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PubMed2003

Electroretinographic evidence for altered phototransduction gain and slowed recovery from photobleaches in albino mice with a MET450 variant in RPE65.

Our purpose was to investigate the physiological phenotype of albino mice with a variation in the Rpe65 gene encoding either methionine or leucine at amino acid #450. Full-field electroretinograms (ERGs) were recorded from C57BL/6J-c(2J) albino mice with MET450 and BALB/cByJ albino mice with LEU450. Recordings from pigmented mice (C57BL/6J) served as controls. Rod ERG a-waves were fitted with a model to estimate parameters of activation. Recovery of function following a photobleach was studied by monitoring the return to pre-bleach a- or b-wave amplitudes of the dark-adapted electroretinogram. The parameter, S, derived from the fit of the rod model, was significantly higher for albino mice compared to pigmented controls. Between the albino mice, S was highest for BALB/cByJ compared to C57BL/6J-c(2J). The parameters t(d) and Rm(P3) were not different across the three strains. The difference in S between the BALB/cByJ and C57BL/6J-c(2J) albino strains is interpreted to reflect differences in intrinsic phototransduction gain. Recovery from a photobleach was also slower for the C57BL/6J-c(2J) albino mice compared with BALB/cByJ albino mice, consistent with prior studies showing slowed rhodopsin regeneration in mice with the RPE65-METH450 variant. ERG recordings show that C57BL/6J-c(2J) albino mice with the MET450 variant of the RPE65 protein have a lower gain of activation and slower recovery from photobleach than do the BALB/cByJ albino mice with LEU450. Both the slower recovery from photobleach and lower gain of activation characteristic of the C57BL/6J-c(2J) strain may contribute to the mechanism by which it is protected from light-induced photoreceptor death relative to BALB/c.

Albinism, Ocular↗
PubMed2004

Photobleaching of photosynthetic pigments in spinach thylakoid membranes. Effect of temperature, oxygen and DCMU.

The time dependence of photobleaching of photosynthetic pigments under high light illumination of isolated spinach thylakoid membranes at 22 and 4 degrees C was investigated. At 22 degrees C, the bleaching at 678, 472 and 436 nm was prominent but lowering the temperature up to 4 degrees C during illumination prevented the pigments from bleaching almost completely. The accelerating effect on pigment photobleaching by the presence of 3-(3,4 dichlorophenyl)-1,1-dimethyl-urea)-(DCMU), a well-known inhibitor of the electron transport and known to prevent photosystem I (PSI) and photosystem II (PSII) against photoinhibitory damage, was also suppressed at low temperature. At 22 degrees C in the presence and absence of DCMU, the decrease of the absorption at 678 and 472 nm was accompanied by a shift to the shorter wavelengths. To check the involvement of reactive oxygen species in the process, pigment photobleaching was followed in anaerobiosis. The effects of the three different environmental factors--light, temperature and DCMU--on the dynamics of photobleaching are discussed in terms of different susceptibility of the main pigment-protein complexes to photoinhibition.

Aerobiosis↗
PubMed2004

Photobleaching, mobility, and compartmentalisation: inferences in fluorescence correlation spectroscopy.

In living cells the transport and diffusion of molecules is constrained by compartments of various sizes. This paper is an attempt to show that the size of these compartments can in principle be estimated experimentally from Fluorescence Correlation Spectroscopy (FCS) combined with the measurement of the photobleaching rate. In this work, confocal fluorescence microscopy experiments have been carried out on giant unilamellar vesicles, a system that mimics cellular compartmentalisation. We have developed numerical and analytical models to describe the fluorescence decay due to photobleaching in this geometry, which has enabled us to point out two regimes depending on the value of the parameter P(B) = sigma(B)P/D (where sigma(B) is the photobleaching cross section of the dye, D its diffusion constant, and P the laser power (in photon/s)). In particular, when P(B) << 1 (i.e. in the fast diffusion regime), the photobleaching rate is independent of the diffusion constant and scales like sigma(B)P/R2, in agreement with the experimental results. On the other hand, the standard diffusion models used to analyse the FCS data do not take into account the effects of the fluorescence decay on the autocorrelation curve. We show here how to correct the raw data for these drawbacks.

