This article reviews the current state of research on the use of molecular contrast agents in optical coherence tomography (OCT) imaging techniques. After a brief discussion of the basic principle of OCT and the importance of incorporating molecular contrast agent usage into this imaging modality, we shall present an overview of the different molecular contrast OCT (MCOCT) methods that have been developed thus far. We will then discuss several important practical issues that define the possible range of contrast agent choice, the design criteria for engineered molecular contrast agent and the implementability of a given MCOCT method for clinical or biological applications. We will conclude by outlining a few areas of pursuit that deserve a greater degree of research and development.

Two-photon excitation of a trifluorophore (6-carboxyfluorescein, N,N,N′,N′-tetramethyl-6-carboxyrhodamine and cyanine-5 monofunctional dye) labeled DNA, which has a scaffold of 26 nucleotides, was achieved using focused laser light of a Q-switched Nd-YAG laser (1064 nm). The observed fluorescence signature (emission ratio from the three fluorophores) of the labeled DNA after two-photon excitation is very different from the fluorescence signatures produced by one-photon excitation at different wavelength. The additional fluorescence signatures produced by two-photon excitation of the fluorescent oligonucleotides will facilitate their use as combinatorial fluorescence energy transfer tags for multiplex genetic analysis.

This article presents the development and characterization of nanoparticles loaded with methylene blue (MB), which are designed to be administered to tumor cells externally and deliver singlet oxygen (¹O₂) for photodynamic therapy (PDT), i.e. cell kill via oxidative stress to the membrane. We demonstrated the encapsulation of MB, a photosensitizer (PS), in three types of sub-200 nm nanoparticles, composed of polyacrylamide, sol–gel silica and organically modified silicate (ORMOSIL), respectively. Induced by light irradiation, the entrapped MB generated ¹O₂, and the produced ¹O₂ was measured quantitatively with anthracene-9,10-dipropionic acid, disodium salt, to compare the effects of different matrices on ¹O₂ delivery. Among these three different kinds of nanoparticles, the polyacrylamide nanoparticles showed the most efficient delivery of ¹O₂, but its loading of MB was low. In contrast, the sol–gel nanoparticles had the best MB loading but the least efficient ¹O₂ delivery. In addition to investigating the matrix effects, a preliminary in vitro PDT study using the MB-loaded polyacrylamide nanoparticles was conducted on rat C6 glioma tumor cells with positive photodynamic results. The encapsulation of MB in nanoparticles should diminish the interaction of this PS with the biological milieu, thus facilitating its systemic administration. Furthermore, the concept of the drug-delivering nanoparticles has been extended to a new type of dynamic nanoplatform (DNP) that only delivers ¹O₂. This DNP could also be used as a targeted multifunctional platform for combined diagnostics and therapy of cancer.

A combination of light, oxygen and a photosensitizer is used to induce death of cancer cells by photodynamic therapy. In this study, we have synthesized several new methyl helianthrone derivatives and compared their phototoxicity with that of hypericin. In contrast to hypericin, methyl helianthrones are soluble in aqueous solutions and have a broad range of light absorbance, which allows the use of polychromatic light. Structural modifications of methyl helianthrone demonstrated that substitution of hydrogen atoms of methyl helianthrone at Positions 2 and 5 with Br atoms or methylation of its phenolic hydroxyls, significantly increases the corresponding singlet oxygen quantum yield and their phototoxicity toward αT3-1, M2R and LNCaP cells. The phototoxicity of some of these compounds was similar to that of hypericin. Methyl helianthrones, like hypericin, accumulated mainly in the perinuclear region as evident by confocal microscopy. Irradiation of cells pretreated with methyl helianthrone derivatives generates intracellular reactive oxygen species and lipid free radicals, as shown by a fluorescentic probe and electron paramagnetic resonance methods, respectively. The phototoxicity of these methyl helianthrones as well as their ability to oxidize membrane lipids were significantly decreased on addition of specific Type-II inhibitors, suggesting the involvement of singlet oxygen as the main oxidant.

Two-photon laser scanning microscopy (TPLSM) of endogenous reduced nicotinamide adenine dinucleotide (NAD(P)H) provides important information regarding the cellular metabolic state. When imaging the punctate mitochondrial fluorescence originating from NAD(P)H in a rat basophilic leukemia (RBL) cell at low laser powers, no morphological changes are evident, and photobleaching is not observed when many images are taken. At higher powers, mitochondrial NAD(P)H fluorescence bleaches rapidly. To assess the limitations of this technique and to quantify the extent of photodamage, we have measured the effect of TPLSM on DNA synthesis. Although previous reports have indicated a threshold power for “safe” two-photon imaging, we find the laser power to be an insufficient indicator of photodamage. A more meaningful metric is a two-photon–absorbed dose that is proportional to the number of absorbed photon pairs. A temporary reduction of DNA synthesis in RBL cells occurs whenever a threshold dose of approximately 2 × 10⁵³ photon² cm⁻⁴ s⁻¹ is exceeded. This threshold is independent of laser intensity when imaging with average powers ranging from 5 to 17 mW at 740 nm. Beyond this threshold, the extent of the reduction is intensity dependent. DNA synthesis returns to control levels after a recovery period of several hours.

