Two-photon excitation (2PE) provides a means of generating reactive oxygen species (ROS) in cells and tissues with a high degree of spatial specificity. In cultured monolayers of human fibroblasts and fibroblast-derived cells treated with the commonly used probe of ROS formation, 5-(and 6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H₂DCFDA), cells irradiated through a microscope objective with 150 fs near-infrared laser pulses became highly fluorescent, reflecting intracellular ROS formation. The fluorescence intensity inside cells increased quadratically with the average power of radiation for pulsed excitation and was unchanged for continuous wave irradiation with the same average power. Single fibroblasts embedded within dermal equivalents were also targeted in this manner and formed ROS, whereas neighboring unirradiated cells were spared. These results demonstrate that ROS can be generated intracellularly in skin cells using 2PE of the metabolic or oxidative products of CM-H₂DCFDA and that formation of ROS can be localized in both cell monolayers and in a tissue equivalent. This technique should be useful in understanding the response of whole tissues such as skin to local generation of ROS and may have applications in photodynamic therapy.

We investigate by X-ray crystallographic techniques the cryotrapped states that accumulate on controlled illumination of the blue light photoreceptor, photoactive yellow protein (PYP), at 110 K in both the wild-type species and its E46Q mutant. These states are related to those that occur during the chromophore isomerization process in the PYP photocycle at room temperature. The structures present in such states were determined at high resolution, 0.95–1.05Å. In both wild type and mutant PYP, the cryotrapped state is not composed of a single, quasitransition state structure but rather of a heterogeneous mixture of three species in addition to the ground state structure. We identify and refine these three photoactivated species under the assumption that the structural changes are limited to simple isomerization events of the chromophore that otherwise retains chemical bonding similar to that in the ground state. The refined chromophore models are essentially identical in the wild type and the E46Q mutant, which implies that the early stages of their photocycle mechanisms are the same.

We are developing a technology based on the combined application of riboflavin (RB) and light for inactivating pathogens in blood products while retaining the biological functions of the treated cells and proteins. Virus and bacteria reduction measured by tissue culture infectivity or colony formation with UV light alone and in combination with RB yield equivalent results. The effects of RB as a sensitizing agent on DNA in white cells, bacteria and viruses in combination with UV light exposure have been evaluated. UV-mediated DNA degradation in Jurkat T cells and leukocytes in plasma as measured by the FlowTACS assay was significantly increased in the presence of RB. Agarose gel electrophoretic analysis of DNA in Escherichia coli and leukocytes in plasma demonstrated enhanced DNA degradation in the presence of RB. UV light in combination with RB prevented the reactivation of lambda phage compared with samples irradiated in the absence of RB. UV-mediated oxidative damage in calf thymus DNA was also enhanced in the presence of RB. These observations clearly demonstrate that the presence of RB and UV light selectively enhances damage to the guanine bases in DNA. These data also suggest that the type and extent of damage to DNA for virus in the presence of RB and light make it less likely to be repaired by normal repair pathways available in host cells.

