Quantum yields for the formation of transients were measured following the quenching of triplet 4-carboxybenzophenone (³CB^*) by methionine-containing peptides in aqueous solutions. Ketyl radicals (CBH^·), ketyl radical anions (CB^·−) and various sulfur radical cations were identified following the triplet-quenching events. The presence of these intermediates indicated that the triplet-quenching mechanism can be characterized as mainly electron-transfer in nature. The quenching rate constants were of the order of 2 × 10⁹ M⁻¹ s⁻¹. There were small, but significant, differences in the triplet-quenching rate constants, and these trends indicate the existence of multiple sulfur targets in the quenchers. The absorption of the transient products was followed in detail by using spectral-resolution analysis. From the absorption data, quantum yields were estimated for the formation of the various transients. There were differences found in the yields of the transient products between the experiments, where the quenchers were the “mixed” stereoisomers of methionylmethionine (l,d and d,l) and experiments where the quenchers were l,l and d,d stereoisomers. Triplet-quenching data from several other methionine-containing small oligopeptides were analyzed in an analogous manner. Systematic variations were observed, and these patterns were discussed in terms of competitive donation of protons to the CB^·− within the charge-transfer complex. The competition was between protons on carbons adjacent to the sulfur-radical center and protons on the protonated amino groups of the radical cation. In addition, there was a competition between the two intramolecular two-centered, three-electron bonded species (S∴S) and (S∴N) that play roles in the secondary kinetics.

The results of a comprehensive investigation of the photophysical properties of the sunscreen agent menthyl anthranilate in various solvent systems are reported. Luminescence studies reveal that this ester is highly fluorescent (Φ_f = 0.64 ± 0.06 in ethanol) and has a solvent-dependent emission maximum in the range of 390–405 nm. Phosphorescence has also been detected in low-temperature glasses with an emission maximum at 445 nm and a lifetime of 2.5 s. Kinetic UV–visible absorption measurements revealed a transient species with absorption maxima at 480 nm and solvent-dependent lifetimes of 26–200 μs that are attributed to the triplet state. The triplet state is efficiently quenched by oxygen, leading to the formation of singlet oxygen in all of the solvent systems studied. The singlet-oxygen quantum yields (Φ_Δ) determined by time-resolved near-infrared luminescence measurements were determined to be in the range 0.09–0.12 for all systems.

Using two-dimensional electrophoresis we investigated the effect of 5-aminolevulinic acid (ALA)–based photodynamic therapy (PDT; induction with 1 mM ALA for 4 h followed by blue light dose of 18 J/cm²) on the protein expression in HL60 leukemia cells. ALA-PDT resulted in extensive qualitative and quantitative changes in the protein pattern of HL60 cell lysates. Of more than 1350 protein spots recognized on the protein maps of ALA-induced cells, seven proteins were enhanced and 17 suppressed following irradiation. Three of these, calreticulin presursor, p58 microsomal protein (ERp57) and protein disulfide isomerase (p55) have been identified by matrix-assisted laser desorption and ionization-mass spectrometry and the pI/molecular weight parameters of the affected proteins were estimated by computer analysis. The findings suggest participation of endoplasmic reticulum Ca² -binding chaperones and/or Ca² signaling in ALA-PDT mediated cytotoxicity.

The crystal structures of 4,6-dimethyltetrahydrobenzoangelicin (THBA), a furocoumarin analog, and of its furan-side cis–syn cycloadduct with thymine formed in the photoreaction with DNA, have been determined. The crystal structure of the latter compound contained only one enantiomeric form corresponding to the addition to a 5′-XpT site. Contrary to most psoralen derivatives studied, THBA showed higher photoreactivity toward synthetic oligonucleotides containing that sequence than toward those with the 5′-TpX sequence.

The capability of the new luminescent probe (dibenzo[h,j]dipyrido[3,2-a:2′,3′-c]phenazine)bis(2,2′-bipyridine)ruthenium(II) dication, (RB2Z), to discriminate live and dead cells has been tested on rat hepatocytes and mouse lymphocytes. RB2Z-stained cells were analyzed using flow cytometry, fluorescence (confocal) microscopy and time-resolved luminescence measurements. The established viability probes propidium iodide (PI) and SYTOX® green (SG) were used as controls. The intense luminescence of RB2Z at 606 nm is localized in the nucleus of nonviable cells. Viability measurements by flow cytometry and fluorescence microscopy using RB2Z as dead-cell marker yield the same results as PI and SG. The luminescence lifetime of RB2Z also displays sensitivity to cell viability (0.45 and 0.82 μs in presence of fully viable and dead cells, respectively). This ruthenium complex is photostable under laser sources and its 200 nm Stokes shift facilitates multicolor labeling experiments in flow cytometry and fluorescence microscopy. Unlike the currently available probes, the long-lived excited state of RB2Z also allows assays based on luminescence decay measurements.

