Dependence Of Fluorescence Lifetime Research Articles

Sensing Temperature in Vitro and in Cells Using a BODIPY Molecular Probe.

Boron dipyrromethene (BODIPY) molecular rotors have shown sensitivity toward viscosity, polarity, and temperature. Here, we report a 1,3,5,7-tetramethyl-8-phenyl-BODIPY modified with a polyethylene glycol (PEG) chain, for temperature sensing and live cell imaging. This new PEG-BODIPY dye presents an increase in nonradiative decay as temperature increases, which directly influences its lifetime. This change in lifetime is dependent on changes in both temperature and viscosity at low viscosity values, but is only dependent on temperature at high viscosity values. The dependence of fluorescence lifetime with temperature allows for temperature monitoring in vitro and in cells, with sub degree resolution. When in contact with cells, the PEG-BODIPY spontaneously penetrates and stains the cell but not the nucleus. Furthermore, no significant cell toxicity was found even at 100 μM concentration. Using fluorescence lifetime imaging microscopy (FLIM), we were able to observe the changes in the lifetime of PEG-BODIPY within the cell at different temperatures. The use of FLIM and molecular probes such as PEG-BODIPY can provide important information about cellular temperature and heat dissipation upon medically relevant stimuli, such as radiofrequency ablation and photodynamic therapy.

Molecular Level Understanding of Sodium Dodecyl Sulfate (SDS) Induced Sol-Gel Transition of Pluronic F127 Using Fisetin as a Fluorescent Molecular Probe.

The thermoreversible sol-gel transition of pluronic F127 is markedly altered even with addition of submicellar concentration of sodium dodecyl sulfate (SDS) surfactant. Multiple fluorescence parameters like fluorescence intensity, fluorescence anisotropy and fluorescence lifetime of both the prototropic forms (anion (A-*) and phototautomer FT*) of the photoprototropic fluorescent probe fisetin has been efficiently used to understand the molecular level properties like polarity and microviscosity of the PF127-SDS system as a function of temperature. The SDS-induced increase in the interfacial hydrophobicity level is seen to affect the sol-gel phase transition of PF127 (21-18 °C). The ET(30) polarity parameter value of anionic emission of fisetin suggests that there is a considerable decrease in the polarity of the PF127 medium with increase in temperature and with the addition of SDS. The microviscosity progressively increases from ∼5 mPa s (sol state, 10 °C) to ∼22.01 mPa s (gel state 35 °C) in aqueous solution of PF127. The variation in microviscosity with addition of SDS in PF127-SDS mixed system is significant in sol phase whereas in gel phase this variation is significantly less. Temperature dependent fluorescence lifetime of FT* indicates that there is heterogeneity in distribution of fisetin molecules at different domains of PF127. This work also show-cases the sensitivity of fisetin toward change in polarity and change in sol-gel transition temperature of copolymer PF127 with variation in temperature (both forward and reverse directions) and SDS.

Preparation and luminescent properties of one-dimensional YVO4:Eu nanocrystals

One-dimensional YVO4:Eu3+ nanofibre and nanospindle samples were fabricated by electrospinning and an ultrasonic chemistry method. The average diameter of the YVO4:Eu3+ nanofibres were 30 nm, and they mainly consisted of nanoparticles arranged in 1-d patterns. The YVO4:Eu3+ nanospindles, with central diameters and lengths of around 50 and 150 nm, were assembled from nanorods with central diameters and lengths of around 10 and 50 nm. Their luminescent characteristics including excitation and emission spectra, and temperature-dependent fluorescence lifetimes, were studied and compared. The luminescence intensities of Eu3+ in YVO4:Eu3+ nanofibres were slightly higher than those in YVO4:Eu3+ nanospindles. Both the dependence of fluorescence lifetimes on the temperature of Eu3+ 5D0 for the YVO4:Eu3+ nanofibres or nanospindles remained quasi-constant, or indeed constant, over the temperature range studied, respectively; however, the lifetimes of Eu3+ 5D0 in YVO4:Eu3+ nanofibres were always shorter than those in YVO4:Eu3+ nanospindles at the same temperature.