The concept of metronomic photodynamic therapy (mPDT) is presented, in which both the photosensitizer and light are delivered continuously at low rates for extended periods of time to increase selective tumor cell kill through apoptosis. The focus of the present preclinical study is on mPDT treatment of malignant brain tumors, in which selectivity tumor cell killing versus damage to normal brain is critical. Previous studies have shown that low-dose PDT using 5-aminolevulinic acid (ALA)–induced protoporphyrin IX (PpIX) can induce apoptosis in tumor cells without causing necrosis in either tumor or normal brain tissue or apoptosis in the latter. On the basis of the levels of apoptosis achieved and model calculations of brain tumor growth rates, metronomic delivery or multiple PDT treatments, such as hyperfractionation, are likely required to produce enough tumor cell kill to be an effective therapy. In vitro studies confirm that ALA-mPDT induces a higher incidence of apoptotic (terminal deoxynucleotidyl transferase–mediated 2′-deoxyuridine 5′-triphosphate, sodium salt nick-end labeling positive) cells as compared with an acute, high-dose regimen (ALA-aPDT). In vivo, mPDT poses two substantial technical challenges: extended delivery of ALA and implantation of devices for extended light delivery while allowing unencumbered movement. In rat models, ALA administration via the drinking water has been accomplished at very high doses (up to 10 times therapeutic dose) for up to 10 days, and ex vivo spectrofluorimetry of tumor (9L gliosarcoma) and normal brain demonstrates a 3–4 fold increase in the tumor-to-brain ratio of PpIX concentration, without evidence of toxicity. After mPDT treatment, histological staining reveals extensive apoptosis within the tumor periphery and surrounding microinvading colonies that is not evident in normal brain or tumor before treatment. Prototype light sources and delivery devices were found to be practical, either using a laser diode or light-emitting diode (LED) coupled to an implanted optical fiber in the rat model or a directly implanted LED using a rabbit model. The combined delivery of both drug and light during an extended period, without compromising survival of the animals, is demonstrated. Preliminary evidence of selective apoptosis of tumor under these conditions is presented.

To collect information on gene expression during the dark period in the luminous dinoflagellate Lingulodinium polyedrum, normalized complementary DNA (cDNA) libraries were constructed from cells collected during the first hour of night phase in a 12:12 h light–dark cycle. A total of 4324 5′-end sequence tags were isolated. The sequences were grouped into 2111 independent expressed sequence tags (EST) from which 433 groups were established by similarity searches of the public nonredundant protein database. Homology analysis of the total sequences indicated that the luminous dinoflagellate is more similar to land plants and animals (vertebrates and invertebrates) than to prokaryotes or algae. We also isolated three bioluminescence-related (luciferase and two luciferin-binding proteins [LBP]) and 37 photosynthesis-related genes. Interestingly, two kinds of LBP genes occur in multiple copies in the genome, in contrast to the single luciferase gene. These cDNA clones and EST sequence data should provide a powerful resource for future genome-wide functional analyses for uncharacterized genes.

The heme in horseradish peroxidase (HRP) was replaced by phosphorescent Pt-mesoporphyrin IX (PtMP), which acted as a phosphorescent marker of oxygen quenching and allowed comparison with another probe, Pd-mesoporphyrin IX (Khajehpour et al. (2003) Proteins 53, 656–666). Benzohydroxamic acid (BHA), a competitive inhibitor of the enzyme, was also used to monitor its effects on phosphorescence quenching. With the addition of BHA, in the presence of oxygen, the phosphorescence intensity of the protein increased. In contrast, the addition of BHA, in the absence of oxygen, reduced the phosphorescence intensity of the protein. K_d = 18 μM when BHA binds to PtMP-HRP. The effect of BHA can be explained by two factors: (1) BHA reduces the accessibility of O₂ to the protein interior and (2) BHA itself quenches the phosphorescence. Consistent with this, the oxygen quenching of the phosphorescence of PtMP-HRP gave a quenching constant of k_q = 234 mm Hg⁻¹ s⁻¹ in the absence of BHA and k_q = 28.7 mm Hg⁻¹ s⁻¹ in the presence of BHA. The quenching rate of BHA is 4000 s⁻¹. The relative quantum yield of the phosphorescence of the Pt derivative is about six times that of the Pd derivative, whereas the phosphorescence lifetime is approximately eight times shorter. The high quantum yield and suitable lifetime make Pt-porphyrins appropriate as sensors of O₂ diffusion and flexibility in heme proteins.