Photophysical investigations of coumarin-7 (C7) dye in different solvents using absorption, steady-state fluorescence and time-resolved fluorescence measurements reveal the behavioral changes of the dye in nonpolar and other solvents. In moderate to higher polarity solvents, the experimental parameters such as fluorescence quantum yield (Φ_f), fluorescence lifetime (τ_f), radiative rate constant (k_f), nonradiative rate constant (k_nr) and Stokes' shift (Δν̄) follow almost linear correlations with the Lippert–Mataga solvent polarity parameter Δf but show unusual deviations in nonpolar solvents. From the observed results, it is inferred that the dye exists in a planar intramolecular charge transfer structure in moderate to higher polarity solvents, but in nonpolar solvents, the dye exists in a nonplanar structure with its 7-NEt₂ group adopting a pyramidal type of configuration. Unlike some of the other coumarin dyes, namely coumarin-120 (C120) (4-CH₃-7-NH₂-1,2-benzopyrone) and coumarin-151 (C151) (4-CF₃-7-NH₂-1,2-benzopyrone), which also show similar structural changes in nonpolar and other solvents, the C7 dye does not show any activation-controlled deexcitation process in nonpolar solvents. This is attributed to the very slow flip–flop motion of the 7-NEt₂ group of the C7 dye in comparison with the very fast flip–flop motion of the 7-NH₂ group in the C120 and C151 dyes. Qualitative potential energy diagrams are presented to rationalize the observed results of C7 dye and to compare these with those of the other dyes such as C120 and C151. A support for the observed results and interpretation has also been obtained from quantum chemical calculations on the structures of the C7 dye.

A series of nano- and micronparticle-grade anatase and rutile titanium dioxide pigments have been prepared with various densities of surface treatments, particle size and surface area. Their photocatalytic activites have been determined in a series of paint films by FTIR, chalking, color, gloss change and weight loss after artifical weathering. The pigments have also been examined by rapid assessment methodologies using photodielectric microwave spectroscopy, 2-propanol oxidation and hydroxyl analysis. The microwave response under light and dark cycles provides an extended timescale probe of charge-carrier dynamics in the pigments. Pigment particle size, surface area and properties clearly play an important role in dispersion and any polymer–pigment interactions. Photooxidation studies on several types of paint films show a clear demarcation between nanoparticle- and pigmentary-grade titanium dioxide, with the former being more active because of their greater degree of catalytic surface activity. The photosensitivity of titanium dioxide is considered to arise from localized sites on the crystal surface (i.e. acidic OH), and occupation of these sites by surface treatments inhibits photoreduction of the pigment by ultraviolet radiation; hence, the destructive oxidation of the binder is inhibited. Coatings containing 2–5% by weight alumina or alumina and silica are satisfactory for general-purpose paints. If greater resistance to weathering is desired, the pigments are coated more heavily to about 7–10% weight. The coating can consist of a combination of several materials, e.g. alumina, silica, zirconia, aluminum phosphates of other metals. For example, the presence of hydrous alumina particles lowers van der Waals forces between pigments particles by several orders of magnitude, decreasing particle–particle attractions. Hydrous aluminum oxide phases appear to improve dispersibility more effectively than most of the other hydroxides and oxides. Coated nanoparticles are shown to exhibit effective light stabilization in various water- and oil-based paint media in comparison with conventional organic stabilizers. Hindered piperidine stabilizers are shown to provide no additional benefits in this regard, often exhibiting strong antagonism. The use of photocatalytic titania nanoparticles in the development of self-cleaning paints and microbiological surfaces is also demonstrated in this study. In the former case, surface erosion is shown to be controlled by varying the ratio of admixture of durable pigmentary-grade rutile (heavily coated) and a catalytic-grade anatase nanoparticle. For environmental applications in the development of coatings for destroying atmospheric pollutants such as nitrogen oxide gases (NO_X), stable substrates are developed with photocatalytic nanoparticle-grade anatase. In this study, porosity of the coatings through calcium carbonate doping is shown to be crucial in the control of the effective destruction of atmospheric NO_X gases. For the development of microbiological substrates for the destruction of harmful bacteria, effective nanoparticle anatase titania is shown to be important, with hydrated high surface area particles giving the greatest activity.

Fluocinolone 16,17-acetonide is a corticosteroid used topically to treat various inflammatory skin diseases. Its photoreactivity was studied under UV-A and UV-B light in aqueous buffer in the presence of oxygen. This drug is photolabile under UV-B light and, to a lesser extent, under UV-A light, which is absorbed far less. In phosphate buffer, approximately 80% of fluocinolone acetonide decomposes after 5 J/cm² of UV-B irradiation, whereas under 30 J/cm² of UV-A light approximately only 20% decomposes. Both the drug and its photoproducts have been evaluated through a battery of in vitro studies and found to cause photohemolysis and induce photodamage to proteins (erythrocyte ghosts, bovine serum albumin) and linoleic acid. In addition, one of the photoproducts (the 17-hydroperoxy derivative) is highly toxic in the dark. Therefore, both loss of therapeutic activity and light-induced adverse effects may be expected when patients expose themselves to sunlight after drug administration. A major mechanism for phototoxicity involves radicals forming from drug breakdown, at least under UV-B, although reactive oxygen species may play a role, particularly under UV-A.