Pyrazole‐substituted Near‐infrared Cyanine Dyes Exhibit pH‐dependent Fluorescence Lifetime Properties

Near-infrared heptamethine cyanine dye is functionalized with pyrazole derivatives at the meso-position to induce pH-dependent photophysical properties. The presence of pyrazole unsubstituted at (1) N-position is essential to induce pH-dependent fluorescence intensity and lifetime changes in these dyes. Replacement of meso-chloro group of cyanine dye IR820 with (1) N-unsubstituted pyrazole resulted in the pH-dependent fluorescence lifetime changes from 0.93ns in neutral media to 1.27ns in acidic media in DMSO. Time-resolved emission spectra (TRES) revealed that at lower pH, the pyrazole consists of fluorophores with two distinct lifetimes, which cor-responds to pH-sensitive and non-pH-sensitive species. In contrast, (1) N-substituted pyrazoles do not exhibit pH response, suggesting excited state electron transfer as the mechanism of pH-dependent fluorescence lifetime sensitivity for this class of compounds.

Fluorescence of Er^3+:AlN polycrystalline ceramic

We report what we believe to be the first preparation and optical spectroscopy of Er3+ doped into bulk AlN ceramic. The material was prepared via hot press sintering of AlN with Er2O3 and [NH4][ErF4], which yielded fully dense, translucent, hexagonal AlN. The Er3+ concentration is a small fraction of a percent, and resides in multiple sites, with one type of center dominant. A number of the energy levels of Er3+ are identified for this center. The temperature dependent fluorescence lifetime is probably radiative, and on that basis the stimulated emission and absorption cross section spectra are inferred for the 4I13/2 ↔ 4I15/2 transitions.

Co-precipitation synthesis, structural and morphological characterization, and luminescence properties of Dy 3+ doped nanocrystal Gd 2(WO 4) 3 and Gd 2WO 6 phosphors

Nanocrystal Gd 2(WO 4) 3 and Gd 2WO 6 phosphors doped with Dy 3+ were prepared via co-precipitation method under different reaction conditions. It was found that the nanocrystal Gd 2(WO 4) 3 and Gd 2WO 6 phosphors exist in the monoclinic phase, and the phosphor particles showed sphere-like and uniform shape. The concentration quenching behavior and the dependence of fluorescence lifetime on the doping concentration were studied. It was found that the electric dipole–dipole interaction is the physical mechanism for the energy transfer between Dy 3+ ions in both the nanocrystal Gd 2(WO 4) 3 and Gd 2WO 6 phosphors.

A Fusion-Spliced Near-Field Optical Fiber Probe Using Photonic Crystal Fiber for Nanoscale Thermometry Based on Fluorescence-Lifetime Measurement of Quantum Dots

We have developed a novel nanoscale temperature-measurement method using fluorescence in the near-field called Fluorescence Near-field Optics Thermal Nanoscopy (Fluor-NOTN). Fluor-NOTN enables the temperature distributions of nanoscale materials to be measured in vivo/in situ. The proposed method measures temperature by detecting the temperature dependent fluorescence lifetimes of Cd/Se Quantum Dots (QDs). For a high-sensitivity temperature measurement, the auto-fluorescence generated from a fiber probe should be reduced. In order to decrease the noise, we have fabricated a novel near-field optical-fiber probe by fusion-splicing a photonic crystal fiber (PCF) and a conventional single-mode fiber (SMF). The validity of the novel fiber probe was assessed experimentally by evaluating the auto-fluorescence spectra of the PCF. Due to the decrease of auto-fluorescence, a six- to ten-fold increase of S/N in the near-field fluorescence lifetime detection was achieved with the newly fabricated fusion-spliced near-field optical fiber probe. Additionally, the near-field fluorescence lifetime of the quantum dots was successfully measured by the fabricated fusion-spliced near-field optical fiber probe at room temperature, and was estimated to be 10.0 ns.

Dependence of fluorescence lifetime in praseodymium-doped silica optical fibres upon strain and temperature

Measurements of the temperature and strain responses of fluorescence lifetimes for two transitions in praseodymium-doped silica optical fibres are presented. These properties were found to be similar to those measured for other rare-earth-doped fibres, except for the strain response. In particular, the fluorescence lifetime of the , 3H6 transition (centred on 870 nm) exhibited a slight decrease with applied strain, rather than an increase. This has implications for our understanding of how strain in rare-earth-doped optical fibres affects fluorescence properties.