Native fluorescence spectroscopy was used for in situ investigations of two lipid-containing bacteriophages from the cystovirus family as well as their Pseudomonad host cells. Both the viruses ϕ6 and ϕ12 and their bacterial host proteins contain the amino acid tryptophan (trp), which is the predominant fluorophore in UV. Within proteins, trp's structural environment differs, and the differences are reflected in their spectroscopic signatures. It was observed that the peak of the trp emission from both viruses was at 330 nm, a significantly shorter wavelength than trp in either the Pseudomonad host cells or the amino acid's chemical form. This allowed us to monitor the viral attachment process and subsequent lytic release of progeny virus particles by measurement of the trp emission spectra during the infection process. This work demonstrates that fluorescence may offer a novel tool to detect viruses and monitor viral infection of cells and may be part of a biodefense application.

Discussions of risks and implications of cutaneous exposure to indoor lighting, including hypothetical contribution to causality of melanoma, have mainly concentrated on ultraviolet (UV) A and B (UVA, UVB) spectral emissions from fluorescent bulbs. Only studies of quartz halogen lamps have suggested that users might sustain UVC-induced injury. Examination of light sources in the home and school of a child with xeroderma pigmentosum revealed that several different types emitted surprising levels of UV. Our purpose was to assess the extent of UV emissions from a variety of commonly used light sources to identify potential dermatological risks. UV and visible spectral emissions of commercially obtained lamps of several types were measured using a calibrated spectral radiometer traceable to the National Institute of Standards and Technology. Indoor light sources including fluorescent, quartz halogen and even tungsten filament incandescent lamps provided UVA, UVB and sometimes UVC emissions. Intensities of some emissions were of similar magnitude to those in sunlight. Chronic exposure to indoor lighting may deliver unexpected cumulative UV exposure to the skin and eyes. Patients with UV-exacerbated dermatoses should be cautioned about potential adverse reactions from indoor lighting.

Light absorption by chromophoric dissolved organic matter (CDOM) plays a number of roles in natural waters, including both control of the underwater light field and the initiation of many photochemical reactions. A multicomponent analysis was used to describe the effects of UV and visible radiation on the optical absorption spectra of two natural water samples, a Suwannee River fulvic acid standard (SRFA) and a Delaware Bay water sample. This analysis used a constrained minimization technique to fit independent spectral components to the observed bleaching behavior of the water samples under monochromatic irradiation. Spectra derived from these fits were used to predict the bleaching behavior of both samples under polychromatic irradiation (λ > 320 nm). This approach reproduces the kinetics and spectral behavior of polychromatic photobleaching very well at times <48 h, but underpredicts the bleaching at longer time periods.

UV-A radiation produces cataract in animals, enhances photoaging of the lens and skin and increases the phototoxicity of drugs. However, the nature of genes that are activated or repressed after cellular exposure to UV-A radiation remains enigmatic. Because lens epithelial cells exposed to UV-A radiation undergo apoptosis 4 h after exposure to the stress, we sought to establish the change in gene expression in cells by UV-A radiation using gene expression profiling using complementary DNA microarrays containing about 12 000 genes. We identified 78 genes abnormally expressed in UV-A–irradiated cells (showing >2.5-fold change at P < 0.05). These genes are implicated in various biological processes, including signal transduction and nucleic acid binding, and genes encoding enzymes. A majority of the genes were downregulated. Our analysis revealed that the expression of genes for the transcription factors ATF-3 and Pilot increased four-fold, whereas the gene for the apoptosis regulator NAPOR-1 decreased five-fold. These changes were confirmed by real-time quantitative reverse transcriptase–polymerase chain reaction. The calpain large polypeptide 3 (CANP3) gene also increased nine-fold after UV-A radiation. In addition, peroxisomal biogenesis factor 7, glucocorticoid receptor-α and tumor-associated calcium signal transducer genes decreased three- to eight-fold. Western blot analysis further confirmed the increase in protein expression of ATF-3 and CANP3 and decreased expression of glucocorticoid receptor-α in the irradiated cells. Surprisingly, most of these genes had not been previously shown to be modulated by UV-A radiation. Our results show that human lens epithelial cells respond to a single dose of UV-A radiation by enhancing or suppressing functionally similar sets of genes, some of which have opposing functions, around the time at which apoptosis occurs. These studies support the intriguing concept that activation of competing pathways favoring either cell survival or death is a means to coordinate the response of cells to UV-A stress.

