SEARCH · PubMed Health

Results for “Photobleaching”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources

PubMed1993

Do photobleached fluorescent microtubules move?: re-evaluation of fluorescence laser photobleaching both in vitro and in growing Xenopus axon.

We previously documented differences in the behavior of microtubules in growing axons of two types of neurons, adult mouse sensory neurons and Xenopus embryonal spinal cord neurons. Namely, the bulk of microtubules was stationary in mouse sensory neurons both by the method of photoactivation of caged-fluorescein-labeled tubulin and photobleaching of fluorescein-labeled tubulin, but the bulk of microtubules did translocate anterogradely by the method of photoactivation. Although these results indicated that the stationary nature of photobleached microtubules in mouse neurons is not an artifact derived from the high levels of energy required for the procedure, it has not yet been settled whether the photobleaching method can detect the movement of microtubules properly. Here we report photobleaching experiments on growing axons of Xenopus embryonal neurons. Anterograde movement of photobleached microtubules was observed at a frequency and translocation rate similar to the values determined by the method of photoactivation. Our results suggest that, under appropriate conditions, the photobleaching method is able to reveal the behavior of microtubules as accurately as the photoactivation method.

Animals↗

PubMed1989

Photoactive yellow protein from the purple phototrophic bacterium, Ectothiorhodospira halophila. Quantum yield of photobleaching and effects of temperature, alcohols, glycerol, and sucrose on kinetics of photobleaching and recovery.

A water-soluble yellow protein from E. halophila was previously shown to be photoactive (Meyer, T. E., E. Yakali, M. A. Cusanovich, and G. Tollin. 1987. Biochemistry. 26:418-423). Pulsed laser excitation in the protein visible absorption band (maximum at 445 nm) causes a rapid bleach of color (k = 7.5 x 10(3) s-1) followed by a slower dark recovery (k = 2.6 s-1). This is analogous to the photocycle of sensory rhodopsin II from Halobacterium (which also has k = 2.6 s-1 for recovery). We have now determined the quantum yield of the photobleaching process to be 0.64, which is comparable with that of bacteriorhodopsin (0.25), and is thus large enough to be biologically significant. Although the photoreactions of yellow protein were previously shown to be relatively insensitive to pH, ionic strength and the osmoregulator betaine, the present experiments demonstrate that temperature, glycerol, sucrose, and various alcohol-water mixtures strongly influence the kinetics of photobleaching and recovery. The effect of temperature follows normal Arrhenius behavior for the bleach reaction (Ea = 15.5 kcal/mol). The rate constant for the recovery reaction increases with temperature between 5 degrees C and 35 degrees C, but decreases above 35 degrees C indicating alternate conformations with differing kinetics. There is an order of magnitude decrease in the rate constant for photobleaching in both glycerol and sucrose solutions that can be correlated with the changes in viscosity. We conclude from this that the protein undergoes a conformational change as a consequence of the photoinduced bleach. Recovery kinetics are affected by glycerol and sucrose to a much smaller extent and in a more complicated manner. Aliphatic, monofunctional alcohol-water solutions increase the rate constant for the bleach reaction and decrease the rate constant for the recovery reaction, each by an order of magnitude. These effects do not correlate with dielectric constant, indicating that the photocycle probably does not involve separation or recombination of charge accessible to the protein surface. However, the effects on both bleaching and recovery correlate well with the relative hydrophobicity(as measured by partition coefficients in detergent/water mixtures), in the order of increasing effectiveness:methanol < ethanol < iso-propanol <n-propanol < n-butanol. We conclude that the change in conformation of the protein induced by light exposes a hydrophobic site to the solvent. This suggests the possibility that light exerts its effect in vivo by exposing a region of the protein for binding to a hydrophobic receptor site in the cell, perhaps to a protein analogous to the chemotactic transducers in the cytoplasmic membranes of enteric bacteria.

Alcohols↗

PubMed1985

Digital imaging fluorescence microscopy: spatial heterogeneity of photobleaching rate constants in individual cells.