In the presence of exciting light, iron and reductants, the singlet oxygen (¹O₂)-generating sensitizer protoporphyrin IX (PpIX) induces free radical lipid peroxidation in membranes, but gradually degrades in the process. We postulated that NO, acting as a chain-breaking antioxidant, would protect PpIX against degradation and consequently prolong its ability to produce ¹O₂. This idea was tested by irradiating PpIX-containing liposomes (LUVs) in the presence of iron and ascorbate, and monitoring the cholesterol hydroperoxides 5α-OOH and 7α/β-OOH as respective ¹O₂ and free radical reporters. 5α-OOH accumulation, initially linear with light fluence, slowed progressively after prolonged irradiation, whereas 7α/β-OOH accumulation only accelerated after an initial lag. The active, but not spent, NO donor spermine NONOate (0.4 mM) virtually abolished 7α/β-OOH buildup as well as 5α-OOH slowdown. Increasing membrane phospholipid unsaturation hastened the onset of rapid chain peroxidation and 5α-OOH slowdown. Accompanying the 5α-OOH effect was a steady decrease in ¹O₂ quantum yield and PpIX fluorescence at 632 nm, both of which were inhibited by NO. An NO-inhibitable decay of PpIX fluorescence was also observed during dark incubation of 5α-OOH–bearing LUVs with iron and ascorbate, confirming a link between chain peroxidation and PpIX loss. By protecting PpIX in irradiated membranes, NO might select for and prolong purely ¹O₂-mediated damage. Supporting this was our observation that ¹O₂-mediated photoinactivation of a nonmembrane target, lactate dehydrogenase, slowed concurrently with 5α-OOH accumulation and that spermine NONOate prevented this. Thus, NO not only protected membrane lipids against PpIX-sensitized free radical damage, but PpIX itself, thereby extending its ¹O₂-generating lifetime. Consistent findings were obtained using porphyrin-sensitized COH-BR1 cells. These previously unrecognized effects of NO could have important bearing on 5-aminolevulinate-based photodynamic therapy in which PpIX is metabolically deposited in tumor cells.

Photodynamic therapy (PDT) is a novel cancer therapy inducing irreversible photodamage to tumor tissue via photosensitizer-mediated oxidative cytotoxicity. The cellular and molecular responses associated with PDT are only partially understood. We have reported previously the generation of several photosensitizer-specific PDT-resistant cell variants of HT29 human colon adenocarcinoma cells by selecting cells from sequential PDT treatment using different photosensitizers. In this report, we describe the use of messenger RNA (mRNA) differential display to identify genes that were differentially expressed in the parental HT29 cells compared with their resistant variants. In comparison with parental HT29 cells, mRNA expression was increased in the PDT-resistant cell variants for BNIP3, estrogen receptor–binding fragment–associated gene 9, Myh-1c, cytoplasmic dynein light chain 1, small membrane protein I and differential dependent protein. In contrast, expression in the PDT-resistant variants was downregulated for NNX3, human HepG2 3′ region Mbol complementary DNA, glutamate dehydrogenase, hepatoma-derived growth factor and the mitochondrial genes coding for 16S ribosomal RNA (rRNA) and nicotinamide adenine dinucleotide (NADH) dehydrogenase subunit 4. The reduction for mitochondrial 16S rRNA in the PDT-resistant variants was confirmed by Northern blotting, and the elevated expression of the proapoptotic BNIP3 in the PDT-resistant variants was confirmed by Northern and Western blotting analysis. We also examined the expression of some additional apoptosis-regulating genes using Western blotting. We show an increased expression of Bcl-2 and heat shock protein 27 and a downregulation of Bax in the PDT-resistant variants. In addition, the mutant p53 levels in the parental HT29 cells were reduced substantially in the PDT-resistant variants. We suggest that the altered expression in several mitochondrial and apoptosis-regulating genes contributes to PDT resistance.

Photosensitized DNA damage participates in solar-UV carcinogenesis, photogenotoxicity and phototoxicity. A chemoprevention of photosensitized DNA damage is one of the most important methods for the above phototoxic effects. In this study, the chemopreventive action of xanthone (XAN) derivatives (bellidifolin [BEL], gentiacaulein [GEN], norswertianin [NOR] and swerchirin [SWE]) on DNA damage photosensitized by riboflavin was demonstrated using [³²P]-5′-end–labeled DNA fragments obtained from genes relevant to human cancer. GEN and NOR effectively inhibited the formation of piperidine-labile products at consecutive G residues by photoexcited riboflavin, whereas BEL and SWE did not show significant inhibition of DNA damage. The four XAN derivatives decrease the formation of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo), an oxidative product of G, by photoexcited riboflavin. The preventive action for the 8-oxodGuo formation of these XAN derivatives increased in the following order: GEN > NOR ≫ BEL > SWE. A fluorescence spectroscopic study and ab initio molecular orbital calculations suggested that the prevention of DNA photodamage is because of the quenching of the triplet excited state of riboflavin by XAN derivatives through electron transfer. This chemoprevention is based on neither antioxidation nor a physical sunscreen effect; rather, it is based on the quenching of a photosensitizer. In conclusion, XAN derivatives, especially GEN, may act as novel chemopreventive agents by the quenching mechanism of an excited photosensitizer.

