Lifetime Of Probe Research Articles

Microscopic Image Resolved by Fluorescence Lifetime of a Fluorescent Probe Incorporated in Cultivated Cancer Cells

Microscopic Image Resolved by Fluorescence Lifetime of a Fluorescent Probe Incorporated in Cultivated Cancer Cells

New insight on beta-lactoglobulin binding sites by 1-anilinonaphthalene-8-sulfonate fluorescence decay.

The fluorescence time decay parameters of the beta-lactoglobulin-1-anilinonaphthalene-8-sulfonate complex have been investigated under physical and chemical perturbations (2 < pH < 8 and added electrolyte 0 < NaCl < 0.5 M) to obtain new insight on the nature of the protein binding interactions. A double exponential decay of the bound probe lifetime has been confirmed by the presence of a longer component, 11 to 14.5 ns, and a shorter component, 2.5 to 3.5 ns. The two lifetimes are ascribed to different binding modes associated also with different exposure to the solvent; in particular, the longer component is attributed to binding inside the hydrophobic beta barrel, while a "surface" site is suggested for the shorter component. A detailed analysis of the lifetime fractional intensities correlates the binding constants with ionic strength and supports the presence of electrostatic effects at both sites. A Debye-Hückel approach, applied to extrapolate the electrostatic free energy contribution vs. pH at vanishing ionic strength, gives interesting clues on the effective charge felt by the ANS ligands in the proximity of each site. In particular, binding is found to parallel the aspartate and glutamate titrations between pH 3 and pH 4.5; the "surface" site mainly responds to the presence of these local titrating charges while the "internal" site more closely follows the overall protein net charge.

Measurement of the Intensity of Long-Lifetime Luminophores in the Presence of Background Signals Using Phase-Modulation Fluorometry.

We describe a method to correct for background fluorescence and to measure the intensity of long-lifetime probes using phase-modulation fluorometry. The theoretically predicted and simulated data were supported by two experiments. The fractional contribution of background fluorescence in the sample was determined by measurement of phase angle and/or modulation at single modulation frequency. In certain experimental situations, where the mean decay times of the background and the long lifetime probe are widely separated, determination of background signals in the sample does not require a blank sample or information about the nature of intensity decay of the background. Hence, phase-modulation fluorometry can be used to directly determine the intensity of the long-lifetime probe in the presence of an unknown short-lifetime background. We also discuss the effects of ambient light (indefinitely long lifetime) and scattered excitation light (zero lifetime) on phase-modulation measurements.

Study of the Effect of Solubilized Gases on the Properties of Microemulsion Droplets

The effect of dissolved gas (nitrogen, argon) on the properties of the droplets in water-in-oil and oil-in-water microemulsions (surfactant aggregation number, microviscosity, and micropolarity) has been investigated by means of time-resolved fluorescence quenching and spectrofluorometry. This study extends a similar one on aqueous micellar solutions (R. G. Alargovaet al., Langmuir14, 1575, 1998). The selected microemulsions were characterized by droplets of fairly large size and high volume fraction, in order to minimize the effect of the curvature of the surfactant layer and maximize the amount of gas that can be solubilized in the system. Within the experimental error, the investigated properties (surfactant aggregation number, intradroplet quenching rate constant which is related to the droplet microviscosity, and fluorescent probe lifetime and micropolarity) were found to be independent on whether the system was degassed, nitrogen-saturated, or argon-saturated, in the temperature range between 10 and 35°C. The results confirm the conclusion reached in the above study; i.e., the effect of solubilized gases on the hydrophobic interaction which controls the formation of surfactant assemblies is extremely small and well below the sensitivity of the fluorescence probing techniques used in this investigation.

Radio-induced structural membrane modifications: a potential bioindicator of ionizing radiation exposure?

Purpose: This study focused on radio-induced membrane alterations in order to assess some related parameters as potential biological indicators of ionizing radiation effects in cases of accidental overexposure. Materials and methods: Radio-induced membrane alterations were assessed after gamma-irradiation of human blood. Biophysical techniques based on fluorescent probe incorporation into isolated living lymphocytes and erythrocytes membranes were applied. Results: Using the technique of fluorescence polarization, the lipophilic phase of the membrane was shown to be more fluid whereas the lipid-protein interface of the membrane was shown to be more rigid after gamma-irradiation. Fluorescent anisotropy modifications showed dose-time effect relationships after radiation exposure. Ionizing radiation induced a decrease in steadystate anisotropy values but did not affect the probe's lifetime as assessed by fluorescence lifetime distribution technique. These data suggest that the anisotropy variations are representative of the local properties of the fluorescent probe's micro-environment. However, the distribution width showed a decrease pointing towards radiation-induced changes of membrane domain organization, probably due not only to membrane water penetration related to lipoperoxidation, but also to compositional changes and redistribution of membrane components. In contrast, the lack of radiation effect observed using the lateral diffusion index technique may be related to the integrated overview of the radio-induced modifications of the membrane provided by this technique, which pointed out radio-induced damage to the membrane in micro-domains. Conclusion: These findings suggest the utility of structural membrane modification measurements as an early bio-indicator of ionizing radiation exposure.