Photobleaching and related photochemical processes are recognized experimental barriers to quantification of fluorescence by microscopy. We have measured the kinetics of photobleaching of fluorophores in living and fixed cells and in microemulsions, and have demonstrated the spatial variability of these processes within individual cells. An inverted fluorescence microscope and a high-sensitivity camera, together with high-speed data acquisition by a computer-controlled image processor, have been used to control precisely exposure time to excitation light and to record images. To improve the signal-to-noise ratio, 32 digital images were integrated. After correction for spatial variations in camera sensitivity and background fluorescence, the images of the relative fluorescence intensities for 0.065 micron2 areas in the object plane were obtained. To evaluate photobleaching objectively, an algorithm was developed to fit a three-parameter exponential equation to 20 images recorded from the same microscope field as a function of illumination time. The results of this analysis demonstrated that the photobleaching process followed first-order reaction kinetics with rate constants that were spatially heterogeneous and varied, within the same cell, between 2- and 65-fold, depending on the fluorophore. The photobleaching rate constants increased proportionally with increasing excitation intensity and, for benzo(a)pyrene, were independent of probe concentration over three orders of magnitude (1.25 microM to 1.25 mM). The propensity to photobleach was different with each fluorophore. Under the cellular conditions used in these studies, the average rates of photobleaching decreased in this order: N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol greater than acridine orange greater than rhodamine-123 greater than benzo(a)pyrene greater than fluorescein greater than tetramethylrhodamine greater than 1,1'dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine. The photobleaching appears to be an oxidation reaction, in that the addition of saturated solutions of Na2S2O5 to mineral oil microemulsions eliminated photobleaching of N-(7-nitrobenz-2-oxa-1,3-diazole)-23,24-dinor-5-cholen-22-amine-3 beta-ol or benzo(a)pyrene. We identified experimental conditions to observe, without detectable photobleaching, fluorophores in living cells, which can not be studied anaerobically. Useful images were obtained when excitation light was reduced to eliminate photobleaching, as determined from zero-time images calculated from the exponential fit routine.(ABSTRACT TRUNCATED AT 400 WORDS)

4-Chloro-7-nitrobenzofurazan↗

PubMed1992

Quantum yields and kinetics of the photobleaching of hematoporphyrin, Photofrin II, tetra(4-sulfonatophenyl)-porphine and uroporphyrin.

Porphyrins used as sensitizers for the photodynamic therapy (PDT) of tumors are progressively destroyed (photobleached) during illumination. If the porphyrin bleaches too rapidly, tumor destruction will not be complete. However, with appropriate sensitizer dosages and bleaching rates, irreversible photodynamic injury to the normal tissues surrounding the tumor, which retain less sensitizer, may be significantly decreased. This paper surveys the quantum yields and kinetics of the photobleaching of four porphyrins: hematoporphyrin (HP), Photofrin II (PF II), tetra(4-sulfonatophenyl)porphine (TSPP) and uroporphyrin I (URO). The initial quantum yields of photobleaching, as measured in pH 7.4 phosphate buffer in air, were: 4.7 x 10(-5), 5.4 x 10(-5), 9.8 x 10(-6), and 2.8 x 10(-5) for HP, PF II, TSPP and URO respectively; thus, the rates of photobleaching are rather slow. Low oxygen concentration (2 microM) significantly reduced the photobleaching yields. However, D2O increased the yields only slightly, and the singlet oxygen quencher, azide, had no effect, even at 0.1 M. Photosensitizing porphyrins in body fluids, cells and tissues may be closely associated with various photooxidizable molecules and electron acceptors and donors. Therefore, selected model compounds in these categories were examined for their effects on porphyrin photobleaching. A number inhibited and/or accelerated photobleaching, depending on the compound, the porphyrin and the reaction conditions. For example, 1.0 mM furfuryl alcohol increased the photobleaching yields of HP and URO more than 5-fold, with little effect on PF II or TSPP. In contrast, the electron acceptor, methyl viologen, increased the photobleaching yield of TSPP more than 10-fold, with little accelerating effect on the other porphyrins. These results suggest that the mechanism(s) of the photobleaching of porphyrin photosensitizers in cells and tissues during PDT may be complex.