On exposure to UV-B, the epidermal component trans-urocanic acid (UCA) is not only photoisomerized into cis-UCA but will also, at least in part, be photooxidized into UCA oxidation products (UOPs). We hypothesized that UOPs can mimic UV-induced systemic immunosuppression comparable to the suppressive properties already established for cis-UCA. A crude mixture of UOPs showed a significant suppression of the sensitization phase of the systemic contact hypersensitivity (CHS) response to picryl chloride (PCl). Three of the UOPs were selected for this study: imidazole-4-carboxylic acid (ImCOOH), imidazole-4-carboxaldehyde (ImCHO) and imidazole-4-acetic acid (ImAc). Effects on the sensitization, elicitation and postelicitation phases of CHS to PCl in BALB/c mice were studied and compared with the effects of cis-UCA. ImCHO was equally effective at suppressing the sensitization phase as cis-UCA. The triplet combination of the imidazoles (1:1:1) showed more pronounced suppression than that induced by cis-UCA. The most effective compounds for the suppression of the elicitation phase appeared to be ImAc and cis-UCA. Significant suppression of the postelicitation phase was only obtained with the triplet combination of ImCHO, ImCOOH and ImAc, the combination that appeared to be effective at all three tested phases. Because these three UOPs are present in UV-B–exposed human stratum corneum, these compounds may play a role in UV-B–induced immunosuppression.

Purpose of this work was to study the effect of UV irradiation on a microecosystem consisting of several interacting species. The system chosen was of a hypersaline type, where all the species present live at high salt concentration; it comprises different bacteria; a producer, the photosynthetic green alga Dunaliella salina; and a consumer, the ciliated protozoan Fabrea salina, which form a complete food chain. We were able to establish the initial conditions that give rise to a self-sustaining microecosystem, stable for at least 3 weeks. We then determined the effect of UV irradiation on this microecosystem under laboratory-controlled conditions, in particular by measuring the critical UV exposure for the two main components of the microecosystem (algae and protozoa) under UV-B irradiances comparable to those of solar irradiation. In our experiments, we varied irradiance, total dose and spectral composition of the actinic light. The critical doses at irradiances of the order of 56 kJ/m² (typical average daily irradiance in a sunny summer day in Pisa), measured for each main component of the microecosystem (algae and ciliates), turned out to be around 70 kJ/m² for ciliates and 50 kJ/m² for D. salina. By exposing microecosystems to daily UV-B irradiances of the order of 8 kJ/m² (typical average daily irradiance in a sunny winter day in Pisa), we found no effect at total doses of the order of the critical doses at high irradiances, showing that the reciprocity law does not hold. We have also measured a preliminary spectral-sensitive curve of the UV effects, which shows an exponential decay with wavelength.

The resonance Raman spectrum of Sepia eumelanin is discussed by analogy to model compounds containing catechol (CAT)–like structural units. These data are then compared with the analogous data on Fe(III)-enriched Sepia eumelanin. In contrast to the natural eumelanin, the Fe(III)-enriched samples exhibit absorption features in the visible and near-IR spectral regions, which are attributed to ligand-to-metal charge-transfer (LMCT) bands. Resonance Raman spectra collected by exciting these LMCT bands reveal bands at 580 and 1470 cm⁻¹; the intensity of these features increases with increasing Fe(III) content. The 580 and 1470 cm⁻¹ bands are assigned to Fe–OR stretching and ring deformation modes, respectively. These data further substantiate that the Fe(III)-melanin–binding site in melanin is composed of CAT-like structural units.

