after passing the SC is converted to PpIX. The layered model is a considerable simplification of real skin, but its successful qualitative and quantitative reproduction of experimental data may encourage further studies to test and refine the model to improve our understanding of the kinetics of ALA and the synthesis of PpIX in the skin.

The fluorescence emission properties of single chlorosomes from the green sulfur photosynthetic bacterium Chlorobium (Chl.) tepidum are studied for the first time, using a total internal reflection fluorescence microscope. The fluorescence peak positions of bacteriochlorophyll (BChl)-c self-aggregates in a single chlorosome of Chl. tepidum were widely distributed in the wavelength region between 750 and 768 nm, and the standard deviation (s.d. = 4.1 nm, n = 51) was larger than that of single chlorosomes of Chloroflexus (Cfl.) (s.d. = 1.9 nm, n = 50). The spectral heterogeneity among single chlorosomes from Chl. tepidum was in sharp contrast to those from Cfl. aurantiacus. The difference of chlorosomal spectral properties between Chl. tepidum and Cfl. aurantiacus at the single-unit level would be ascribed to the homolog composition of BChl-c—chlorosomes of Chl. tepidum have BChl substituted with various alkyl groups at both the 8- and 12-positions, whereas light-harvesting BChl-c molecules in Cfl. chlorosomes have the same substituents at the 8- (ethyl group) and 12- (methyl group) positions.