Mechanism of Interaction between the General Anesthetic Halothane and a Model Ion Channel Protein, II: Fluorescence and Vibrational Spectroscopy Using a Cyanophenylalanine Probe

We demonstrate that cyano-phenylalanine (PheCN) can be utilized to probe the binding of the inhalational anesthetic halothane to an anesthetic-binding, model ion channel protein hbAP-PheCN. The Trp to PheCN mutation alters neither the α-helical conformation nor the 4-helix bundle structure. The halothane binding properties of this PheCN mutant hbAP-PheCN, based on fluorescence quenching, are consistent with those of the prototype, hbAP1. The dependence of fluorescence lifetime as a function of halothane concentration implies that the diffusion of halothane in the nonpolar core of the protein bundle is one-dimensional. As a consequence, at low halothane concentrations, the quenching of the fluorescence is dynamic, whereas at high concentrations the quenching becomes static. The 4-helix bundle structure present in aqueous detergent solution and at the air-water interface, is preserved in multilayer films of hbAP-PheCN, enabling vibrational spectroscopy of both the protein and its nitrile label (-CN). The nitrile groups' stretching vibration band shifts to higher frequency in the presence of halothane, and this blue-shift is largely reversible. Due to the complexity of this amphiphilic 4-helix bundle model membrane protein, where four PheCN probes are present adjacent to the designed cavity forming the binding site within each bundle, all contributing to the infrared absorption, molecular dynamics (MD) simulation is required to interpret the infrared results. The MD simulations indicate that the blue-shift of -CN stretching vibration induced by halothane arises from an indirect effect, namely an induced change in the electrostatic protein environment averaged over the four probe oscillators, rather than a direct interaction with the oscillators. hbAP-PheCN therefore provides a successful template for extending these investigations of the interactions of halothane with the model membrane protein via vibrational spectroscopy, using cyano-alanine residues to form the anesthetic binding cavity.

Mechanism Of Interaction Between The Volatile Anesthetic Halothane And A Model Ion Channel Protein: Fluorescence And Infrared Spectroscopy Employing A Cyano-phenylalanine Probe And Molecular Dynamics Simulation

We demonstrate that cyano-phenylalanine (PheCN) can be utilized to probe the binding of the inhalational anesthetic halothane to a model ion channel protein hbAP-PheCN possessing a designed binding cavity. The Trp to PheCN mutation adjacent the cavity alters neither the α-helical conformation nor the 4-helix bundle structure. The halothane binding properties of hbAP-PheCN, based on fluorescence quenching, are consistent with those of the Trp-prototype, hbAP1. The dependence of fluorescence lifetime on halothane concentration implies a one-dimensional diffusion of halothane along the nonpolar core of the protein bundle.

Solubility and fluorescence lifetime of 2,5-diphenyloxazole and 1,4-bis(5-phenyl-oxazolyl-2)benzene in water–ethanol and water–acetone solvent systems

The solubility of two luminophores, 2,5-diphenyl-1,3-oxazole (PPO) and 1,4-bis(5-phenyl-oxazolyl-2)benzene (POPOP), has been determined in water–acetone and water–ethanol solvent systems at 25.0 °C. The excited states of PPO and POPOP have been studied in these mixed solvents. The dependence of fluorescence lifetime and solubility on mixed solvents composition is discussed in terms of solvation of luminophore molecules. The multiple linear regression analysis of fluorescence lifetime and solubility dependences on solvent polarity parameters has been carried out. Equations, describing the dependence of solubility and fluorescence lifetime of luminophores on solvent parameters, are presented. It has been revealed that both solubility of PPO and POPOP and their fluorescence lifetimes are controlled mainly by the energy of cavity formation.

Emission wavelength dependence of fluorescence lifetimes of bacteriological spores and pollens

Concern about biological terrorism has greatly increased in the 21st century, and correspondingly, so has the need for accurate detection and identification of biological hazards, such as Bacillus anthracis. Optical techniques have been shown to be useful for this purpose. Use of fluorescence lifetimes as a function of emission wavelength for different materials using point- detection methods appears to be an additional viable option. Although the lifetimes range only between 2 and 6 ns, most biological materials tested in this study were distinguishable. A preliminary database has been compiled for use in a possible future detection system.