Dihematoporphyrin Ether↗

PubMed1996

Reversible photobleaching of fluorescein conjugates in air-saturated viscous solutions: singlet and triplet state quenching by tryptophan.

Fluorescence recovery after photobleaching (FRAP) measurements on air-saturated aqueous solutions of fluorescein made viscous with glycerol or sucrose revealed a rapid component of fluorescence recovery with exponential time constants of 30-120 microseconds at viscosities of 15-300 cP. The rapid recovery process was not related to fluorophore translational diffusion and was insensitive to fluorophore concentration and the additive used to increase solution viscosity. At constant viscosity, the rate of reversible photobleaching recovery increased 2.5-fold in an O2- vs N2-saturated solution. The relative efficiency of reversible-to-irreversible photobleaching decreased with increasing photobleaching time and/or beam intensity. Reversible photobleaching was also detected for conjugates of fluorescein with dextrans and proteins in viscous media. In screening triplet state quenchers that might influence the reversible recovery, it was found that tryptophan enhanced the rate of reversible photobleaching recovery (two-fold increase at 8 mM) and quenched the fluorescein singlet state (Stern-Volmer constant, 12 M-1). Analysis of fluorescein lifetimes and photobleaching parameters for a series of fluorescein-labeled proteins with different numbers of tryptophans were also carried out. The results provide evidence for an oxygen-dependent, reversible photobleaching mechanism for the fluorescein chromophore involving triplet state relaxation. The identification of reversible fluorescein photobleaching has important implications for FRAP measurements of rapid solute diffusion in biological systems.

Fluoresceins↗

PubMed1998

Fluorescence photobleaching of ALA-induced protoporphyrin IX during photodynamic therapy of normal hairless mouse skin: the effect of light dose and irradiance and the resulting biological effect.

The photobleaching of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HR1 hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of photobleaching and on the corresponding PDT-induced damage were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photobleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photobleaching is not a simple function of fluence rate but is dependent on the initial concentration of sensitizer. Also, at high fluence rates (50-150 mW/cm2, 514 nm) oxygen depletion is shown to have a significant effect. The rate of photobleaching with respect to light dose and the corresponding PDT damage both increase with decreasing fluence rate. We therefore suggest that the definition of a bleaching dose as the light dose that causes a 1/e reduction in fluorescence signal is insufficient to describe the dynamics of photobleaching and PDT-induced damage. We have detected the formation of PpIX photoproducts during the initial period of irradiation that were themselves subsequently photobleached. In the absence of oxygen, PpIX and its photoproducts are not photobleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.

Aminolevulinic Acid↗

PubMed1993

Photobleaching of mono-L-aspartyl chlorin e6 (NPe6): a candidate sensitizer for the photodynamic therapy of tumors.

Most sensitizers used for the photodynamic therapy (PDT) of tumors photobleach on illumination. Thus, it is of interest to examine the photobleaching behavior of new sensitizers proposed for use in PDT. This report surveys the quantum yields and kinetics of the photobleaching of mono-L-aspartyl chlorin e6(NPe6), a hydrophilic chlorin that has many of the photoproperties desirable in a sensitizer for clinical PDT. It is a very effective sensitizer for the PDT of several types of model tumors in animals and is now in Phase I clinical trials. The quantum yield of NPe6 photobleaching in pH 7.4 phosphate buffer in air was 8.2 x 10(-4); this is greater than the yields for typical porphyrin photosensitizers. For example, the yields for hematoporphyrin and uroporphyrin are 4.7 x 10(-5) and 2.8 x 10(-5), respectively. The yield decreased significantly in organic solvents of low dielectric constant. The Sn derivative of NPe6 was more light stable than NPe6 (yield = 5.7 x 10(-6), while the Zn derivative was more sensitive (yield = 1.9 x 10(-2). Oxygen appeared to be necessary for the photobleaching of NPe6; however, bleaching was not inhibited by 100 mM azide, an efficient quencher of singlet oxygen. The photooxidizable substrates cysteine, dithiothreitol and furfuryl alcohol increased the quantum yield of photobleaching two- to four-fold, while the electron acceptor, metronidazole, increased it almost six-fold. Photobleaching yields for several other chlorins were also measured.

