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Henripierre Lassalle - One of the best experts on this subject based on the ideXlab platform.
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photodegradation and phototransformation of 5 10 15 20 tetrakis m hydroxyphenyl bacteriochlorin m thpbc in solution
Photochemical and Photobiological Sciences, 2004Co-Authors: Henripierre Lassalle, Lina Bezdetnaya, Vladimir Iani, Asta Juzeniene, Francois Guillemin, Johan Emelian MoanAbstract:The kinetics of Photobleaching and formation of photoproducts upon irradiation (735 nm) of 5,10,15,20-tetrakis(m-hydroxyphenyl)bacteriochlorin (m-THPBC) in phosphate buffer saline (PBS) supplemented with human serum albumin (HSA) were studied by means of absorption and steady-state fluorescence spectroscopy. Measurements were performed either immediately after the dye was dissolved in the HSA solution (0 h) or after six hours incubation in the HSA solution (6 h). Spectroscopic studies indicated that the dye was mainly present as aggregates in freshly prepared solutions, whereas incubation favored monomerisation. Irrespective to incubation time, the rates of Photobleaching obtained by fluorescence measurements were higher than those obtained from absorbance measurements. Photobleaching of freshly prepared m-THPBC can be described by a single exponential decay, while the absorbance and fluorescence decays of the incubated dye solutions better fit a bi-exponential decay. Two Photobleaching rates probably reflect differences in the photosensitivity of monomer (bound to proteins) and aggregated (non-bound) forms. Irradiation of the freshly prepared m-THPBC solution led to phototransformation of 50% of the bleached m-THPBC into 5,10,15,20-tetrakis(m-hydroxyphenyl)chlorin (m-THPC), a clinically used second generation photosensitizer. For irradiation 6 h after dissolving m-THPBC, different kinetics of m-THPC formation were found. A rapid decrease in concentration of m-THPBC was accompanied by a slow formation of m-THPC. The quantum yield of this process was small since only 5% of m-THPBC was transformed to m-THPC. The kinetics characteristics of m-THPBC Photobleaching reported in the present study, together with the different kinetics of photoproduct formation during m-THPBC Photobleaching, may provide important indications in the m-THPBC-based PDT dosimetry.
Claus A. M. Seidel - One of the best experts on this subject based on the ideXlab platform.
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analysis of Photobleaching in single molecule multicolor excitation and forster resonance energy transfer measurements
Journal of Physical Chemistry A, 2006Co-Authors: Christian Eggeling, Jerker Widengren, Leif Brand, J Schaffer, Suren Felekyan, Claus A. M. SeidelAbstract:Dye Photobleaching is a major constraint of fluorescence readout within a range of applications. In this study, we investigated the influence of Photobleaching in fluorescence experiments applying multicolor laser as well as Forster resonance energy transfer (FRET) mediated excitation using several red-emitting dyes frequently used in multicolor experiments or as FRET acceptors. The chosen dyes (cyanine 5 (Cy5), MR121, Alexa660, Alexa680, Atto647N, Atto655) have chemically distinct chromophore systems and can be excited at 650 nm. Several fluorescence analysis techniques have been applied to detect Photobleaching and to disclose the underlying photophysics, all of which are based on single-molecule detection: (1) fluorescence correlation spectroscopy (FCS) of bulk solutions, (2) fluorescence cross-correlation of single-molecule trajectories, and (3) multiparameter fluorescence detection (MFD) of single-molecule events. The maximum achievable fluorescence signals as well as the survival times of the red dyes were markedly reduced under additional laser irradiation in the range of 500 nm. Particularly at excitation levels at or close to saturation, the 500 nm irradiation effectively induced transitions to higher excited electronic states on already excited dye molecules, leading to a pronounced bleaching reactivity. A theoretical model for the observed laser irradiance dependence of the fluorescence brightness of a Cy5 FRET acceptor dye has been developed introducing the full description of the underlying photophysics. The model takes into account acceptor as well as donor Photobleaching from higher excited electronic states, population of triplet states, and energy transfer to both the ground and excited states of the acceptor dye. Also, photoinduced reverse intersystem crossing via higher excited triplet states is included, which was found to be very efficient for Cy5 attached to DNA. Comparing continuous wave (cw) and pulsed donor excitation, a strong enhancement of acceptor Photobleaching by a factor of 5 was observed for the latter. Thus, in the case of fluorescence experiments utilizing multicolor pulsed laser excitation, the application of the appropriate timing of synchronized green and red laser pulses in an alternating excitation mode can circumvent excessive Photobleaching. Moreover, important new single-molecule analysis diagnosis tools are presented: (1) For the case of excessive acceptor Photobleaching, cross-correlation analysis of single- molecule trajectories of the fluorescence signal detected in the donor and acceptor detection channels and vice versa shows an anticorrelated exponential decay and growth, respectively. (2) The time difference, Tg - Tr, of the mean observation times of all photons detected for the donor and acceptor detection channels within a single-molecule fluorescence burst allows one to identify and exclude molecules with an event of acceptor Photobleaching. The presented single-molecule analysis methods can be constrained to, for example, FRET- active subpopulations, reducing bias from FRET-inactive molecules. The observations made are of strong relevance for and demand a careful choice of laser action in multicolor and FRET experiments, in particular when performed at or close to saturation.