Functional optical detection based on pH dependent fluorescence lifetime

Detection of possible alterations of physiological parameters (e.g., pH and temperature), resulting from malignant transformation of initially healthy tissue, can be a powerful diagnostic tool for earlier cancer detection. Such variations can be observed by comparing these parameters with those of healthy tissue surrounding the abnormality. Time-resolved spectroscopy of specifically targeted fluorescent labeled antibodies can be sensitive to such variations and provide a high resolution functional image of the region of interest. The goal of this study was to establish a forward experimental setup for calibration of the lifetime dependencies of near-IR fluorescent dyes on physiological parameters, and to develop analytical solutions, taking into account the effects of light propagation in turbid media (e.g., tissue), that was able to extract an original lifetime fluorescence signal from time-of-flight intensity distributions, measured in vivo from a deeply embedded live organ for further analysis. Tissue-like phantoms with embedded fluorescent dyes and background optical properties simulating those of live tissues were designed and created. Fluorescence decay curves were measured for different fluorophore positions, and pH values. Those measurements were made with a system based on a time-correlated single photon counting (TCSPC) instrument and a tunable femtosecond Ti-Sapphire system built by our group. Decay curves were recorded for fluorophore depths of up to 5 mm and source-detector separation of 7 mm. It was shown that a forward model, based on the random walk theory, adequately described the experimental data. Measured pH dependencies of the fluorescence lifetime were characterized for two different dyes. Good correlation between experimental data and predictions of the theoretical model allows the use of close-form analytical solutions to separate the effects of photon time delays due to multiple scattering in tissues from the original intensity fluorescence time decay curve, determined by the fluorophore itself and its immediate surroundings. It is the latter dependence that can be diagnostically important. Experimentally obtained scaling between lifetime and a parameter of interest can be used in vivo to obtain a map of physiological parameter changes which can serve as a base for an in vivo specific diagnostic system.

Concentration and Temperature Dependence of Spectroscopic Properties of Highly-Doped Nd:YAG Crystal Grown by Temperature Gradient Technique (TGT)

The high resolution absorption spectra of Nd:YAG crystals grown by temperature gradient technique (TGT) were measured for Nd ion concentration in the range of 1.0 to 3.0 at% at low temperature (8 K). The measurement of emission spectra for different Neodymium dopant concentrations at low temperatures suggested that the 2.0 at% Nd:YAG crystals possessed the highest fluorescence intensity. The emission decays of the 4 F 3 / 2 level of Nd 3 + in YAG by pumping into the 4 G 5 / 2 level were measured at temperatures from 6 to 300 K for different Neodymium dopant concentrations. The nonexponential luminescence decays and concentration quenching were observed from 1.0 to 3.0 at% Nd:YAG even at the low temperatures (6K), due to the cross-relaxation and energy migration process. The dependence of fluorescence lifetime on temperature for highly doped Nd: YAG is ascribed to the interactions between Nd ions and the increasing rate of energy transfer.

Effects of gel material on fluorescence lifetime detection of dyes and dye-labeled DNA primers in capillary electrophoresis

Investigations of fluorescence lifetimes of the dye 6-[ N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]hexanoic acid (NBD-HA) and of DNA M13 primers labeled with NBD-HA, Cy3, rhodamine green and 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY-FL) dyes in polyacrylamide gels of various degrees of crosslinking and different crosslinkers, and in a cellulose sieving buffer with different organic modifiers, are described. The dependence of fluorescence lifetime on gel matrix and on experimental conditions was studied in order to identify which factors may be important for optimization of multiplex fluorescence lifetime detection. Lifetimes were determined in both batch solution and on-the-fly, on-column in CE. Results show that lifetimes of the primer-attached dyes remain constant in gels of different composition. Additionally, multiexponential fluorescence decays are observed for primer-attached dyes in batch solutions of the cellulose sieving buffers but are reduced to monoexponential decays when measured on-the-fly, on-column in CE. Lifetime detectability can be improved by addition of an organic modifier to the gel matrix.

Solvation dynamics study of 3-aminophthalimide in n-butanol solution at different temperatures

We have studied the solvation dynamics of 3-aminophthalimide (3-AP) in n-butanol. The fluorescence lifetimes and the rotational correlation times of the molecule were measured at several wavelengths over the emission spectrum at six temperatures. Solvation time correlation functions were evaluated from wavelength dependent fluorescence lifetime distributions at four temperatures for 3-AP in n-butanol solution. The solvation dynamics of 3-AP was strongly temperature dependent. The solvation times of 3-AP were compared to the dielectric relaxation times (τL and τD) of n-butanol and to the solvation times of 4-amino-N-methyl-phthalimide (4-ANMP) studied earlier in n-butanol and n-decanol solutions. The solvation time of 3-AP molecule in n-butanol at room temperature was much closer to the longitudinal dielectric relaxation time than to the Debye time. At low temperatures the solvation times of 3-AP and 4-ANMP approach the Debye times of n-butanol. The solvation dynamics of 3-AP occurs on a time scale roughly given by the longitudinal relaxation time as predicted by the simple continuum theory.