We have analyzed the tryptophan (trp) fluorescence-decay kinetics of single trp mutants of the Tet repressor protein in the free, the tet operator and anhydrotetracycline (atc)-bound states. The position of the single trp varies between residues 164 and 171, in close proximity to one entrance of the tetracycline-binding pocket. A good fit of the trp fluorescence decay needed generally three exponentials. The decay times vary with detection wavelength, the extent of this variation being correlated to the variation of the emission maximum. Quenching experiments with neutral (acrylamide), cationic (N-methylpyridinium chloride) and anionic quencher (KI) support the interpretation of the three fluorescence components within a conformer model. Operator and atc binding change the ratio of the relative amplitudes of the medium- and long-lived component, thus pointing to structural changes as indicated also by the changes in decay time. Since the fluorescence decay is different between the free, atc- and operator-bound states we conclude that the protein structure is different in each of these three states. The fluorescence quenching constants reflect not only the variation in solvent exposure with position, but also the fact that the net surface charge in this region is negative, because the quenching constants by the cationic quencher are up to 10-fold higher. The atc fluorescence appears to decay monoexponentially with about the same decay time for all mutants, except W170, in which the trp residue sterically interferes with atc.

A series of cationic porphyrins with 1–4 positive charges are studied: mono(N-methyl-4-pyridyl)triphenylporphine chloride [Mono], cis(N-methyl-4-pyridyl)diphenylporphine chloride [Cis], tri(N-methyl-4-pyridyl)monophenylporphine chloride [Tri] and tetra(N-methyl-4-pyridyl)porphine chloride [Tetra]. Their photophysical properties are measured in small unilamellar vesicles and compared with those in homogeneous solution. Liposomes of l-α-dimyristoyl-phosphatidylcholine (100 nm diameter) and l-α-dipalmitoyl-phosphatidylcholine (50 nm diameter) in phosphate-buffered saline (pH = 7.4) or D₂O 0.15 M NaCl were used. The effect of the medium microheterogeinity is discussed. The triplet quantum yields in liposomes for all the porphyrins are about 0.7, similar to the value obtained for Tetra in aqueous media. The singlet molecular oxygen quantum yields for the hydrophilic compounds Tri and Tetra are greater than those of the hydrophobic ones, Mono and Cis. Also, association constants (K_L) of the dyes to liposomes and their localization within the membranes are determined from fluorescence and fluorescence polarization measurements, respectively. K_L values are in the range of 10⁴–10⁵ M⁻¹ for all the compounds, indicating that hydrophobic and coulombic interactions between porphyrins and liposomes are responsible for the dye association. Fluorescence polarization experiments indicate that Mono and Cis can penetrate into the lipidic phase, and that Tri and Tetra are located near the polar heads of the lipidic molecules.

Liposomes containing high concentrations of the anticancer drug doxorubicin, prepared by active-loading techniques, have been intensively investigated as potential agents for chemotherapy. The present study investigates the possibility of active uptake and photoinduced release of such solutes from liposomes incorporating a photoisomerizable lipid. The active loading of acridine orange and doxorubicin was investigated using liposomes containing entrapped ammonium sulfate. The liposomes were prepared with dipalmitoyl-l-α-phosphatidyl choline (DPPC) and a photochromic lipid, (1,2-(4′-n-butylphenyl)azo-4′-(γ-phenylbutyroyl))-glycero-3-phosphocholine (Bis-Azo PC), which isomerizes on exposure to near-UV light with resulting changes in membrane permeability to solutes. The rate of loading of the vesicles below the phase transition temperature of DPPC was investigated as a function of Bis-Azo PC and cholesterol concentrations in the liposome. The rate of doxorubicin uptake was found to be greatly decreased in the presence of cholesterol, while below 30°C the rate of acridine orange uptake was increased in the presence of cholesterol. On exposure to a single UV laser pulse, actively loaded acridine orange was rapidly released from liposomes containing Bis-Azo PC at a rate similar to that found for the indicator dye calcein. However while cholesterol had previously been shown to greatly enhance the rate of photoinduced calcein leakage, it had no significant effect on the rate of acridine orange release. After active loading into DPPC vesicles containing Bis-Azo PC, doxorubicin was also released after exposure to a single laser pulse, but at a rate slower than for acridine orange and calcein. The difference in behavior between these systems is ascribed to the interactions of acridine orange and doxorubicin with the liposome bilayer. Photoinduced release of pharmacologically active materials from sensitized liposomes might provide a useful adjunct or alternative to conventional photodynamic therapy.