Animals↗

PubMed2001

Photobleaching of hypocrellin B and its butylamino-substituted derivative in solutions.

The photobleaching of hypocrellin B (HB) and its derivative butylamino-substituted hypocrellin B (BAHB), both of which are potent sensitizers for photodynamic therapy (PDT), were investigated by studies of absorption spectra and quenching experiments and by the determination of photoproducts. Control experiments indicated that the sensitizer, oxygen and light were essential for the photobleaching of HB and BAHB, which suggested that it was a photodynamic process, e.g. the photobleaching processes of both HB and BAHB were mainly self-sensitized photooxidations. The illumination of HB with visible light in oxygenated nonpolar solvents generated singlet oxygen efficiently [phi(1O2) = 0.76] which in turn attacked the sensitizer HB with the subsequent formation of an endoperoxide product. The endoperoxide of HB is unstable at room temperature and undergoes loss of singlet oxygen with regeneration of the parent HB. The singlet oxygen released from the endoperoxide of HB was detected with chemical trapping experiments. The quenching experiments indicated that in increasingly polar solvents the superoxide anion mechanism (type I) as well as the singlet oxygen mechanism (type II) contributed to the photobleaching of HB. The introduction of the electron-donating butylamino group not only enhanced the yield of the superoxide anion generation but also altered the position of attack in the BAHB molecule by the activated oxygen species. No endoperoxide product was observed, and no singlet oxygen released from the photobleaching process of BAHB was detected. The photobleaching process of BAHB was more complex. Both the singlet oxygen and superoxide anion mechanism played important roles in the photobleaching of BAHB in all organic solvent used here, even in aerobic nonpolar solvents such as CHCl3.

Electron Transport↗

PubMed1999

Photobleaching of arterial fluorescent compounds: characterization of elastin, collagen and cholesterol time-resolved spectra during prolonged ultraviolet irradiation.

To study the photobleaching of the main fluorescent compounds of the arterial wall, we repeatedly measured the time-resolved fluorescence of elastin, collagen and cholesterol during 560 s of excitation with nitrogen laser pulses. Three fluence rate levels were used: 0.72, 7.25 and 21.75 microW/mm2. The irradiation-related changes of the fluorescence intensity and of the time-resolved fluorescence decay constants were characterized for the emission at 390, 430 and 470 nm. The fluorescence intensity at 390 nm decreased by 25-35% when the fluence delivered was 4 mJ/mm2, a common value in fluorescence studies of the arterial wall. Cholesterol fluorescence photobleached the most, and elastin fluorescence photobleached the least. Photobleaching was most intense at 390 nm and least intense at 470 nm such that the emission spectra of the three compounds were markedly distorted by photobleaching. The time-resolved decay constants and the fluorescence lifetime were not altered by irradiation when the fluence was below 4 mJ/mm2. The spectral distortions associated with photobleaching complicate the interpretation of arterial wall fluorescence in terms of tissue content in elastin, collagen and cholesterol. Use of the time-dependent features of the emission that are not altered by photobleaching should increase the accuracy of arterial wall analysis by fluorescence spectroscopy.

Animals↗

PubMed1997

Influence of fluorochrome labeling density on the photobleaching kinetics of fluorescein in microscopy.