Four spectroradiometers located at latitudes from 55° to 90°S conducted near-continuous measurements of ground-level solar ultraviolet irradiance from 1990 through 2001. The behavior during months from October through December is of special interest because this period includes the springtime loss in column ozone and the naturally large irradiances of early summer. Monthly integrated irradiances using biological weightings for erythema and damage to DNA show a distortion of the normal annual cycle in irradiance, with enhanced values occurring in October and November. In some cases, these irradiances exceed those near summer solstice in December. Changes in local cloudiness and column ozone both contribute significantly to interannual variability in erythemal irradiance. This is particularly the case at Palmer Station, near 65°S, where the monthly integrated erythemal irradiance in November 1997 was more than double that observed 5 years earlier. In general, at sites on the Antarctic continent, interannual variability in monthly integrated erythemal irradiance is greatest in November, when the observation for any given year can fall 40% above or below the multiyear mean. Near the tip of South America, interannual variability is approximately half that seen in Antarctica.

Thyronine derivatives are essential indicators of thyroid gland diseases in clinical diagnosis and are currently used as standards for developing ordinary biochemical assays. Photooxidation of gland hormones of the thyronine (TN) family and structurally related compounds (TN, 3,5-diiodothyronine,3,3′,5-triiodothyronine and 3,3′,5,5′-tetraiodothyronine or thyroxine) was studied using rose bengal, eosin and perinaphthenone (PN) as dye sensitizers. Tyrosine (Tyr) and two iodinated derivatives (3-iodotyrosine and 3,5-diiodotyrosine) were also included in the study for comparative purposes. Irradiation of aqueous solutions of substrates containing xanthene dyes with visible light triggers a complex series of competitive interactions, which include the triplet excited state of the dye (³Xdye^*) and singlet molecular oxygen [O₂(¹Δ_g)]–mediated and superoxide ion–mediated reactions. Rate constants for interaction with the ³Xdye^*, attributed to an electron transfer process, are in the order of 10⁸–10⁹ M⁻¹ s⁻¹ depending on the dye and the particular substrate. The photosensitization using PN follows a pure Type-II (O₂(¹Δ_g) mediated) mechanism. The presence of the phenolic group in Tyr, TN and iodinated derivatives dominates the kinetics of photooxidation of these compounds. The reactive rate constants, k_r, and the quotient between reactive and overall rate constants (k_r/k_t values, in the range of 0.7–0.06) behave in an opposite fashion compared with the overall rate constants and oxidation potentials. This apparent inconsistency was interpreted on the basis of an internal heavy atom effect, favoring the intersystem-crossing deactivation route within the encounter complex with the concomitant reduction of effective photooxidation.

The suitability of a new technology single-monochromator diode array spectroradiometer for UV-radiation safety measurements, in particular for sunbed measurements, was evaluated. The linearity, cosine response, temperature response, wavelength scale, stray-light and slit function of the spectroradiometer were determined and their effects on the measurement accuracy evaluated. The main error sources were stray-light and nonideal cosine response, for which correction methods are presented. Without correction, the stray-light may reduce the accuracy of the measurement excessively, particularly in the UV-B range. The expanded uncertainty of the corrected UV measurements is estimated to be 14%, which is confirmed with the comparative measurements carried out with a well-characterized double-monochromator spectroradiometer. The measurement accuracy is sufficient for sunbed measurements, provided that all corrections described above have been done and the user of the instrument has a good understanding of the instrument's operating principles and potential error sources. If these requirements are met, the tested spectroradiometer improves and facilitates market surveillance field measurements of sunbeds.

WST11 is a novel negatively charged water-soluble palladium-bacteriochlorophyll derivative that was developed for vascular-targeted photodynamic therapy (VTP) in our laboratory. The in vitro results suggest that WST11 cellular uptake, clearance and phototoxicity are mediated by serum albumin trafficking. In vivo, WST11 was found to clear rapidly from the circulation (t_1/2 = 1.65 min) after intravenous bolus injection in the mouse, whereas a longer clearance time (t_1/2 = 7.5 min) was noted in rats after 20 min of infusion. The biodistribution of WST11 in mouse tissues indicates hepatic clearance (t_1/2 = 20 min), with minor (kidney, lung and spleen) or no intermediary accumulation in other tissues. As soon as 1 h after injection, WST11 had nearly cleared from the body of the mouse, except for a temporal accumulation in the lungs from which it cleared within 40 min. On the basis of these results, we set the VTP protocol for a short illumination period (5 min), delivered immediately after WST11 injection. On subjecting M2R melanoma xenografts to WST11-VTP, we achieved 100% tumor flattening at all doses and a 70% cure with 9 mg/kg and a light exposure dose of 100 mW/cm². These results provide direct evidence that WST11 is an effective agent for VTP and provide guidelines for further development of new candidates.