A model of UV-induced DNA damage in oceanic bacterioplankton was developed and tested against previously published and novel measurements of cyclobutane pyrimidine dimers (CPD) in surface layers of the ocean. The model describes the effects of solar irradiance, wind-forced mixing of bacterioplankton and optical properties of the water on net DNA damage in the water column. The biological part includes the induction of CPD by UV radiation and repair of this damage through photoreactivation and excision. The modeled damage is compared with measured variability of CPD in the ocean: diel variation in natural bacterioplankton communities at the surface and in vertical profiles under different wind conditions (net damage as influenced by repair and mixing); in situ incubation of natural assemblages of bacterioplankton (damage and repair, no mixing); and in situ incubation of DNA solutions (no repair, no mixing). The model predictions are generally consistent with the measurements, showing similar patterns with depth, time and wind speed. A sensitivity analysis assesses the effect on net DNA damage of varying ozone thickness, colored dissolved organic matter concentration, chlorophyll concentration, wind speed and mixed layer depth. Ozone thickness and mixed layer depth are the most important factors affecting net DNA damage in the mixed layer. From the model, the total amplification factor (TAF; a relative measure of the increase of damage associated with a decrease in ozone thickness) for net DNA damage in the euphotic zone is 1.7, as compared with 2.1–2.2 for irradiance weighted for damage to DNA at the surface.

The effect of leaf temperature (T), between 23 and 4°C, on the chlorophyll (Chl) fluorescence spectral shape was investigated under moderate (200 μE m⁻² s⁻¹) and low (30–35 μE m⁻² s⁻¹) light intensities in Phaseolus vulgaris and Pisum sativum. With decreasing temperature, an increase in the fluorescence yield at both 685 and 735 nm was observed. A marked change occurred at the longer emission band resulting in a decrease in the Chl fluorescence ratio, F685/F735, with reducing T. Our fluorescence analysis suggests that this effect is due to a temperature-induced state 1–state 2 transition that decreases and increases photosystem II (PSII) and photosystem I (PSI) fluorescence, respectively. Time-resolved fluorescence lifetime measurements support this interpretation. At a critical temperature (about 6°C) and low light intensity a sudden decrease in fluorescence intensity was observed, with a larger effect at 685 than at 735 nm. This is probably linked to a modification of the thylakoid membranes, induced by chilling temperatures, which can alter the spillover from PSII to PSI. The contribution of photosystem I to the long-wavelength Chl fluorescence band (735 nm) at room temperature was estimated by both time-resolved fluorescence lifetime and fluorescence yield measurements at 685 and 735 nm. We found that PSI contributes to the 735 nm fluorescence for about 40, 10 and 35% at the minimal (F₀), maximal (F_m) and steady-state (F_s) levels, respectively. Therefore, PSI must be taken into account in the analysis of Chl fluorescence parameters that include the 735 nm band and to interpret the changes in the Chl fluorescence ratio that can be induced by different agents.

The spectroscopic properties of photoactive (i.e. flash-transformable) and nonphotoactive protochlorophyll(ide)s (Pchl(ide)) were reinvestigated during the development of bean leaves in darkness. Two phases in the process of Pchl(ide) accumulation were apparent from quantitative measurements of pigment content: a lag phase (first week) during which photoactive Pchl(ide) accumulated faster than nonphotoactive Pchl(ide); and a fast phase (second week), showing parallel accumulation of both types of Pchl(ide). ‘Flashed-minus-dark’ absorbance difference spectra recorded in situ at 77 K showed that P650–655 was the predominant form of photoactive protochlorophyllide regardless of developmental stage. Quantitative analysis of energy migration processes between the Pchl(ide) forms showed the existence of energy transfer units containing a 1:8 ratio of nonphotoactive and photoactive Pchl(ide)s during development. Gaussian deconvolution of in situ 77 K fluorescence spectra indicated that the 633 nm band of nonphotoactive Pchl(ide) was made of four bands, at 625, 631, 637 and 643 nm, whose relative amplitudes only slightly changed during development. The emission band of photoactive Pchlide was also analyzed using the same method. Three components were found at 644, 652 and 657 nm. The emission band of P650–655 included the last two components, which become predominant only in fully etiolated plants. Photoactive Pchlide with an emission maximum at 653 nm was detected in the light during development of leaves of photoperiodically grown plants.