Enzymatic Flow-Injection Analysis of Metabolites Using New Type of Oxygen Sensor Membranes and Phosphorescence Phase Measurements

ABSTRACT A simple system for enzymatic flow-injection analysis of metabolites is described, which is based on the phosphorescence lifetime based detection of molecular oxygen using phase-modulation techniques and a simple instrument - phosphorescence phase detector equipped with a fibre-optic probe. The phase detector is connected to the oxygen sensor membrane and allows real-time continuous monitoring of the phosphorescence phase shift. This parameter is related to the phosphorescence lifetime of the oxygen probe, therefore giving a measure of the dissolved oxygen concentration, and its changes as a result of the enzymatic oxidative reaction with the substrate. The sensor membrane is positioned in a compact integrated flow-through cell and exposed to the flow stream. Using glucose as a test analyte and glucose oxidase enzyme, two different sensor setups were tested: 1) the membrane type biosensor in which the enzyme is immobilized directly on the oxygen sensor membrane; 2) the microcolumn type biosensor in which the enzyme is immobilized separately, on a microparticle sorbent (controlled pore glass) and put into a microcolumn with the oxygen sensor membrane placed at the column outlet. In either case a new type of oxygen sensitive material was used, which provides a number of advantages over the existing materials. In this material the oxygen-sensitive coating was applied on a microporous scattering support, the latter comprised of a layer of cellulose particles on polyester support. Performance and main working characteristics for the two setups and the new oxygen sensor membranes were investigated and compared.

Interactions between positively charged starburst dendrimers and Ru(4,7-(SO 3C 6H 5) 2-phen) 3posttaggered4−

The emission intensity and emissive lifetimes of *Ru(4,7-(SO 3C 6H 5) 2-phen) 3 poststaggered4− in solutions containing various cationic starburst dendrimers (SBD) in the presence and absence of potential quenchers were investigated. The binding constants of *Ru(4,7-(SO 3C 6H 5) 2-phen) 3 poststaggered4− to SBD were determined using changes in the probe's excited state lifetime as a function of the SBD concentration and generation. An observed increase in the probe's lifetime in the presence of SBD is attributed to a lower O 2 concentration at the dendrimer's surface than in water. The quenching constants between *Ru(4,7-(SO 3C 6H 5) 2-phen) 3 poststaggered4− and methylviologen (MV 2+) and K 4Fe(CN) 6 and K 3Fe(CN) 6 were determined by laser flash-photolysis in the presence of various generations of SBD.

Model-Independent Estimation of the Photokinetic Transfer Matrix from Matrix-Formatted Frequency-Domain Fluorescence

In this paper, the mathematical relationships between matrix-formatted frequency-domain fluorescence decay data and the photokinetic transfer matrix are derived. These relationships reveal that procedures for analyzing the fluorescence spectra and photokinetic mechanism of excited state reactions without model assumptions can be developed when the fluorescence decay is described by first-order differential equations with constant coefficients and when the spectral emission profiles of the decaying fluorophores are distinct. The linear structure of wavelength-dependent frequency-domain decay data permits the simultaneous estimation of the emission spectra, relative initial concentrations, and the photokinetic transfer matrix describing the decay and interaction of multiple fluorophores. Data matrices acquired using a single sample are amenable to this approach, eliminating the necessity to measure probe lifetimes separately at low fluorophore concentration. Most importantly, this method does not require that the analyst choose a kinetic model describing fluorophore emission. Instead, the kinetic mechanism is revealed in the structure of the transfer matrix. Statistical methods can be used to estimate the number of emitting components contributing to a data matrix, so that every aspect of the analysis can be pursued without a priori assumptions. Other analytical tools, including a procedure for matrix partitioning to estimate the spectra of sample fluorophores when the spectra are unknown and graphical tools for evaluating prospective spectra and decays, also are described.