The objective of this study was to identify, through kinetic analysis of individual elementary reactions, the conditions under which a simple first-order photobleaching kinetic model is sufficient for quantitative fluorescence measurements, and those under which more complex photobleaching kinetics must be considered. Three model systems of various fluorophore densities and distributions were employed to verify the kinetic analysis. The results showed that the photobleaching kinetics of free fluorescein at concentrations lower than 5 microM corresponded closely to a single exponential function and therefore involved predominantly simple unimolecular or pseudounimolecular photochemical reactions. When fluorescein was bound to polyvinyl alcohol (PVA) molecules, the photobleaching kinetics of the densely labeled PVA deviated more from a single-exponential function than sparsely labeled PVA. When fluorescein was bound to a DNA probe, the photobleaching kinetics were more complex and deviated significantly from a single-exponential function, due to one or more bimolecular processes with apparent concentration-dependent photobleaching rate constants. The practical applications of time-integrated fluorescence emission are discussed in the context of simple and complex photobleaching kinetics.

Centromere↗

PubMed2002

High-order photobleaching of green fluorescent protein inside live cells in two-photon excitation microscopy.

Combination of green fluorescent protein (GFP) and two-photon excitation fluorescence microscopy (TPE) has been used increasingly to study dynamic biochemical events within living cells, sometimes even in vivo. However, the high photon flux required in TPE may lead to higher-order photobleaching within the focal volume, which would introduce misinterpretation about the fine biochemical events. Here we first studied the high-order photobleaching rate of GFP inside live cells by measuring the dependence of the photobleaching rate on the excitation power. The photobleaching rate under one- and two-photon excitation increased with 1-power and 4-power of the incident intensity, respectively, implying the excitation photons might interact with excited fluorophore molecules and increase the probability of photobleaching. These results suggest that in applications where two-photon imaging of GFP is used to study dynamic molecular process, photobleaching may ruin the imaging results and attention should be paid in interpreting the imaging results.

Diagnostic Imaging↗

PubMed2000

Photobleaching in two-photon excitation microscopy.

The intensity-squared dependence of two-photon excitation in laser scanning microscopy restricts excitation to the focal plane and leads to decreased photobleaching in thick samples. However, the high photon flux used in these experiments can potentially lead to higher-order photon interactions within the focal volume. The excitation power dependence of the fluorescence intensity and the photobleaching rate of thin fluorescence samples ( approximately 1 microm) were examined under one- and two-photon excitation. As expected, log-log plots of excitation power versus the fluorescence intensity and photobleaching rate for one-photon excitation of fluorescein increased with a slope of approximately 1. A similar plot of the fluorescence intensity versus two-photon excitation power increased with a slope of approximately 2. However, the two-photon photobleaching rate increased with a slope > or =3, indicating the presence of higher-order photon interactions. Similar experiments on Indo-1, NADH, and aminocoumarin produced similar results and suggest that this higher-order photobleaching is common in two-photon excitation microscopy. As a consequence, the use of multi-photon excitation microscopy to study thin samples may be limited by increased photobleaching.

Biophysical Phenomena↗

PubMed1995

Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy.

An investigation on the photobleaching behavior of fluorescein in microscopy was carried out through a systematic analysis of photobleaching mechanisms. The individual photochemical reactions of fluorescein were incorporated into a theoretical analysis and mathematical simulation to study the photochemical processes leading to photobleaching of fluorescein in microscopy. The photobleaching behavior of free and bound fluorescein has also been investigated by experimental means. Both the theoretical simulation and experimental data show that photobleaching of fluorescein in microscopy is, in general, not a single-exponential process. The simulation suggests that the non-single-exponential behavior is caused by the oxygen-independent, proximity-induced triplet-triplet or triplet-ground state dye reactions of bound fluorescein in microscopy. The single-exponential process is a special case of photobleaching behavior when the reactions between the triplet dye and molecular oxygen are dominant.

Fluorescein↗

PubMed1997

Mathematical description of photobleaching in vivo describing the influence of tissue optics on measured fluorescence signals.