We present in vivo fluorescent, near-infrared (NIR), reflectance images of indocyanine green (ICG) and carotene-conjugated 2-devinyl-2-(1-hexyloxyethyl) pyropheophorbide (HPPH-car) to discriminate spontaneous canine adenocarcinoma from normal mammary tissue. Following intravenous administration of 1.0 mg kg⁻¹ ICG or 0.3 mg kg⁻¹ HPPH-car into the canine, a 25 mW, 778 nm or 70 mW, 660 nm laser diode beam, expanded by a diverging lens to approximately 4 cm in diameter, illuminated the surface of the mammary tissue. Successfully propagating to the tissue surface, ICG or HPPH-car fluorescence generated from within the tissue was collected by an image-intensified, charge-coupled device camera fitted with an 830 or 710 nm bandpass interference filter. Upon collecting time-dependent fluorescence images at the tissue surface overlying both normal and diseased tissue volumes, and fitting these images to a pharmacokinetic model describing the uptake (wash-in) and release (wash-out) of fluorescent dye, the pharmacokinetics of fluorescent dye was spatially determined. Mapping the fluorescence intensity owing to ICG indicates that the dye acts as a blood pool or blood persistent agent, for the model parameters show no difference in the ICG uptake rates between normal and diseased tissue regions. The wash-out of ICG was delayed for up to 72 h after intravenous injection in tissue volumes associated with disease, because ICG fluorescence was still detected in the diseased tissue 72 h after injection. In contrast, HPPH-car pharmacokinetics illustrated active uptake into diseased tissues, perhaps owing to the overexpression of LDL receptors associated with the malignant cells. HPPH-car fluorescence was not discernable after 24 h. This work illustrates the ability to monitor the pharmacokinetic delivery of NIR fluorescent dyes within tissue volumes as great as 0.5–1 cm from the tissue surface in order to differentiate normal from diseased tissue volumes on the basis of parameters obtained from the pharmacokinetic models.

There is no satisfactory mechanism to detect premalignant lesions in the upper aero-digestive tract. Fluorescence spectroscopy has potential to bridge the gap between clinical examination and invasive biopsy; however, optimal excitation wavelengths have not yet been determined. The goals of this study were to determine optimal excitation–emission wavelength combinations to discriminate normal and precancerous/cancerous tissue, and estimate the performance of algorithms based on fluorescence. Fluorescence excitation–emission matrices (EEM) were measured in vivo from 62 sites in nine normal volunteers and 11 patients with a known or suspected premalignant or malignant oral cavity lesion. Using these data as a training set, algorithms were developed based on combinations of emission spectra at various excitation wavelengths to determine which excitation wavelengths contained the most diagnostic information. A second validation set of fluorescence EEM was measured in vivo from 281 sites in 56 normal volunteers and three patients with a known or suspected premalignant or malignant oral cavity lesion. Algorithms developed in the training set were applied without change to data from the validation set to obtain an unbiased estimate of algorithm performance. Optimal excitation wavelengths for detection of oral neoplasia were 350, 380 and 400 nm. Using only a single emission wavelength of 472 nm, and 350 and 400 nm excitation, algorithm performance in the training set was 90% sensitivity and 88% specificity and in the validation set was 100% sensitivity, 98% specificity. These results suggest that fluorescence spectroscopy can provide a simple, objective tool to improve in vivo identification of oral cavity neoplasia.

Subcellular localization of photosensitizers is thought to play a critical role in determining the mode of cell death after photodynamic treatment (PDT) of leukemia cells. Using confocal laser scanning microscopy and fluorescent organelle probes, we examined the subcellular localization of merocyanine 540 (MC540) in the murine myeloid leukemia M1 and WEHI 3B (JCS) cells. Two patterns of localization were observed: in JCS cells, MC540 was found to localize on the plasma membrane and mitochondria; and in M1 leukemia cells, MC540 was found to localize on lysosomes. The relationship between subcellular localization of MC540 and PDT-induced apoptosis was investigated. Apoptotic cell death, as judged by the formation of apoptotic nuclei, was observed 4 h after irradiation in both leukemia cell lines. Typical ladders of apoptotic DNA fragments were also detected by DNA gel electrophoresis in PDT-treated JCS and M1 cells. At the irradiation dose of 46 kJ/m² (LD₉₀ for JCS and LD₈₆ for M1 cells), the percentage of apoptotic JCS and M1 cells was 78 and 38%, respectively. This study provided substantial evidence that MC540 localized differentially in the mitochondria, and the subsequent photodamage of the organelle played an important role in PDT-mediated apoptosis in myeloid leukemia cells.