Application of fluorescence lifetime imaging microscopy to the investigation of intracellular PDT mechanisms

The potential for the application of fluorescence lifetime imaging (FLIM) microscopy to studies of photosensitization mechanisms in photodynamic therapy (PDT) has been investigated. The fluorescence microscope incorporates a standard inverted optical microscope, a picosecond pulsed dye-laser excitation source, and an intensified CCD camera detector capable of being gated on a sub-nanosecond timescale. Fluorescence lifetime images resulting from multi-component analysis of sub-nanosecond gated fluorescence images of monolayer V79-4 Chinese hamster lung fibroblasts stained with disulphonated aluminium phthalocyanine (AlPcS2), a photosensitizer used in PDT, are presented. The results of these measurements are discussed in terms of the intracellular localization of the sensitizer. Preliminary results from multi-component FLIM of V79-4 cells multiply stained with AlPcS2 and a potential intracellular pH lifetime probe, 5(+6)-carboxynaphthofluorescein, are also presented.

Outer leaflet-packing defects promote poly(ethylene glycol)-mediated fusion of large unilamellar vesicles.

Poly(ethylene glycol)-induced fusion of two different vesicle systems has been examined: dipalmitoylphosphatidylcholine (DPPC) large unilamellar vesicles (LUV) and cardiolipin (CL)/dioleoylphosphatidylcholine (DOPC) (1:10) LUVs. A slight perturbation was established in the outer leaflets of DPPC LUVs by hydrolyzing 0.8% of the outer leaflet lipid with phospholipase A2 to produce lysophosphatidylcholine and palmitate which were then removed by bovine serum albumin. Similarly, 5 mM Ca2+ was added to the external compartment of CL/DOPC LUVs to alter the shape of the CL molecule and thereby create a perturbation in the outer leaflet packing of these vesicles. Contents mixing assays showed that both vesicle systems fused only when the outer leaflets of both contacting vesicles were perturbed as described. Two fluorescent probes (C6-NBD-PC and TMA-DPH) were used to detect changes in outer leaflet molecular packing between nonfusing and fusing systems. The steady-state fluorescence intensity of C6-NBD-PC added externally to either fusing system was enhanced relative to that of nonfusing vesicles. Phase-resolved measurements of probe lifetime showed that this was due mainly to enhanced partitioning of probe from a micellar state into fusing versus nonfusing membranes. Similarly, TMA-DPH was found to undergo more rapid motion when incorporated into fusing as opposed to nonfusing vesicles. The effects of deuterium exchange on probe lifetime also indicated that C6-NBD-PC and TMA-DPH penetrated more deeply into fusing than into nonfusing membranes. These results suggest that the fusogenic perturbations produced in these two very different lipid systems took the form of altered outer leaflet packing. We conclude that, for the two model lipid bilayers examined, small perturbations in lipid packing within contacting bilayer leaflets are necessary and probably sufficient to promote membrane fusion.

Electrical Properties of Silver Impurities and their Annealing Behaviour in p-Type Fz Silicon

The electrical activity of silver as well as its annealing properties in 10 Ω cm p-type Fz silicon substrate are studied by means of the four-point probe and minority carrier lifetime measurements. Silver atom concentration in the range 10 14 to 10 15 cm -3 consistently showed a donor type behaviour in the material and its presence led to a reduction of up to two orders of magnitude in the lifetime of minority carriers by the formation of deep-level traps. Isochronal annealing of silver contaminated specimens showed some gettering of the Ag impurities with resulting temperature dependent changes in the resistivity as well as the minority carrier lifetime values. Analysis of our results shows that a large fraction of the silver impurity atoms present forms the deep level defects and both the deep- and donor-levels appear to originate from the same source.

Amperometric ammonium ion and urea determination with enzyme-based probes

Amperometric enzyme probes for ammonium and urea have been assembled and evaluated using immobilized glutamate dehydrogenase and urease enzymes coupled with platinum electrodes. Analytical parameters such as pH, buffer, temperature, probe life-time, enzyme immobilization, cofactor concentration and response time have been optimized. Ammonium was detected in the range 10 −5−3 × 10 −4 mol l −1. Better reproducibility and stability were achieved using the enzyme GLDH type III and NADH at a concentration of 10 −3 mol l −1. Urea has been determined in the range 10 −5−3 × 10 −4 mol l −1 using the enzyme urease first in solution and then immobilized on nylon net. The analysis was based on an amperometric measurement which gives a linear relationship between current and analyte concentration. This considerably improved the sensitivity of the analysis when compared with the potentiometric-based procedures. Moreover, this method does not suffer from the potassium ion interference which affects the potentiometric nonactin-based NH 4 + electrodes. Analysis of ammonium and urea were carried out in standard solutions and in saliva samples. Results compared with a spectrophotometric reference procedure correlated well.