The observed decrease in the fluorescence signal during photodynamic therapy (PDT) may contain dosimetric information as this photobleaching provides direct information on the photodynamic processes occurring in the tissue. A correct interpretation of the photobleaching signal, however, is crucial for its use in dosimetry. In this study the influence of scattering and absorption phenomena in tissue on the emitted fluorescence signal are described mathematically. Analytical solutions of the resulting expression show a difference from the single-decaying-exponential function generally used for describing photobleaching signals. The solutions are a function of the fluence rate at the inner side of tissue boundary psi(0*), the photobleaching dose constant beta, the incident irradiation power I0 and time. The accuracy of the results was investigated by comparison of the analytic solutions with numerical calculations using fluence rate profiles and escape functions obtained by Monte Carlo (MC) simulations. Good resemblance is observed when the value for psi(0*) calculated by the MC simulations is used in the analytical solutions. Experimental results in this study indicate the photobleaching dose constant of ALA-induced PpIX to be 33 +/- 3 J cm-2. Determination of beta for different types of photosensitizer and the development of an accurate method to determine psi(0*) can make monitoring of photobleaching during PDT valuable for dosimetry.

Aminolevulinic Acid↗

PubMed1999

Protoporphyrin IX fluorescence photobleaching during ALA-mediated photodynamic therapy of UVB-induced tumors in hairless mouse skin.

Fluorescence photobleaching of protoporphyrin IX (PpIX) during superficial photodynamic therapy (PDT), using 514 nm excitation, was studied in UVB-induced tumor tissue in the SKH-HR1 hairless mouse. The effects of different irradiance and light fractionation regimes upon the kinetics of photobleaching and the PDT-induced damage were examined. Results show that the rate of PpIX photobleaching (i.e., fluorescence intensity vs fluence) and the PDT damage both increase with decreasing irradiance. We have also detected the formation of fluorescent PpIX photoproducts in the tumor during PDT, although the quantity recorded is not significantly greater than generated in normal mouse skin, using the same light regime. The subsequent photobleaching of the photoproducts also occurs at a rate (vs fluence) that increases with decreasing irradiance. In the case of light fractionation, the rate of photobleaching increases upon renewed exposure after the dark period, and there is a corresponding increase in PDT damage although this increase is smaller than that observed with decreasing irradiance. The effect of fractionation is greater in UVB-induced tumor tissue than in normal tissue and the damage is enhanced when fractionation occurs at earlier time points. We observed a variation in the distribution of PDT damage over the irradiated area of the tumor: at high irradiance a ring of damage was observed around the periphery. The distribution of PDT damage became more homogeneous with both lower irradiance and the use of light fractionation. The therapeutic dose delivered during PDT, calculated from an analysis of the fluorescence photobleaching rate, shows a strong correlation with the damage induced in normal skin, with and without fractionation. The same correlation could be made with the data obtained from UVB-induced tumor tissue using a single light exposure. However, there was no such correlation when fractionation schemes were employed upon the tumor tissue.

Aminolevulinic Acid↗

PubMed1998

Singlet oxygen- versus nonsinglet oxygen-mediated mechanisms of sensitizer photobleaching and their effects on photodynamic dosimetry.

We report the effects of singlet oxygen (1O2) and non-1O2-mediated sensitizer photobleaching on oxygen consumption and dosimetry during photodynamic therapy (PDT) of sensitized multicell tumor spheroids. We develop a theoretical model for the description of non-1O2-mediated photobleaching resulting from irreversible reactions of the excited singlet or triplet sensitizer populations with cell substrate. We show that the fluence-dependent simple exponential decay expression of sensitizer degradation is not consistent with these mechanisms and, therefore, with any reasonable mechanism that we consider, because we have shown previously that 1O2-mediated photobleaching cannot be described by a simple exponential with a constant photobleaching coefficient (I. Georgakoudi et al., Photochem. Photobiol. 65, 135-144, 1997). Analysis of oxygen microelectrode measurements performed at the edge of Nile blue selenium (EtNBSe)- and protoporphyrin IX (PpIX)-sensitized spheroids during PDT demonstrates that the former drug photobleaches via a non-1O2-mediated mechanism, while the latter is degraded via a 1O2-mediated mechanism. Comparisons of the cytotoxic effects of EtNBSe with those of Photofrin (a drug that is degraded via a 1O2-mediated mechanism) indicate that the lower threshold 1O2 dose and the higher extinction coefficient and 1O2 yield for EtNBSe do not necessarily result in improved photodynamic effects, thus emphasizing the importance of the sensitizer photobleaching mechanism for dosimetry.