Artifacts↗
PubMed2004

Photodynamic therapy of actinic keratosis at varying fluence rates: assessment of photobleaching, pain and primary clinical outcome.

BACKGROUND: Although photodynamic therapy (PDT) is becoming an important treatment method for skin lesions such as actinic keratosis (AK) and superficial basal cell carcinoma, there are still discussions about which fluence rate and light dose are preferable. Recent studies in rodents have shown that a low fluence rate is preferable due to depletion of oxygen at high fluence rates. However, these results have not yet been verified in humans. OBJECTIVES: The objective was to investigate the impact of fluence rate and spectral range on primary treatment outcome and bleaching rate in AK using aminolaevulinic acid PDT. In addition, the pain experienced by the patients has been monitored during treatment. PATIENTS/METHODS: Thirty-seven patients (mean age 71 years) with AK located on the head, neck and upper chest were treated with PDT, randomly allocated to four groups: two groups with narrow filter (580-650 nm) and fluence rates of 30 or 45 mW cm(-2), and two groups with broad filter (580-690 nm) and fluence rates of 50 or 75 mW cm(-2). The total cumulative light dose was 100 J cm(-2) in all treatments. Photobleaching was monitored by fluorescence imaging, and pain experienced by the patients was registered by using a visual analogue scale graded from 0 (no pain) to 10 (unbearable pain). The primary treatment outcome was evaluated at a follow-up visit after 7 weeks. RESULTS: Our data showed a significant correlation between fluence rate and initial treatment outcome, where lower fluence rate resulted in favourable treatment response. Moreover, the photobleaching dose (1/e) was found to be related to fluence rate, ranging from 4.5 +/- 1.0 J cm(-2) at 30 mW cm(-2), to 7.3 +/- 0.7 J cm(-2) at 75 mW cm(-2), indicating higher oxygen levels in tissue at lower fluence rates. After a cumulative light dose of 40 J cm(-2) no further photobleaching took place, implying that higher doses are excessive. No significant difference in pain experienced by the patients during PDT was observed in varying the fluence rate from 30 to 75 mW cm(-2). However, the pain was found to be most intense up to a cumulative light dose of 20 J cm(-2). CONCLUSIONS: Our results imply that the photobleaching rate and primary treatment outcome are dependent on fluence rate, and that a low fluence rate (30 mW cm(-2)) seems preferable when performing PDT of AK using noncoherent light sources.

Aged↗
PubMed2004

Photobleaching-corrected FRET efficiency imaging of live cells.

Fluorescent resonance energy transfer (FRET) imaging techniques can be used to visualize protein-protein interactions in real-time with subcellular resolution. Imaging of sensitized fluorescence of the acceptor, elicited during excitation of the donor, is becoming the most popular method for live FRET (3-cube imaging) because it is fast, nondestructive, and applicable to existing widefield or confocal microscopes. Most sensitized emission-based FRET indices respond nonlinearly to changes in the degree of molecular interaction and depend on the optical parameters of the imaging system. This makes it difficult to evaluate and compare FRET imaging data between laboratories. Furthermore, photobleaching poses a problem for FRET imaging in timelapse experiments and three-dimensional reconstructions. We present a 3-cube FRET imaging method, E-FRET, which overcomes both of these obstacles. E-FRET bridges the gap between the donor recovery after acceptor photobleaching technique (which allows absolute measurements of FRET efficiency, E, but is not suitable for living cells), and the sensitized-emission FRET indices (which reflect FRET in living cells but lack the quantitation and clarity of E). With E-FRET, we visualize FRET in terms of true FRET efficiency images (E), which correlate linearly with the degree of donor interaction. We have defined procedures to incorporate photobleaching correction into E-FRET imaging. We demonstrate the benefits of E-FRET with photobleaching correction for timelapse and three-dimensional imaging of protein-protein interactions in the immunological synapse in living T-cells.

Animals↗
PubMed2000

Reversible loss of crystallinity on photobleaching purple membrane in the presence of hydroxylamine.