Time-Resolved Fluorescence in Micellar Systems: A Critical Application to the Partitioning of Naphthalene in Aqueous Sodium Dodecyl Sulfate

Static and dynamic aspects of the partitioning of naphthalene into the hydrocarbon phase in aqueous sodium dodecyl sulfate micelles have been critically examined by nanosecond time-resolved pulse fluorometry. It is shown that, in such a system, a properly detailed analysis of the excited-state decay behavior as a function of progressive partitioning of the solute into increasing concentrations of micelles is capable of revealing and directly quantitating the underlying kinetics of the reversible transfer of naphthalene in its excited state between aqueous and micellar phases. The time-resolved data indicate that, in the aqueous sodium dodecyl sulfate system, the partition coefficient for excited-state naphthalene can differ only marginally from that for the ground state. From the estimate obtained for the rate coefficient for entry of excited-state naphthalene into micelles, it would appear that any barrier to the crossing of excited naphthalene into the micelle, given that a collision has taken place, is likely to be rather small. The contrasting time-resolved behavior of a system in which the basis lifetime of the fluorescence probe in the micellar environment is shorter than that in the bulk environment rather than longer, as in the case examined experimentally, is also modeled. The effects of quenching of the fluorescence at the interface between the micellar and aqueous phases are also examined and discussed in the context of the aqueous cetyltrimethylammonium bromide system.

The role of polymer flexibility on the interaction with surfactant micelles: poly(vinyl alcohol) and sodium dodecyl sulphate aqueous micelle interactions studied by dynamic fluorescence quenching

The interactions between poly(vinyl alcohol) and the surfactant sodium dodecyl sulphate in aqueous solutions have been investigated by means of static and dynamic fluorescence measurements. By static fluorescence measurements the onset of interaction, the critical aggregation concentration, is determined to be 6.5 mM at 20 °C and 7.0 mM at 40 °C. By dynamic fluorescence measurements without added quencher the influence on the lifetime of the fluorescent probe, pyrene, by the presence of the polymer is investigated. From measurements with an added quencher, dimethylbenzophenone, the surfactant aggregation numbers of the micellar aggregates formed upon interaction between the surfactant and the polymer and the quenching rate constant are obtained. At the two polymer concentrations, i.e., 0.2% and 2.0%, the surfactant aggregation numbers at low surfactant concentration are very small, but increase rapidly with increasing surfactant concentration. Comparisons are made with results from a system where poly(ethylene oxide) instead of poly(vinyl alcohol) is the polymer. Key words: dynamic fluorescence quenching, polymer–surfactant interactions, aggregation numbers.

Cholesterol protects the phospholipid bilayer from oxidative damage

The measurement of fluorescence lifetime distribution of 1,6-diphenyl-1,3,5-hexatriene is used for the detection of oxidative damage produced in phospholipid membranes by ionizing radiation. The recently developed method is based on the linear relationship between the width of the probe lifetime distribution and the logarithm of the dose. The molecular origin of the damage resides in the production of hydroperoxide residues at the level of acyl chains double bonds. A chemiluminescence assay was used to quantitate the amount of produced hydroperoxides. Consequences of the produced damages include an increased disorder in the upper portion of the bilayer, accompanied by the penetration of water molecules. In the presence of the physiological concentration of cholesterol in phospholipid bilayers, the amount of hydroperoxides produced by ionizing radiation is dramatically reduced. The packing effect of cholesterol in phospholipid bilayers is well recognized, as well as its influence on the reduction of water concentration in the bilayer. The dramatic reduction of hydroperoxides concentration observed when irradiation is performed in the presence of cholesterol probably originates from a steric hindrance to the radical chain reaction through the unsaturated lipids due to the presence of cholesterol.