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Photobleaching of fluorescent dyes under conditions used for single molecule detection evidence of two step photolysis
Analytical Chemistry, 1998Co-Authors: Christian Eggeling, Jerker Widengren, Rudolf Rigler, Claus A. M. SeidelAbstract:The photostability of fluorescent dyes is of crucial importance for the statistical accuracy of single-molecule detection (SMD) and for the image quality of scanning confocal microscopy. Concurrent results for the photostability were obtained by two different experimental techniques. First, the photostabilities of several coumarin and rhodamine derivatives in aqueous solution were obtained by monitoring the steady-state fluorescence decay in a quartz cell. Furthermore, an epi-illuminated microscope, continuous wave (CW) excitation at 514.5 nm, and fluorescence correlation spectroscopy (FCS) with a newly developed theory were used to study the Photobleaching characteristics of rhodamines under conditions used for SMD. Depending on the rhodamine structure, the probability of Photobleaching, pb, is in the order of 10-6−10-7 for irradiances below 103 W/cm2. However, a considerable increase of pb for irradiances above this level was observed which can only be described by Photobleaching reactions from higher e...
Willem M Star - One of the best experts on this subject based on the ideXlab platform.
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protoporphyrin ix fluorescence Photobleaching during ala mediated photodynamic therapy of uvb induced tumors in hairless mouse skin
Photochemistry and Photobiology, 1999Co-Authors: Dominic J Robinson, Henriette S De Bruijn, Nynke Van Der Veen, M R Stringer, Stanley B Brown, Willem M StarAbstract:— 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.
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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
Photochemistry and Photobiology, 1998Co-Authors: Dominic J Robinson, Henriette S De Bruijn, M R Stringer, Stanley B Brown, Nynke Van Der Veen, Willem M StarAbstract:The Photobleaching of 5-aminolaevulinic acid (ALA)-induced protoporphyrin IX (PpIX) was investigated during superficial photodynamic therapy (PDT) in normal skin of the SKH HRt hairless mouse. The effects of light dose and fluence rate on the dynamics and magnitude of Photobleaching and on the corresponding PDT-induced dam-age were examined. The results show that the PDT damage cannot be predicted by the total light dose. Photo-bleaching was monitored over a wide range of initial PpIX fluorescence intensities. The rate of PpIX photo-bleaching 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 dam-age. 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 photo-bleached. We present a method of calculating a therapeutic dose delivered during superficial PDT that demonstrates a strong correlation with PDT damage.
Michael Schaefer - One of the best experts on this subject based on the ideXlab platform.
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reversible Photobleaching of enhanced green fluorescent proteins
Biochemistry, 2005Co-Authors: Daniel Sinnecker, Nicole Hellwig, Philipp Voigt, Michael SchaeferAbstract:Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after Photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible Photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible Photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible Photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25−58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible Photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based F...
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Reversible Photobleaching of enhanced green fluorescent proteins
Biochemistry, 2005Co-Authors: Daniel Sinnecker, Nicole Hellwig, Philipp Voigt, Michael SchaeferAbstract:Color variants of green fluorescent protein (GFP) are increasingly used for multicolor imaging, fluorescence resonance energy transfer (FRET), and fluorescence recovery after Photobleaching (FRAP). Here we show that experimental settings commonly used in these imaging experiments may induce an as yet uncharacterized reversible Photobleaching of fluorescent proteins, which is more pronounced at acidic pH. Whereas the reversible Photobleaching spectrum of eCFP corresponds to its absorption spectrum, reversible Photobleaching spectra of yellow variants resemble absorption spectra of their protonated states. Fluorescence intensities recover spontaneously with time constants of 25-58 s. The recovery of eCFP can be further accelerated by illumination. The resulting steady-state fluorescence reflects a variable equilibrium between reversible Photobleaching, spontaneous recovery, and light-induced recovery. These processes can cause significant artifacts in commonly applied imaging techniques, photobleach-based FRET determinations, and FRAP assays.
Jean-françois Roch - One of the best experts on this subject based on the ideXlab platform.
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Enhancing single-molecule photostability by optical feedback from quantum jump detection
Applied Physics Letters, 2008Co-Authors: Vincent Jacques, John D. Murray, François Marquier, Dominique Chauvat, Frédéric Grosshans, François Treussart, Jean-françois RochAbstract:We report an optical technique that yields an enhancement of single-molecule photostability by greatly suppressing Photobleaching pathways which involve photoexcitation from the triplet state. This is accomplished by dynamically switching off the excitation laser when a quantum jump of the molecule to the triplet state is optically detected. The resulting improvement in photostability unambiguously confirms the importance of photoexcitation from the triplet state in Photobleaching dynamics and will allow the investigation of new phenomena at the single-molecule level.
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Enhancing single-molecule photostability by optical feedback from quantum-jump detection
2007Co-Authors: Vincent Jacques, François Marquier, Dominique Chauvat, Frédéric Grosshans, François Treussart, John Murray, Jean-françois RochAbstract:We report an optical technique that yields an enhancement of single-molecule photostability, by greatly suppressing Photobleaching pathways which involve photoexcitation from the triplet state. This is accomplished by dynamically switching off the excitation laser when a quantum-jump of the molecule to the triplet state is optically detected. This procedure leads to a lengthened single-molecule observation time and an increased total number of detected photons. The resulting improvement in photostability unambiguously confirms the importance of photoexcitation from the triplet state in Photobleaching dynamics, and may allow the investigation of new phenomena at the single-molecule level.