Structural changes of purple membrane during photobleaching in the presence of hydroxylamine were monitored using atomic force microscopy (AFM). The process of bleaching was associated with the disassembly of the purple membrane crystal into smaller crystals. Imaging steps of the photobleaching progress showed that disassembly proceeds until the sample is fully bleached and its crystallinity is almost lost. As revealed from high resolution AFM topographs, the loss of crystallinity was initiated by loss of lattice forming contact between the individual bacteriorhodopsin trimers. The bacteriorhodopsin molecules, however, remained assembled into trimers during the entire photobleaching process. Regeneration of the photobleached sample into intact purple membrane resulted in the reassembly of the bacteriorhodopsin trimers into the trigonal lattice of purple membrane. The data provide novel insights into factors triggering purple membrane formation and structure.

Bacteriorhodopsins↗
PubMed1985

Characterization of the photosynthetic electron transport chain in normal and photobleached Anabaena cylindrica by flash spectroscopy.

Electron transport of normal and photobleached Anabaena cylindrica was studied using spectral and kinetic analyses of absorbance transients induced by single turnover flashes. Between 500 and 600 nm two positive bands (approximately 540 and approximately 566 nm) and two negative bands (approximately 515 and approximately 554 nm) were found. Absorbance changes at 515 and 540 nm were partly characterized. None of these absorbance changes represent an electrochromic shift. Absorbance changes at 554 and 566 nm correspond to the oxidation of cytochrome f and the reduction of cytochrome b563, respectively. We found a very slight 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) sensitivity of cytochrome f in normal cells, while DCMU was completely ineffective for cytochrome f reduction in photobleached cells. The absorbance change of cytochrome b563 increased, while the absorbance change of cytochrome f was smaller than in normal cells. The increased O2 evolution in photobleached cells and the negligible electron transport via cytochrome f suggest the participation of other electron acceptor(s) in the electron-transport chain of photobleached Anabaena cylindrica.

Cyanobacteria↗
PubMed1991

Photobleaching of astaxanthin and canthaxanthin. Quantum-yields dependence of solvent, temperature, and wavelength of irradiation in relation to packaging and storage of carotenoid pigmented salmonoids.

The quantum yield for the photobleaching of astaxanthin (the carotenoid of wild salmonoids) and of canthaxanthin (the closely related carotenoid used as a feeding additive for farmed salmonoids) has been determined for monochromatic light at different wavelengths and in different solvents. Astaxanthin is less sensitive to light than canthaxanthin. The photobleaching is strongly wavelength dependent, and the quantum yield for astaxanthin dissolved in chloroform at 22 degrees C is 3.2 x 10(-1) mol.Einstein-1 at 254 nm, 3.1 x 10(-2) at 313 nm, and 1.6 x 10(-6) at 436 nm, respectively. The quantum yields are less dependent on the nature of the solvent and show no simple correlation with oxygen solubility, i.e. for 366 nm excitation of astaxanthin the quantum yields are 6.1 x 10(-5) mol.Einstein-1 in acetone, 1.2 x 10(-4) in saturated vegetable oil, 1.9 x 10(-4) in chloroform, and 3.4 x 10(-4) solubilized in water, respectively. The photobleaching quantum yield provides an objective measure of the light sensitivity of the carotenoids in relation to the discolouration of carotenoid-pigmented salmonoids. The quantum yield was also found to be independent of the carotenoid concentration and, in a homogenous solution, of light intensities. For astaxanthin solubilized in water, the quantum yield increases for low light intensities. Excitation of astaxanthin solubilized in water using visible light shows that the photobleaching quantum yield is independent of temperature, while excitation at 313 nm shows an increase in the quantum yield with increasing temperatures, corresponding to an energy of activation of 28 kJ.mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1998

Interactions of Photobleaching and Inorganic Nutrients in Determining Bacterial Growth on Colored Dissolved Organic Carbon.