Animals↗

PubMed2002

The photobleaching of disulfonated aluminium phthalocyanine in microbial systems.

Front-face illumination spectroscopic techniques were used to study photobleaching of disulfonated aluminium phthalocyanine (AlPcS2) in the bacteria and yeast cells Escherichia coil, Porphyromonas gingivalis (Gram-negative bacteria), Streptococcus mutans (a Gram-positive bacterium), and the yeast Candida albicans. The photobleaching of AlPcS2 with the microbes studied is complex, involving reactive species generated by type I and type II processes with the rate dependent on the relative contributions of the different photobleaching mechanisms. The average rate of photobleaching of the AlPcS2 monomer in the presence of both E. coli and C. albicans increases as the fluence of the illumination source decreases. The effects of oxygen and sensitiser concentration on the rate of AlPcS2 photobleaching in these systems are also investigated.

Candida albicans↗

PubMed2002

Studies on singlet oxygen formation and UVA light-mediated photobleaching of the yellow chromophores in human lenses.

The protein-bound chromophores, which increase with aging in the human lens, act as UVA sensitizers, producing almost exclusively singlet oxygen in vitro. Direct irradiation of whole, aged human lenses with high intensity UVA light (200 mW cm(-2) for 24 hr), however, failed to produce singlet oxygen damage, as evidenced by the lack of either His or Trp photodestruction. Total homogenates of human lenses prepared in a cuvette under air did show destruction of His and Trp residues by UVA light, but no destruction was seen when equivalent homogenates were prepared under argon. These data are consistent with the idea that the low oxygen levels in the lens prevent singlet oxygen damage in vivo.UVA irradiation of aged human lenses in culture caused an extensive photobleaching of the yellow chromophores. A time course indicated that the photobleaching increased with time, with significant color loss apparent after 6 hr. Homogenization of the irradiated and dark control lenses in 6 M guanidine-HCl, followed by determination of the difference spectrum, showed approximately 50% bleaching of compounds with a lambda(max) at 355 nm. Similarly, fluorophores with a lambda(max) for excitation of 355 nm and for emission of 420 nm were 50% destroyed by the UVA light. Similar results were obtained in vitro by the anaerobic irradiation of a sonication-solubilized WI fraction from type II brunescent cataracts and from aged human lenses. In this system, there was an initial bleaching of 15% after 30 min of irradiation, followed by a slow increase over the next 6 hr to a final bleaching of 30%. The addition of 1.0 m M ascorbic acid, but not 1.0 m M glutathione (GSH), increased the photobleaching to 60% under argon, and the loss of ascorbate could be detected under these anaerobic conditions. In the presence of air, UVA light produced no photobleaching, but rather caused a three-fold increase in absorbance at 345 nm, which was prevented by the inclusion of 1.0 m M ascorbic acid and almost 50% inhibited by 1.0 m M GSH. The data are consistent with the conversion of the triplet state of the sensitizers to anion and cation radicals in the absence of oxygen. Photobleaching may occur either by dismutation of the anion radical or by reduction of the anion radical by ascorbate via type I chemistry. UVA irradiation of an enriched fraction of sensitizers from a proteolytic digest from type II cataract lenses produced a 63% bleaching at 330 nm in the absence of oxygen, and the almost complete loss of the A(330) absorbing and 350/450 nm fluorescent peaks upon HPLC separation. This loss correlated with the loss of the ability of the irradiated fraction to produce singlet oxygen in vitro upon subsequent UVA irradiation.

Aged↗