The treatment of cutaneous vascular lesions (port wine stains etc.) using lasers has been guided by theories based on the “cold” or room-temperature optical properties of the hemoglobin target chromophore. We have recently presented evidence showing that under the influence of laser irradiation, the optical properties of blood in vitro are time and temperature dependent. Such complications are not currently subsumed into the in vivo theory. Here, we study the time-domain optical properties of blood undergoing photocoagulation in vitro using two newly developed time-resolved techniques. We also study the asymptotic effect of laser photocoagulation on the chemical and structural properties of the components of the blood matrix. We present evidence showing that the photocoagulation process involves significant changes in the optical absorption and scattering properties of blood, coupled with photothermally induced chemical and structural changes. We demonstrate the first use of a laser to deliberately generate magnetic resonance imaging contrast in vitro. We show that this technique offers significant potential advantages to in vivo intravenous chemical contrast agent injection.

Fluorescence spectroscopy of the endogenous emission of brain tumors has been researched as a potentially important method for the intraoperative localization of brain tumor margins. We investigated the use of time-resolved, laser-induced fluorescence spectroscopy for demarcation of primary brain tumors by studying the time-resolved spectra of gliomas. The fluorescence of human brain samples (glioblastoma multiforme, cortex and white matter: six patients, 23 sites) was induced ex vivo with a pulsed nitrogen laser (337 nm, 3 ns). The time-resolved spectra were detected in a 360–550 nm wavelength range using a fast digitizer and gated detection. Parameters derived from both the spectral- (intensities from narrow spectral bands) and the time domain (average lifetime) measured at 390 and 460 nm were used for tissue characterization. We determined that high-grade gliomas are characterized by fluorescence lifetimes that varied with the emission wavelength (>3 ns at 390 nm, <1 ns at 460 nm) and their emission is overall longer than that of normal brain tissue. Our study demonstrates that the use of fluorescence lifetime not only improves the specificity of fluorescence measurements but also allows a more robust evaluation of data collected from brain tissue. Combined information from both the spectral- and the time domain can enhance the ability of fluorescence-based techniques to diagnose and detect brain tumor margins intraoperatively.

Experimental results on various photophysical properties of coumarin-30 (C30) dye, namely, Stokes' shift (Δν), fluorescence quantum yield (Φ_f), fluorescence lifetime (τ_f), radiative rate constant (k_f) and nonradiative rate constant (k_nr), as obtained using absorption and fluorescence measurements have been reported. Though in most of the solvents the properties of C30 show more or less linear correlation with the solvent polarity function, Δf = {(ϵ − 1)/(2ϵ 1) − (n² − 1)/(2n² 1)}, they show unusual deviations in nonpolar solvents at one end and in high-polarity protic solvents at the other end. From the solvent polarity and temperature effect on the photophysical properties of the dye, following inferences have been drawn: (1) in nonpolar solvents, the dye exists in a nonpolar structure, where its 7-NEt₂ substituent adopts a pyramidal configuration and the amino lone pair is out of resonance with the benzopyrone π cloud; (2) in medium to higher polarity solvents, the dye exists in a polar intramolecular charge transfer structure, where the 7-NEt₂ group and the 1,2-benzopyrone moiety are in the same plane and the amino lone pair is in resonance with the benzopyrone π cloud; (3) in protic solvents, the dye–solvent intermolecular hydrogen bonding influences the photophysical properties of the dye; and (4) in high-polarity protic solvents, the excited C30 undergoes a new activation-controlled nonradiative deexcitation process because of the involvement of a twisted intramolecular charge transfer (TICT) state. Contrary to most other TICT molecules, the activation barrier for this deexcitation process in C30 is observed to increase with solvent polarity. A rational for this unusual behavior has been given on the basis of the solvent polarity–dependent stabilization and crossing of relevant electronic states and the relative propensity of interconversion among these states.