Abstract Bacteria are key organisms in the processing of dissolved organic carbon (DOC) in aquatic ecosystems. Their growth depends on both organic substrates and inorganic nutrients. The importance of allochthonous DOC, usually highly colored, as bacterial substrate can be modified by photobleaching. In this study, we examined how colored DOC (CDOC) photobleaching, and phosphorus (P) and nitrogen (N) availability, affect bacterial growth. Five experiments were conducted, manipulating nutrients (P and N) and sunlight exposure. In almost every case, nutrient additions had a significant, positive effect on bacterial abundance, production, and growth efficiency. Sunlight exposure (CDOC photobleaching) had a significant, positive effect on bacterial abundance and growth efficiency. We also found a significant, positive interaction between these two factors. Thus, bacterial use of CDOC was accelerated under sunlight exposure and enhanced P and N concentrations. In addition, the accumulation of cells in sunlight treatments was dependent on nutrient availability. More photobleached substrate was converted into bacterial cells in P- and N-enriched treatments. These results suggest nutrient availability may affect the biologically-mediated fate (new biomass vs respiration) of CDOC.

Journal Article↗
PubMed1994

Quantitation of fluorescence energy transfer between cell surface proteins via fluorescence donor photobleaching kinetics.

We describe practical aspects of photobleaching fluorescence energy transfer measurements on individual living cells. The method introduced by T. M. Jovin and co-workers (see, most recently, Kubitscheck et al. 1993. Biophys. J. 64:110) is based on the reduced rate of irreversible photobleaching of donor fluorophores when acceptor fluorophores are present. Measuring differences in donor photobleaching rates on cells labeled with donor only (fluorescein isothiocyanate-conjugated proteins) and with both donor and acceptor (tetramethylrhodamine-conjugated proteins) allows calculation of the fluorescence energy transfer efficiency. We assess possible methods of data analysis in light of the underlying processes of photobleaching and energy transfer and suggest optimum strategies for this purpose. Single murine B lymphocytes binding various ratios of donor and acceptor conjugates of tetravalent concanavalin A (Con A) and divalent succinyl Con A were examined for interlectin energy transfer by these methods. For Con A, a maximum transfer efficiency of 0.49 +/- 0.02 was observed. Under similar conditions flow cytometric measurements of donor quenching yielded a value of 0.54 +/- 0.03. For succinyl Con A, the maximum transfer efficiency was 0.36. To provide concrete examples of quantities arising in such energy transfer determinations, we present examples of individual cell data and kinetic analyses, population rate constant distributions, and error estimates for the various quantities involved.

Animals↗
PubMed1989

Interpretation of fluorescence correlation spectroscopy and photobleaching recovery in terms of molecular interactions.

The theoretical basis and experimental implementation of FCS and FPR measurements are now well established. Because of the requirements for system stability and long data acquisition times FCS is relatively rarely used. But FCS can provide unique information, especially about extents of aggregation or polymerization and therefore is a useful supplement to FPR for certain applications. FPR measurements are now carried out routinely in many laboratories in a variety of formats using different beam profiles, optical systems, and analytical schemes. A particular version may be better adapted to a specific application. The spot photobleaching approach, however, seems simplest and most versatile for cellular studies and is now most often used. Important experimental considerations in setting up a spot photobleaching instrument are discussed in detail in Chapter 10 by Wolf (this volume) and elsewhere (Petersen et al., 1986a). In interpreting FPR measurements it is also important to take into account the possibility of systematic errors from a number of sources. In Chapter 10 in this volume, Wolf discusses many factors that must be properly controlled in carrying out FPR measurements. Additional consideration of some of these points is presented by Petersen et al. (1986a). One potentially troublesome type of error arises from the possibility that chemical reactions initiated by the photobleaching pulse or during the measurement of recovery could significantly perturb the system. Evidence from a variety of sources [summarized, for example, in Petersen et al. (1986a)] indicates that photobleaching fluorophores can induce chemical cross-linking of cellular proteins under some conditions. But measurements in a number of different systems have demonstrated that, even if these types of reactions occur in FPR measurements, nevertheless they do not perturb the measured mobilities. If possible, however, this point should be checked for each new system because variations in structure or environmental conditions could enhance the chemical cross-linking reactions mediated by photogenerated free radicals. In practice, the principal difficulty in carrying out FPR measurements on cells is frequently the low intensity of the fluorescent signal which can be obtained from specifically labeled cell surface ligands or microinjected components. This low intensity results from the typically low capacity of an individual cell for the specifically labeled macromolecule. Even in the absence of systematic errors, low emission intensity will reduce the accuracy of measurements due to shot noise. This is an important practical limitation on measuring accuracy. Low measurement accuracy severely limits the extent to which the data can be interpreted mechanistically. Precision can be improved by averaging many recovery experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Diffusion↗
PubMed2002

Investigation of photobleaching of hypocrellin B in non-polar organic solvent and in liposome suspension.