A novel continuous-flow photocatalytic reactor was designed to decompose toluene by using porous nickel sheets that were coated with a Fe-doped TiO₂ catalyst. While locating the UV lamp at the central axis, the catalyst sheets were located along the inner wall and positioned vertically with an equal space of 50 mm along the reactor. This geometry ensures better use of UV light, and a zigzag flow pattern of gas between the vertically located sheets provides for better mass transfer. The X-ray diffraction, scanning electron microscope and electric field–induced surface photovoltage spectra characterizations showed that Fe³ ions were embedded effectively and distributed evenly throughout the TiO₂ crystal lattice and an optimum molar ratio of Fe:Ti was 0.007. The reactor was used to investigate the factors that affect toluene degradation. The results showed that inlet toluene concentration, relative humidity and gas flow rate significantly affect toluene decomposition. The conversion decreases as inlet concentration increases. Degradation efficiencies of more than 95% can be achieved provided that the toluene concentration is kept below 3200 mg/m³. The conversion is affected little when oxygen content exceeds 21%. The optimal relative humidity is 25%. From the experimental data, a rate constant k of 131 mg/(m³·min) and Langmuir adsorption coefficient K of 0.0175 m³/mg were obtained.

Chromophore-assisted laser inactivation (CALI) is a light-mediated technique used to selectively inactivate proteins of interest to elucidate their biological function. CALI has potential applications to a wide array of biological questions, and its efficiency allows for high-throughput application. A solid understanding of its underlying photochemical mechanism is still missing. In this study, we address the CALI mechanism using a simplified model system consisting of the enzyme β-galactosidase as target protein and the common dye fluorescein. We demonstrate that protein photoinactivation is independent from dye photobleaching and provide evidence that the first singlet state of the chromophore is the relevant transient state for the initiation of CALI. Furthermore, the inactivation process was shown to be dependent on oxygen and likely to be based on photooxidation of the target protein via singlet oxygen. The simple model system used in this study may be further applied to identify and optimize other CALI chromophores.

Melanocytes play a central role in the response of skin to sunlight exposure. They are directly involved in UV-induced pigmentation as a defense mechanism. However, their alteration can lead to melanoma, a process where the role of sun overexposure is highly probable. The transformation process whereby UV damage may result in melanoma initiation is poorly understood, especially in terms of UV-induced genotoxicity in pigmented cells, where melanin can act either as a sunscreen or as a photosensitizer. The aim of this study was to analyze the behavior of melanocytes from fair skin under irradiation mimicking environmental sunlight in terms of spectral power distribution. To do this, normal human Caucasian melanocytes in culture were exposed to simulated solar UV (SSUV, 300–400 nm). Even at relatively high doses (until 20 min exposure, corresponding to 12 kJ/m² UV-B and 110 kJ/m² UV-A), cell death was limited, as shown by cell viability and low occurrence of apoptosis (caspase-3 activation). Moreover, p53 accumulation was three times lower in melanocytes than in unpigmented cells such as fibroblasts after SSUV exposure. However, an important fraction of melanocyte population was arrested in G2–M phase, and this correlated well with a high induction level of the gene GADD45, 4 h after exposure. Among the genes involved in DNA repair, gene XPC was the most inducible because its expression increased more than two-fold 15 h after a 20 min exposure, whereas expression of P48 was only slightly increased. In addition, an early induction of Heme Oxygenase 1 (HO1) gene, a typical response to oxidative stress, was also observed for the first time in melanocytes. Interestingly, this induction remained significant when melanocytes were exposed to UV-A radiation only (320–400 nm), and stimulation of melanogenesis before irradiation further increased HO1 induction. These results were obtained with normal human cells after exposure to SSUV radiation, which mimicked natural sunlight. They provide new data related to gene expression and suggest that melanin in light skin could contribute to sunlight-induced genotoxicity and maybe to melanocyte transformation.

The photochemistry of lapachol and other 1,4-naphthoquinone (NQ) derivatives, e.g. 2-methoxy-1,4-naphthoquinone (MeONQ), 2-hydroxy-1,4-naphthoquinone (2-HONQ) or 5-hydroxy-1,4-naphthoquinone (5-HONQ) and 2-methyl-5-hydroxy-1,4-naphthoquinone (P-NQ) in solution at room temperature was studied by ultraviolet–visible spectroscopy after nanosecond laser pulses at 248 nm. The triplet state and semiquinone radicals were observed for MeONQ, HONQ and P-NQ, whereas for lapachol, intramolecular H-atom and charge transfer processes take place, as in the case of vitamin K₁. The photoinduced reaction of NQ into HONQ is initiated by nucleophilic water addition to the triplet state, and for the secondary reactions, a modified mechanism is proposed.