Optical dynamics of excitons in J aggregates of a carbocyanine dye

Results of temperature and wavelength dependent fluorescence lifetime and accumulated photon-echo experiments on the J aggregate of the dye 5,5′,6,6′-tetrachloro-1,1′-diethyl-3,3′-di(4–sulfobutyl) -benzimidazolocarbocyanine (TDBC) in an ethylene glycol/water glass are reported. Additionally, the temperature dependent relative fluorescence quantum yield was determined. Using two-color pump–probe spectroscopy, the intersite correlation of the frequency disorder and the size of the coherence domains were estimated. Both the phase and the population relaxation times are frequency dependent in the echo and the single-photon-counting experiments. The dependence of the fluorescence lifetime on detection wavelength is restricted to temperatures below 80 K, indicating a temperature activated process. From our experiments and by comparison with previously published results we conclude that the dispersive nature of both the dephasing and the depopulation is caused by intraband relaxation processes. At higher temperatures this wavelength dependence vanishes due to thermalization. The temperature dependence of the relative fluorescence quantum yield of TDBC is equal to the one of pseudoisocyanine (PIC). Using a motional narrowing model for disordered molecular aggregates with consideration of intersite correlation, at 1.5 K, the two-color pump–probe experiments indicate a very high correlation in the frequency disorder in TDBC J aggregates with a correlation length of several hundred molecules. From pump–probe experiments the delocalization length of the exciton is determined to be between 30 and 45 molecules at 1.5 K.

Fluorescence lifetime imaging of intracellular calcium in COS cells using Quin-2

We describe the first fluorescence lifetime images of cells. To demonstrate this new capability we measured intracellular images of Ca 2+ in COS cells based on the Ca 2+-dependent fluorescence lifetime of Quin-2. Apparent fluorescence lifetimes were measured by the phase-modulation method using a gain-modulated image intensifier and a slow-scan CCD camera. We describe methods to correct the images for photobleaching during acquisition of the data, and to correct for the position-dependent response of the image intensifier. The phase angle Quin-2 images were found to yield lower than expected Ca 2+ concentrations, which appears to be the result of the formation of fluorescent photoproducts by Quin-2. Fluorescence lifetime imaging (FLIM) does not require wavelength-ratiometric probes and appears to provide new opportunities for chemical imaging of cells.

A New Fluorescence Sensor for Quantification of Atmospheric Humidity

A new fiber‐optic sensor (optrode) for humidity has been developed. The sensor utilizes a fluorescent dye entrapped within a perfluorinated ionomer matrix. The fluorescence intensity increases strongly and linearly with increasing watervapor partial pressure even though the lifetime of fluorescence is simultaneously lowered. The response time of the optrode is approximately 1s and the presence of has no detectable effect on the determination of humidity. Apparently, the immobilized fluorescent dye, rhodamine 6G, associates with water to form a complex with a higher absorptivity. The dependence of fluorescence lifetime on emission wavelength revealed the coexistence of multiple excited states for the water‐dye system.

Characterization of carboxylate sites in surface oxidized polyethylene films by fluorescence techniques

Oxidized reactive sites introduced by controlled chemical reactions in non-crystalline regions of semi-crystalline polyolefin films are located mainly on the film surface or in adjacent subsurface regions. The so-formed reactive sites in low density polyethylene (LDPE) blown films were labelled by fluorescent groups and investigated by various fluorescence techniques. The surface or subsurface location of the labelled site is indicated by a solvatochromic spectral shift and confirmed by the polarity dependent fluorescence lifetime of the labelled LDPE film immersed in a polar liquid. Fluorescence concentration quenching and concentration depolarization demonstrate the high density of oxidized sites near the film surface. The measurement of rotational mobility and orientation during film stretching adds further experimental tools to distinguish reactive oxidized sites in the surface or in the bulk.