Hypocrellin B (HB) is a natural pigment with a promising application in the photodynamic therapy (PDT) for anticancer treatment. The photobleaching of HB in non-polar organic solvents and in liposomes in aqueous solution were investigated by the measurements of absorption spectra, quenching experiments and determination of photoproducts. Control experiments indicated that the sensitizer, oxygen and light were all essential for the photobleaching of HB, which suggested that it was mainly self-sensitized photooxidation. The illumination of HB with visible light in aerobic non-polar solvent generated singlet oxygen efficiently [Phi(1O(2))=0.76] which then attacked the sensitizer HB with formation of an endoperoxide product. The endoperoxide of HB was unstable at room temperature and underwent predominantly loss of singlet oxygen with regeneration of parent HB. The singlet oxygen released from the endoperoxide of HB was detected with chemical trapping experiments. When HB was embedded in EPC liposomes, no endoperoxide product and no singlet oxygen release from the photobleaching process of HB were detected. The quenching experiments indicated that the singlet oxygen mechanism (type II) played an important role in the non-polar solvent and the free radical mechanism (type I) was predominant in liposomal aqueous solution for the photobleaching of HB.

Liposomes↗
PubMed2000

Monitoring electroosmotic flow by periodic photobleaching of a dilute, neutral fluorophore

Electroosmotic flow has been monitored in a capillary using a method based on periodic photobleaching of a neutral, fluorescent buffer additive. Rhodamine B was determined to be neutral between pH 6.0 and 10.8 and was added to the running buffer at a concentration of 400 nM. Rhodamine B was photobleached by opening a shutter under computer control for 250 ms every 5.00 s, to expose the dye to a laser beam and create a photobleached zone. The time was measured for the photobleached zone to migrate 6.13 mm to a downstream laser-induced fluorescence detector, to determine the rate of electroosmotic flow in the entire capillary. The flow rate was sampled every 5.00 s, and the precision of the flow measurements was 0.7% or better. Three fluorescent compounds were separated and detected by capillary electrophoresis with laser-induced fluorescence detection, while simultaneously monitoring the electroosmotic flow rate.

Journal Article↗
PubMed2002

Multiphoton versus confocal high resolution z-sectioning of enhanced green fluorescent microtubules: increased multiphoton photobleaching within the focal plane can be compensated using a Pockels cell and dual widefield detectors.

Multiphoton excitation was originally projected to improve live cell fluorescence imaging by minimizing photobleaching effects outside the focal plane, yet reports suggest that photobleaching within the focal plane is actually worse than with one photon excitation. We confirm that when imaging enhanced green fluorescent protein, photobleaching is indeed more acute within the multiphoton excitation volume, so that whilst fluorescence increases as predicted with the square of the excitation power, photobleaching rates increase with a higher order relationship. Crucially however, multiphoton excitation also affords unique opportunities for substantial improvements to fluorescence detection. By using a Pockels cell to minimize exposure of the specimen together with multiple nondescanned detectors we show quantitatively that for any particular bleach rate multiphoton excitation produces significantly more signal than one photon excitation confocal microscopy in high resolution Z-axis sectioning of thin samples. Both modifications are readily implemented on a commercial multiphoton microscope system.

Bacteria↗
PubMed2004

Nonexponential statistics of fluorescence photobleaching.

In this paper, I consider theoretical models of the decay via photobleaching of a sample of surface-immobilized fluorescent molecules excited by a spatially varying laser intensity profile. I show that, with mild restrictions on the photobleaching mechanism, the fluorescence decay measured in a nonuniform excitation profile is always nonexponential. Under the same conditions, the fluorescence decay can always be approximated by a discrete sum of exponentials. A particular example is given in which a homogeneous population of fluorophores with a single (intensity-dependent) photobleaching lifetime, when illuminated by a Gaussian laser, exhibits power law fluorescence decay at long times. These results indicate that the observation of multiple exponentials in single molecule or ensemble photobleaching lifetime measurements can arise solely as an artifact of a spatially varying laser profile and is not necessarily indicative of heterogeneity in molecular internal states, conformation, or local environment.