The effect of ultraviolet radiation (UVR) on inhibition of photosynthesis was studied in two species of marine picoplankton with different carbon concentration mechanisms: Nannochloropsis gaditana Lubián possesses a bicarbonate uptake system and Nannochloris atomus Butcher a CO₂ active transport system. Biological weighting functions (BWFs) for inhibition of photosynthesis by UVR and photosynthesis vs irradiance (PI) curves for photosynthetically active radiation (PAR) were estimated for both species grown with an enriched CO₂ supply (high dissolved inorganic carbon [DIC]: 1% CO₂ in air) and in atmospheric CO₂ levels (low DIC: 0.03% CO₂). The response to UVR and PAR exposures was different in each species depending on the DIC treatment. Under PAR exposure, rates of maximum photosynthesis were similar between treatments in N. gaditana. However, the cultures growing in high DIC had lower sensitivity to UVR than the low DIC cultures. In contrast, N. atomus had higher rates of photosynthesis under PAR exposure with high DIC, but the BWFs were not significantly different between treatments. The results suggest that one or more processes in N. gaditana associated with HCO₃⁻ transport are target(s) for UV photodamage because there was relatively less UV inhibition of the high DIC-grown cultures in which inorganic carbon fixation is supplied by passive CO₂ diffusion. Time courses of photochemical efficiency in PAR, during UV exposure and during subsequent recovery in PAR, were determined using a pulse amplitude modulated fluorometer. The results were consistent with the BWFs. In all time courses, a steady state was obtained after an initial decrease, consistent with a dynamic balance between damage and repair as found for other phytoplankton. However, the relationship of response to exposure showed a steep decline in activity that is consistent with a constant rate of repair. A novel feature of a model developed from a constant repair rate is an explicit threshold for photosynthetic response to UV.

The kinetics of accumulation of protoporphyrin IX (PpIX) after topical application of 5-aminolevulinic acid (ALA) and its methylester (5-aminolevulinic acid methylester [ALA-Me]) was studied on rat oral mucosa. The accumulation of PpIX in mucosa and skin after intravenous injection of ALA and ALA-Me was also studied. The elimination rate of PpIX was dependent on drug and dose as well as on administration route. Application of ALA on rat oral mucosa and skin caused a systemic effect with PpIX building up in remote skin sites not exposed to the drugs. No such systemic effect was seen after application of ALA-Me either in mucosa or on skin. Intravenous injection of the drugs (0.2 g/kg) leads to more fluorescence in the skin than topical application of the drug (20%). For mucosa, the opposite is true. Maximal PpIX fluorescence appeared later after application of high concentrations of the drugs (around 8 h for 5% and 20% wt/wt) than after application of low concentrations (around 3–5 h for 1% and 2% wt/wt).

The photoprotector role of pigment dispersion in the melanophores of the crab, Chasmagnathus granulata, against DNA and oxidative damages caused by UV-A and UV-B was investigated. Intact and eyestalkless crabs were used. In eyestalkless crabs, the dorsal epidermis of the cephalothorax (dispersed melanophores) and the epidermis of pereiopods (aggregated melanophores) were analyzed. Intact crabs showed only dispersed melanophores in the two epidermis. Antioxidant enzymes activity and lipoperoxidation content were analyzed after UV-A (2.5 J/cm²) or UV-B (8.6 J/cm²) irradiation. DNA damage was analyzed by single cell electrophoresis (comet) assay, after exposure to UV-B (8.6 J/cm²). UV-A radiation increased the glutatione-S-transferase activity in the pereiopods epidermis of eyestalkless crabs (P < 0.05). UV-B radiation induced DNA damage in the dorsal epidermis of eyestalkless crabs (P < 0.05). In pereiopod epidermis of eyestalkless crabs, there was no significant difference between control and UV-B–exposed crabs. In the pereiopods epidermis of eyestalkless, the control group showed higher scores of DNA damage and ∼50% of cellular viability. Because in eyestalkless and irradiated crabs the cellular viability was ∼5%, it was not possible to observe nuclei for determination of DNA damage. The findings show that melanophores can play a role in the defense against harmful effects of a momentary exposure to UV radiation.

The photochemistry of the dinucleoside monophosphate thymidylyl-(3′-5′)-5-methyl-2′-deoxycytidine (Tpm⁵dC) has been studied in aqueous solution using both 254 nm and UV-B radiation. A variety of dinucleotide photoproducts containing 5-methylcytosine (m⁵C) have been isolated and characterized. These include two cyclobutane dimers (CBD) (the cis-syn [c,s]and trans-syn forms), a (6-4) adduct and its related Dewar isomer, and two isomers of a product in which the m⁵C moiety was converted into an acrylamidine. Small amounts of thymidylyl-(3′-5′)-thymidine (TpT) were also formed, presumably as a secondary photoreaction product. In addition, a photoproduct was characterized in which the m⁵C moiety was lost, thus generating 3′-thymidylic acid esterified with 2′-deoxyribose at the 5-hydroxyl on the sugar moiety. The c,s CBD of Tpm⁵dC readily undergoes deamination to form the corresponding CBD of TpT. The kinetics of this deamination process has been studied; the corresponding enthalpy and entropy of activation for the reaction have been evaluated at pH 7.4 as being, respectively, 73.4 kJ/mol and −103.5 J/K mol. Deamination was not observed for the other characterized photoproducts of Tpm⁵dC.