Relation between micellar structure of model bile and activity of esterase

In a model bile solution composed of lecithin (L)-bile salt (B), the solubilization of lipid and the accessibility of enzyme to the lipid were examined by observation of EPR spectra and measurement of enzyme activity. The lifetime of the spin probe in the micellar phase was estimated to be approx. 1 μs by means of line shape analysis. Both population and lifetime increased with temperature and the molar ratio of lecithin to bile salt (L/B). The EPR data indicated that simple micelle of bile salt, mixed disk micelle of bile salt-lecithin, and multi-lamellar mixed disk micelle can exist in a model bile solution, depending on the L/B molar ratio across a range from 0 to 1.5. The maximal power of the mixed disk micelle to solubilize cholesteryl ester in the model bile at a L/B molar ratio of 1:1 was confirmed by EPR measurement of cholesteryl 12-DOXYL-stearate. Observation of the enzyme activity on a mixture of model bile and substrate at 37°C revealed selective accessibility of cholesterol esterase (bovine pancreas) to mixed disk micelle, of cholesterol oxidase ( Streptomyces cinnamomeus) to both simple and mixed disk micelle, and of pancreatic lipase (porcine pancreas) to both simple micelle and an oil droplet of substrate. The temperature-dependent activity of cholesterol oxidase to cholesterol in mixed disk micelle can be explained in terms of mesomorphic phase transition of lecithin side chains followed with fluidity of liquid crystal phase. Regarding phospholipase C from Bacillus cereus, though the selective accessibility to the micelles was not observed at 37°C, a decrease in activity for mixed disk micelle could be found at lower temperatures.

Determination of the intersubband lifetime in Si/SiGe quantum wells

A direct pump and probe lifetime determination in the ps regime has been made in quantum wells of Si/Si1−xGex. We have used an rf linac-pumped free-electron laser to determine the relaxation time associated with intersubband absorption in Si/SiGe quantum wells with a subband separation smaller than the optical phonon energy. This measurement yields a lifetime of T1=30 ps.

Effect of PGML excipient mixture in a transdermal system on the in vitro transport of estradiol across skin.

To measure the effect of a combination of excipients from a silicone based pressure sensitive adhesive (PSA) on drug transport across skin. Partitioning of propylene glycol monolaurate (PG-ML) from silicone PSA and a solution formulation into the stratum corneum (SC) was measured using radiolabeled PG-ML. Transport of a model drug, estradiol, as well as PG-ML across skin were measured in vitro using heat separated epidermis from human cadaver skin. The PG-ML partitioning into SC showed a saturation and was independent of the formulation. The local dielectric of the lipid bilayers of the SC showed an increase as a result of PG-ML uptake, as determined by the decrease in fluorescence lifetime of a lipophilic probe incorporated into the SC. However, there was no alteration of lipid packing in SC. The PG-ML and estradiol transport showed a good correlation over 3 days, suggesting that the two species are co-transported across the epidermis.

Fluorescence lifetime imaging of free calcium in single cells

It is shown here that the intensity probe CalciumGreen is suitable for quantitative Ca2+ concentration determinations using fluorescence lifetime imaging. The probe is characterized by two distinct fluorescence lifetimes over a large Ca2+ concentration range. This indicates that the probe exists in two forms, one free and one bound to Ca2+. This behaviour is exploited for the determination of the absolute free Ca2+ concentration from fluorescence lifetime measurements. Fluorescence lifetime confocal imaging affords the determination of an effective lifetime of the probe in buffers and single rat myocytes. The images are used for the quantification of the local concentrations of free Ca2+. We find CalciumGreen to be a useful lifetime probe at physiological Ca2+ concentrations. A similar photophysical behaviour has also been found for the intensity probe Fluo-3. However, it is less suitable for the determination of Ca2+ concentrations in cells.

Relationship of extracellular dopamine in striatum of newborn piglets to cortical oxygen pressure.

The present studies describes the relationship between extracellular dopamine in striatum of newborn piglets and cortical oxygen pressure. The extracellular level of dopamine was measured by in vivo microdialysis and the oxygen pressure in the cortex was measured by phosphorescence lifetime of oxygen probe in the blood. Controlled, graded levels of hypoxic insult to the brain of animals were generated by decreasing of the oxygen fraction in the inspired gas (FiO2) from 21% to 14%, 11%, and 9%. This resulted in decrease in the cortical oxygen pressure from 31-35 Torr to about 24 Torr, 15 Torr and 4 Torr, respectively. The changes in extracellular level of dopamine, DOPAC and HVA were dependent on changes in cortical oxygen pressure. Stepwise decrease in the cortical oxygen pressure (see above) caused increases in extracellular dopamine of about 80%, 200% and 550%, respectively. The levels of DOPAC and HVA progressively decreased and when cortical oxygen decreased to 4-6 Torr were about 50% and 70% of control, respectively. After return of FiO2 to control (21%), the cortical oxygen pressure rapidly increased to above normal, then returned to control values. The extracellular levels of dopamine, DOPAC, and HVA recovered more slowly, attaining control values in about 30 minutes. The data show that extracellular levels of dopamine increase with even very small decreases in oxygen pressure. Thus, there is no "oxygen reserve" which protects dopamine release and metabolism from decrease in oxygen pressure.