We report on the emission spectra and emission quantum yields of a newly synthesized hypocrellin dye, 2-demethoxy-2,3-ethylenediamino hypocrellin B (EDAHB), and its parent HB in different solvents of varying polarity. Our results demonstrate that EDAHB is one of the few dyes that exhibit highly solvent polarity–dependent fluorescence in the useful region (680–730 nm). Therefore, it offers some applications in the biomedical field as a fluorescent probe molecule. The solvatochromic effect of EDAHB is proposed to be due to a distinct change in the dipole moment of the dye on excitation. A photoinduced intramolecular proton transfer and a photoinduced intramolecular electron transfer process are considered relevant for the fluorescence properties of HB and EDAHB, respectively.

In the cuticle of live social hornets, such as Vespa orientalis (Hymenoptera, Vespinae), endogenous electric effects are encountered, i.e. voltages of 100–200 mV under illumination and currents amounting to several microamperes on its subjection to darkness—clearly a process of charging and discharging. Of the various wavelengths of sunlight, UV was found to be the most contributory to the active cuticular voltage generation. Throughout the warm season of the year—the active period in colonies of social hornets and wasps—colony members exit from the dark nest during the daytime and fly to the field under the hot sun for various foraging purposes, ultimately returning to the nest. Thus, each hornet, be it queen, worker or drone, probably undergoes daily cyclical process of electric charge and discharge in the exterior part of their integument, cuticle, which lasts up to 30–40 min. Such photoelectric phenomenon was detected in both live, ether-anaesthetized hornets and dead hornets, albeit in the latter the electric values recorded were lower. The present study addresses the possible impact of the phenomenon on vespan daily life and also compares it with a parallel occurrence in electric fish.

Effects of solvent, pH and hydrogen bonding with N-methylimidazole (MIm) on the photophysical properties of 1-hydroxyfluorenone (1HOF) have been studied. Fluorescence lifetime, fluorescence quantum yield and triplet yield measurements demonstrated that intersystem crossing was the dominant process in apolar media and its rate constant significantly diminished with increasing solvent polarity. The acceleration of internal conversion in alcohols paralleled the strength of intermolecular hydrogen bonding. The faster energy dissipation from the singlet-excited state in cyclohexane was attributed to intramolecular hydrogen bonding. The pK_a of 1HOF decreased from 10.06 to 5.0 on light absorption, and H₃O quenched the singlet-excited molecules in a practically diffusion-controlled reaction. On addition of MIm in toluene, dual fluorescence was observed, which was attributed to reversible formation of excited hydrogen-bonded ion pair. Rate constants for the various deactivation pathways were derived from the combined analysis of the steady-state and the time-resolved fluorescence results.

Stratospheric ozone depletion has caused an increase in the amount of ultraviolet-B (UV-B) radiation reaching the earth's surface. Numerous investigations have demonstrated that the effect of UV-B enhancements on plants includes reduction in grain yield, alteration in species competition, susceptibility to disease and changes in plant structure and pigmentation. Many experiments examining UV-B radiation effects on plants have been conducted in growth chambers or greenhouses. It has been questioned whether the effect of UV-B radiation on plants can be extrapolated to field responses from indoor studies because of the unnaturally high ratios of UV-B/ultraviolet-A radiation (320–400 nm) and UV-B/photosynthetically active radiation (PAR) in many indoor studies. Field studies on UV-B radiation effect on plants have been recommended to use the UV and PAR irradiance provided by natural light. This study reports the growth and yield responses of a maize crop exposed to enhanced UV-B radiation and the UV-B effects on maize seed qualities under field conditions. Enhanced UV-B radiation caused a significant reduction in the dry matter accumulation and the maize yield in turn was affected. With increased UV-B radiation the flavonoid accumulation in maize leaves increased and the contents of chlorophyll a, b and (a b) of maize leaves were reduced. The levels of protein, sugar and starch of maize seed decreased with enhanced UV-B radiation, whereas the level of lysine increased with enhanced UV-B radiation.