Steady-state photophysics of norharmane (NHM), a bioactive alkaloid, has been studied in the presence of a model transport protein, bovine serum albumin (BSA). The emission spectrum undergoes a remarkable change upon addition of BSA to the aqueous solution of NHM in buffer. Addition of BSA leads to a marked increase in the fluorescence anisotropy of the neutral species of NHM, although the fluorescence anisotropy for the cationic species is almost invariant to BSA addition, suggesting that the neutral species is located in a motionally restricted environment of BSA, whereas the cationic species remains in the bulk aqueous phase. The binding constant (K) and free energy change (ΔG) for the probe–protein binding have been calculated from the fluorescence data. Light has been thrown on the action of urea on protein-bound NHM. The denaturation study suggests that the protein, in its native form, binds with NHM. Polarity of the microenvironment around the probe has been determined from a comparison of the fluorescence properties of the two prototropic species of NHM in water–dioxane mixture with varying composition.

The 4a-hydroperoxy-4a,5-dihydroFMN intermediate (II or HFOOH) of Vibrio harveyi luciferase is known to transform from a low quantum yield II_x to a high quantum yield (λ_max 485 nm, uncorrected) II_y fluorescent species on exposure to excitation light. Similar results were observed with II prepared from the αH44A luciferase mutant, which is very weak in bioluminescence activity. Because of the rapid decay of the αH44A II, its true fluorescence was obscured by the more intense 520 nm fluorescence (uncorrected) from its decay product oxidized flavin mononucleotide (FMN). Potassium iodide (KI) at 0.2 M was effective in quenching the FMN fluorescence, leaving the 485 nm fluorescence of II from both the wild-type (WT) and αH44A luciferase readily detectable. For both II species, the luciferase-bound peroxyflavin was well shielded from KI quenching. KI also enhanced the decay rates of both the WT and αH44A II. For αH44A, the transformation of II_x to II_y can be induced by KI in the dark, and it is proposed to be a consequence of a luciferase conformational change. The WT II formed a bioluminescence-inactive complex with KI, resulting in two distinct decay time courses based on absorption changes and decreases of bioluminescence activity of II.

Changes in the room-temperature emission spectrum of chlorophyll (Chl) were analyzed using fast diode-array recordings during the Kautsky effect in mature and in greening barley leaves. In mature leaves, the comparison of F_O (basal level of fluorescence yield at transient O) and F_M (maximum level of fluorescence yield at transient M) spectra showed that the relative amplitude of total variable fluorescence was maximal for the 684 nm Photosystem II (PSII) band and minimal for the 725 nm Photosystem I band. During the increase from F_O to F_M, a progressive redshift of the spectrum of variable fluorescence occurred. This shift reflected the different fluorescence rise kinetics of different layers of chloroplasts inside the leaf. This was verified by simulating the effect of screening on the emission spectrum of isolated chloroplasts and by experiments on greening leaves with low Chl content. In addition, experiments performed at different greening stages showed that the presence of uncoupled Chl at early-greening stages and light-harvesting complex II (LHCII) at later stages have detectable but minor effects on the shape of room-temperature emission spectra. When strong actinic light was applied to mature green leaves, the slow fluorescence yield, which declined from F_M to F_T (steady-state level of fluorescence yield at transient T), was accompanied by a slight redshift of the 684 nm PSII band because of nonphotochemical quenching of short-wavelength–emitting Chl ascribed to LHCII.

A 1 week UV-exposure measurement and outdoor-activity pattern survey was conducted for elementary school children for four seasons at five sites in Japan, i.e. Sapporo (43°05′ N, altitude 40 m), Tsukuba (36°05′ N, 20 m), Tokyo (35°40′ N, 45 m), Miyazaki (31°60′ N, 40 m) and Naha (26°10′ N, 5 m), and UV exposure was measured directly and estimated using outdoor-activity records. The study site with largest UV exposure was Miyazaki, a southern rural area. Comparing the results for boys and girls, UV exposure was larger in boys. UV exposure was large in spring and summer and small in winter. The total amount of UV exposure in spring and summer contributed 57.7–73.4% of total exposure for the year. As a whole, 8.1% and 1.8% of the schoolchildren were exposed to more than 1 minimum erythemal dose (MED) and 2 MED of solar UV in a day, respectively. The estimated yearly UV exposure ranged from 49 207 J/m² in Miyazaki to 31 520 J/m² in Tsukuba. The actual UV exposure correlated to potential UV exposure, estimated using outdoor-activity records and ambient UV irradiance, but the ratio differed by season and site. The yearly average of percent UV exposure to ambient UV on a horizontal plane ranged from 9.9% in Tokyo to 4.0% in Naha. In the questionnaire survey on outdoor-activity pattern, a short question “How long did you spend time outdoors between 0900 and 1500 h?” gives the best estimates of UV exposure.