Journal Article↗
PubMed1996

Spectroscopic and biological testing of photobleaching of porphyrins in solutions.

The photobleaching of protoporhyrin IX (PP IX) and hematoporphyrin derivative (HpD) solutions was followed using three different methods: spectrophotometry, fluorometry and photodynamically induced cytotoxicity. The latter entails photoirradiation of HT29 human colon adenocarcinoma cells in the presence of preirradiated solutions of HpD and PP IX (lambda < or = 415 nm). The highest cytotoxicity was observed in the presence of unirradiated dye and decreased with the time of preirradiation. This decay in photocytotoxicity was further used to determine the porphyrin photobleaching kinetics in solution. For both sensitizers, quantum yields of photobleaching obtained by matching fluorescence were higher than that obtained from absorbance measurements (10 and 11 times for HpD and PP IX, respectively). This difference reflects preferential photobleaching of photolabile monomeric forms compared to aggregated. The highest quantum yield was obtained in the biological test (decay in cytotoxicity) which was 14 times higher for HpD and 30 times higher for PP IX than the quantum yield obtained from absorbance measurements. The absence of correlation between biological and fluorescence measurements has to be taken into account in the in vivo situation. Dark storage of preirradiated sensitizers (37 degrees C, 24 h) completely restored photocytotoxity for PP IX but only partially for HpD, whereas fluorescence patterns were partially restored for both sensitizers.

Fluorometry↗
PubMed2001

Effects of fluence rate on cell survival and photobleaching in meta-tetra-(hydroxyphenyl)chlorin-photosensitized Colo 26 multicell tumor spheroids.

We report the influence of fluence rate on the photobleaching and cell survival in Colo 26 multicell spheroids photosensitized by meta-tetra-(hydroxyphenyl)chlorin (mTHPC). Photosensitizer degradation and therapeutic efficacy increased dramatically and progressively when the fluence rate was reduced over the range from 90 to 5 mW cm-2. These experimental results were compared to a mathematical model of photobleaching based on self-sensitized singlet oxygen reactions with the photosensitizer ground state. This model incorporates photophysical parameters obtained from microelectrode measurements of oxygen depletion at the surface of mTHPC-sensitized spheroids and was refined by including the inhomogeneous distribution of mTHPC in spheroids and oxygen depletion in the bulk medium. Since the model is consistent with the experimental data we conclude that the fluence rate dependence of the cell survival and of mTHPC photobleaching is due to photochemical oxygen consumption and a predominantly singlet oxygen-mediated mechanism of mTHPC photobleaching. The threshold dose of reacting singlet oxygen was calculated to be 7.9 +/- 2.2 mM in this system.

Animals↗
PubMed2002

Intracellular photobleaching of 5,10,15,20-tetrakis(m-hydroxyphenyl) chlorin (Foscan) exhibits a complex dependence on oxygen level and fluence rate.

The understanding of photosensitizer photobleaching is important not only for mechanistic studies, but also for the development of monitoring techniques for clinical dosimetry in photodynamic therapy. In this study, we investigated the intracellular photobleaching of 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin (mTHPC, Foscan) in the murine macrophage cell line J774A.1, using quantitative fluorescence imaging microscopy, microspectrofluorometry and microspectrophotometry. Using 652 nm laser irradiation, it was found that mTHPC exhibits oxygen- and fluence rate-dependent intracellular photobleaching. The kinetics showed an inverse dose-rate behavior, i.e. a reduction of fluence rate resulted in more photobleaching at comparable fluences. The effect of deoxygenation was found to be more complex, with decreased bleaching at low fluence rates and increased bleaching at higher fluence rates. The intracellular formation of reactive oxygen species was measured using 2',7'-dichlorodihydrofluorescein diacetate. The results are analyzed in terms of competitive Type-I and Type-II mechanisms.

Animals↗