The Rose Bengal–sensitized photooxidations of the dipeptides l-tryptophyl-l-phenylalanine (Trp-Phe), l-tryptophyl-l-tyrosine (Trp-Tyr) and l-tryptophyl-l-tryptophan (Trp-Trp) have been studied in pH 7 water solution using static photolysis and time-resolved methods. Kinetic results indicate that the tryptophan (Trp) moiety interacts with singlet molecular oxygen (O₂(¹Δ_g)) both through chemical reaction and through physical quenching, and that the photooxidations can be compared with those of equimolecular mixtures of the corresponding free amino acids, with minimum, if any, influence of the peptide bond on the chemical reaction. This is not a common behavior in other di- and polypeptides of photooxidizable amino acids. The ratio between chemical (k_r) and overall (k_t) rate constants for the interaction O₂(¹Δ_g)-dipeptide indicates that Trp-Phe and Trp-Trp are good candidates to suffer photodynamic action, with k_r/k_t values of 0.72 and 0.60, respectively (0.65 for free Trp). In the case of Trp-Tyr, a lower k_r/k_t value (0.18) has been found, likely as a result of the high component of physical deactivation of O₂(¹Δ_g) by the tyrosine moiety. The analysis of the photooxidation products shows that the main target for O₂(¹Δ_g) attack is the Trp group and suggests a much lower accumulation of kynurenine-type products, as compared with free Trp. This is possibly because of the occurrence of another accepted alternative pathway of oxidation that gives rise to 3a-oxidized hydrogenated pyrrolo[2,3-b]indoles.

In aquatic ecosystems, the UV and visible radiation environment is strongly influenced by variation in the chemical and physical parameters of the ecosystems. In shallow lakes, highly heterogeneous water characteristics produce a wide variety of optical environments. Such ecosystems require analysis approaches that consider a potential variability. In this study, 77 stations were used to characterize the optical properties of a shallow lake (open water surface 54 km²). The vertical attenuation of solar radiation at 305, 313, 320 and 340 nm and at photosynthetically active radiation was measured during the seasonal cycle. Dissolved organic matter (DOM), turbidity, fluorescence, pH, temperature, conductance and dissolved oxygen were simultaneously measured. The spatial variation of the extinction spectra of the dissolved fraction at each sampling station was also measured and analyzed between 270 and 400 nm. The spatial heterogeneity of the lake was examined by determining the distributions of the attenuation coefficients and biooptical parameters at high spatial resolution and describing the distributions in a series of maps. The methodology permitted a quantitative description of the interaction between solar radiation and aquatic ecosystems as well as a spatial classification of the dominating processes within the lake. This included the determination of the role played by DOM loading and changing chemical properties within the lake optical environment.

The Bacillus subtilis protein YtvA, related to plant phototropins (phot), binds flavin mononucleotide (FMN) within the N-terminal light, oxygen and voltage (LOV) domain. The blue light–triggered photocycle of YtvA and phot involves the reversible formation of a covalent photoadduct between FMN and a cysteine (cys) residue. YtvA contains a single tryptophan, W103, localized on the LOV domain and conserved in all phot-LOV domains. In this study, we show that the fluorescence parameters of W103 in YtvA-LOV are markedly different from those observed in the full-length YtvA. The fluorescence quantum yields are ca 0.03 and 0.08, respectively. In YtvA-LOV, the maximum is redshifted (ca 345 vs 335 nm) and the average fluorescence lifetime shorter (2.7 vs 4.7 ns). These data indicate that W103 is located in a site of tight contact between the two domains of YtvA. In the FMN–cys adduct, selective excitation of W103 at 295 nm results in minimal changes of the fluorescence parameters with respect to the dark state. On 280 nm excitation, however, there is a detectable decrease in the fluorescence emitted from tyrosines, with concomitant increase in W103 fluorescence. This effect is reversible in the dark and might arise from a light-regulated energy transfer process from a yet unidentified tyrosine to W103.