The effect of the acidity of the environment on the topography and photophysics of sensitizer molecules in homogeneous solutions, and when embedded in a lipid microenvironment, was studied. Four hematoporphyrin (HP) analogs were studied, which have chemical “spacers” of varying lengths between the chromophoric tetrapyrrole and the carboxylate moiety. These derivatives have essentially the same chemical attributes and reactivity as the parent compound, HP IX, which is used in clinical procedures of photodynamic therapy. The binding constants of these HP derivatives to membrane model systems increase with the length of carboxylate chain in the pH range 3.0–6.6. This effect of chain length is attributed to an increase in the hydrophobicity of the molecule on elongation of the alkyl chains. A strong pH dependence of the quenching efficiency of the porphyrins' fluorescence by iodide ions was observed in aqueous solution and is attributed to a unique electrostatic interaction between the fluorophore and the quencher. The quenching efficiency in liposomes, relative to the quenching in buffer, as a function of pH, shows that porphyrins in the neutral form penetrate deeper inside the lipid bilayer and are less exposed to external quenching than when negatively charged at the carboxylic moiety. This vertical displacement in the membrane is also evidenced in the effect of pH on the photosensitized oxidation efficiency of a membrane-bound chemical target. Increasing the pH causes a significant decrease in the sensitization efficiency in liposomes. This trend is attributed to the vertical localization, and protonation of the carboxylic groups on lowering the pH leads to sinking of the sensitizer into the lipid bilayer and to a consequent generation of singlet oxygen at a deeper point. This increases the dwell time of singlet oxygen within the bilayer, which results in greater photodamage to a membrane-residing singlet oxygen target.

A major concern raised about photostability studies of sunscreen products is that the photodegradation of sunscreens does not readily translate into changes in product performance. This study examines the correlation between photochemical degradation of sunscreen agents and changes in protection provided by sunscreen films. Films of a commercial sunscreen product containing avobenzone, oxybenzone and octinoxate were irradiated using a fluorescent UV-A phototherapy lamp with additional UV-B blocking filter. Periodically, during irradiation the transmittances of the films were measured and samples collected for chemical analysis of the sunscreen agents using high-performance liquid chromatography techniques. The results show that UV-induced changes in UV transmittance of sunscreen films correlate with changes in concentration of sunscreen agents. In a parallel experiment, we also irradiated a thin film of the same product in the cavity of an electron spin resonance (ESR) spectrometer. We report the concomitant photolysis of avobenzone and octinoxate that predominates over expected E/Z photoisomerization and that irradiation of a film of this product produced free radicals detected by ESR spectroscopy that persisted even after exposure had ended.

Altered collagen and elastin content correlates closely with remodeling of the arterial wall after injury. Optical analytical approaches have been shown to detect qualitative changes in plaque composition, but the capacity for detection of quantitative changes in arterial collagen and elastin content in vivo is not known. We have assessed fluorescence spectroscopy for detection of quantitative changes in arterial composition in situ, in rabbit models of angioplasty and stent implant. Fluorescence emission intensity (FEI) recorded at sites remote from the primary implant site was correlated with immunohistochemical (IH) analysis and extracted elastin and collagen. FEI was significantly decreased (P < 0.05) after treatment with anti-inflammatory agents, and plaque area decreased on comparison with saline-treated rabbits after stent implant or angioplasty (P ≤ 0.013). Excellent correlations for FEI with elastin and collagen I, III and IV content measured by IH (R² ≥ 0.961) analysis were detected by multiple regression (MR) analysis. Good correlations also were found for FEI with elastin and collagen measured by high-performance liquid chromatography; MR analysis provided highly predictive values for collagen and elastin (R² ≥ 0.994). Fluorescence spectroscopic analysis detects quantitative compositional changes in arterial connective tissue in vivo, demonstrating changes at sites remote from primary angioplasty and stent implant sites.

Molecular beacons (MB) are becoming more common as sequence-selective detectors of nucleic acids. Although they can easily detect single-base mismatches, they have never been used to directly detect DNA or RNA damage. To measure the degree of ultraviolet (UV) light damage in oligonucleotides, we report a novel MB approach for general detection of photoproducts in UV-irradiated rU₁₇ and dT₁₇ oligonucleotides. With monochromatic UV light irradiation at ca 280 nm under anoxic conditions, the oligonucleotide absorption decays with a single-exponential time constant of 123 ± 1 min for rU₁₇ and with double-exponential time constants of 78 ± 0.5 min (99%) and 180 ± 5 min (0.05%) for dT₁₇ oligonucleotides. Under the same conditions, the MB fluorescence decays more quickly, with single-exponential time constants of 19 ± 2 and 26 ± 3 min for rU₁₇ and dT₁₇, respectively. Similar kinetics were observed with broadband UV light irradiation of oligonucleotides. The differences in the UV damage kinetics of dT₁₇ and rU₁₇ and their detection by absorption and fluorescence techniques will be discussed in the context of differential instabilities introduced in the nucleic acid–MB duplex by the different photoproducts formed.

The Action Spectrum of riboflavin (RB) sensitized inactivation of lambda phage was determined between 266 and 575 nm. Below 304 nm, RB depresses the phage reduction by screening phage from radiation that it would otherwise absorb directly. Between 308 and 525 nm, RB sensitizes the inactivation of phage. Enhanced phage reduction is observed at 320 and 500 nm because of binding of RB to the phage and the shifting of the absorption curve of the phage-bound flavin relative to free flavin in phosphate-buffered saline. Enhanced inactivation at 320 and 500 nm and depressed phage inactivation between 360 and 410 nm is also influenced by the inner filter effect.