Lutetium (III) texaphyrin photosensitizes postirradiation or “delayed” photohemolysis (DPH) of human and bovine red blood cells at 730 nm by a Type-2 pathway mediated by singlet molecular oxygen. The DPH rate increases with increasing incubation temperature and with the second power of the incident fluence. The experimental DPH curves are in good agreement with a multihit kinetics model based on target theory.

The response of human glioma spheroids to 5-aminolevulinic acid (ALA)–mediated photodynamic therapy (PDT) is investigated. A two-photon fluorescence microscopy technique is used to show that human glioma cells readily convert ALA to protoporphyrin IX throughout the entire spheroid volume. The central finding of this study is that the response of human glioma spheroids to ALA-mediated PDT depends not only on the total fluence, but also on the rate at which the fluence is delivered. At low fluences (≤50 J cm⁻²), lower fluence rates are more effective. At a fluence of 50 J cm⁻², near-total spheroid kill is observed at fluence rates of as low as 10 mW cm⁻². The fluence rate effect is not as pronounced at higher fluences (>50 J cm⁻²), where a favorable response is observed throughout the range of fluence rates investigated. The clinical implications of these findings are discussed.

We have recently shown that UVB radiation activates epidermal growth factor receptor (EGFR)/extracellular regulated kinase 1 and 2 (ERK1/2) and p38 signaling pathways in keratinocytes. However, the functional relevance of these processes for downstream signaling and cell survival remains to be determined. The specific EGFR inhibitor PD153035 markedly decreased UVB-induced phosphorylation of EGFR, ERK1/2 and shc, whereas p38 activation was unaffected. PD153035 pretreatment followed by UVB reduced clonogenic potential and enhanced peroxide production, apoptosis and cell death. Our data suggest that ligand-independent phosphorylation of EGFR and likely dependent downstream signaling pathways regulate cellular defense mechanisms important for cell survival following oxidative stress.

Phoborhodopsin (pR or sensory rhodopsin II, sRII) or pharaonis phoborhodopsin (ppR or pharaonis sensory rhodopsin II, psRII) has a unique absorption maximum (λ_max) compared with three other archaeal rhodopsins: λ_max of pR or ppR at ca 500 nm and others at 560–590 nm. Alignment of amino acid sequences revealed three sites characteristic of the shorter wavelength–absorbing pigments. The amino acids of these three sites are conserved completely among archaeal rhodopsins having longer λ_max, and are different from those of pR or ppR. We replaced these amino acids of ppR with amino acids corresponding to those of bacteriorhodopsin, Val-108 to Met, Gly-130 to Ser and Thr-204 to Ala. The λ_max of V108M mutant was 502 nm with a slight redshift. G130S and T204A mutants had λ_max of 503 and 508 nm, respectively. Thus, each site contributes only a small effect to the color tuning. We then constructed three double mutants and one triple mutant. The opsin-shifts of these mutants suggest that Val-108 and Thr-204 or Gly-130 are synergistic, and that Gly-130 and Thr-204 work additively. Even in the triple mutant, the λ_max was 515 nm, an opsin-shift only ca 30% of the shift value from 500 to 560 nm. This means that there is another yet unidentified factor responsible for the color tuning.

Raman spectroscopy (RS) has potential for disease classification within the gastrointestinal tract (GI). A near-infrared (NIR) fiber-optic RS system has been developed previously. This study reports the first in vivo Raman spectra of human gastrointestinal tissues measured during routine clinical endoscopy. This was achieved by using this system with a fiber-optic probe that was passed through the endoscope instrument channel and placed in contact with the tissue surface. Spectra could be obtained with good signal-to-noise ratio in 5 s. The effects on the spectra of varying the pressure of the probe tip on the tissue and of the probe-tissue angle were determined and shown to be insignificant. The limited set of spectra from normal and diseased tissues revealed only subtle differences. Therefore, powerful spectral-sorting algorithms, successfully implemented in prior ex vivo studies, are required to realize the full diagnostic potential of RS for tissue classification in the GI.