Long-term luminescence of sensibilizers in tissues at the conditions of oxygen deficiency due to photodynamic effect

Long-term luminescence of organic dyes (xanthene dyes, halogen substituted fluoroscein) was used for an in vitro study of the photodynamic effect of exogenic probes in malignant tumors and healthy tissues of mice. It is shown that the photodynamic activity of oxygen and the dynamics of its concentration in tissues can be estimated from the delayed fluorescence of exogenic probes caused by singlet–triplet annihilation of singlet oxygen and excited triplet states of the molecules of photosensitizer dyes. It is found that quenching of long-term luminescence of photosensitizers significantly differs in tumors and normal tissues.

     

强荧光背景环境中荧光涨落谱--蒙特卡罗模拟研究

荧光涨落谱方法(Fluorescence Fluctuation Spectroscopy)通过分析微小探测区域内的荧光涨落信号,获得粒子亮度、扩散系数以及溶液浓度等信息。利用Monte Carlo模拟方法,研究了溶液中自荧光背景和系统噪声对荧光涨落谱的影响。结果表明,利用双组分光子计数统计方法,可以有效去除低亮度、高浓度背景组分自荧光和均匀分布系统噪声产生的影响。本结果为利用荧光涨落谱方法测量细胞体系复杂环境中的蛋白质相互作用提供帮助。     

Characterization of ultra-weak fluorescence using picosecond non-collinear optical parametric amplifier

We report a technique for characterization of ultra-weak fluorescence based on a 355-nm pumped picosecond non-collinear optical parametric amplifier (OPA). In the experiment, we effectively reduced the strong super-fluorescence background by using a series of methods. With the picosecond OPA as the pre-amplifier and the gating pulse, the decay of the fluorescence of Rhodamine 6G dye in ethanol was measured and the fluorescence lifetime was found to be about 941 ps. The gain factor of this parametric fluorescence amplifier was measured to be ∼4.2 × 10⁶, while the energy detection limit was ∼160 aJ per pulse within a 15-ps gating pulse.     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术，对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究，尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明，利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团，利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具，对眼底细胞随年龄增长的衰老机理的研究具有重要的意义.     

眼底视网膜色素上皮层细胞脂褐素及氧化黑色素自体荧光寿命成像研究

利用一种基于时间相关单光子计数器的双光子激发荧光寿命显微成像技术,对猪眼底视网膜色素上皮层细胞内的脂褐素和氧化黑色素颗粒的空间分布及其荧光寿命特性进行了研究,尤其对于这些色素颗粒在光致氧化环境中的荧光寿命差异进行了分析.结果表明,利用荧光寿命测量能有效区分视网膜色素上皮层细胞中的多组分荧光团,利用荧光寿命的衰减参数可分辨正常及异常的荧光现象.该方法有望发展成为一种用于眼科临床诊断及病理学研究的高灵敏度的工具,对眼底细胞随年龄增长的衰老机理的研究具有重要的意义. 关键词： 双光子激发荧光 荧光寿命成像 视网膜色素上皮层     

Investigation of effect of thiophene-2-acetic acid as an electron anchoring group for a photovoltaic device

Different anchoring groups such as thiophene-2-acetic and malonic acid were investigated for synthesis of new photosensitizers. The new dyes (photosensitizers) were made pure and determined by various analytical techniques. The chemical structure of synthesized materials was certified by analytical studies. UV-Visible and fluorescence spectra revealed intense fluorescence and absorption for organic photosensitizers. The cyclic voltammetry results showed that the two photosensitizers were suitable for dye sensitized solar cell preparation. The work electrode was gathered using tin (IV) oxide nanoparticles in dye-sensitized solar cells structure. The new photosensitizers and tin (IV) oxide were used for photovoltaic devices preparation. The power conversion efficiency was obtained as about 4.12 and 4.29% for Dye 1 and Dye 2, respectively.     

Annihilation kinetics for triplet excitations in organic glasses

The results of investigations of delayed luminescence decay are presented for disordered phenanthrene in the microsecond, millisecond, and second time ranges. At liquid nitrogen temperature, the highly non-exponential decay of both the phosphorescence and the annihilation delayed fluorescence is observed. This character of decay is caused by the relaxation of the electron-excitation energy in the system of energy-disordered centers when a quantity kT is much less than the width of the distribution for the excited-state energy. At the same time, the analysis of the time dependence for the triplet-triplet-annihilation rate exhibits two time intervals in which different kinetics is observed for the triplet excitations annihilation. In the microsecond range, the classical relationship between the phosphorescence and the delayed fluorescence takes place for the system under investigation; i.e., the triplet-triplet-annihilation rate is constant. At large times, the reaction-rate time dependence is described by the power law characteristic of inhomogeneous and low-dimensional systems. When the temperature increases, a transition to the classical behavior is observed throughout the entire time interval.     

Effect of Temperature on the Rate of Triplet–Triplet Annihilation of 1,2-Benzanthracene in a Polymer Matrix

The process of triplet–triplet annihilation (TTA) of 1,2-benzanthracene (1,2-BA) incorporated into polymer films of polyvinyl butyral has been investigated in the temperature interval 80–360 K. An analysis of the kinetics of the decay of delayed annihilation fluorescence (DAF) of 1,2-BA has shown that the process of the triplet excitation energy transfer in a disperse medium of the polymer at distant times of the DAF decay can be described using the approximation of random walks of the triplet energy in the inhomogeneous medium of the polymer. At low temperatures, at the initial times of DAF decay the TTA process is described with the aid of a model of static annihilation.     

Kinetics of long-lived luminescence of eosin in Langmuir-Blodgett films

The spectral and kinetic properties of photoluminescence of mixed Langmuir-Blodgett (LB) films of eosin decyl ether and palmitic acid on a solid substrate are studied. The electronic absorption and fluorescence spectra of the films are identical to the spectra of the dye in ethanol. An increase in the dye concentration in a monolayer results in the appearance of a dimer absorption band, quenching of fluorescence of monomers, and the red shift of the spectral bands. At 90 K, the distinct phosphorescence and delayed fluorescence bands of LB films were observed. The decay kinetics of phosphorescence and delayed fluorescence is nonexponential. It is shown that the decay curve of delayed fluorescence is determined by triplet-triplet annihilation (TTA) and T ₁→S₁ triplet-singlet intersystem crossing (IS). The initial nonexponential phosphorescence decay is caused the dominant contribution of TTA to the decay of triplet molecules. The experimental data are interpreted based on the mechanisms of exchange-resonance and inductive-resonance annihilation.     

Kinetics of annihilation of triplet excitationsand decay of delayed fluorescence for isolated 1,12-benzoperylene pairs

We studied the annihilation kinetics for triplet excitations in isolated pairs of organic molecules and their delayed fluorescence decay using a new mathematical model for the process. According to this model, the intensity of delayed annihilation fluorescence (DAF) for isolated pairs is directly proportional to the number of pairs in which both molecules are found in the excited triplet state. We show that the decrease in such pairs and the decay of their delayed annihilation fluorescence occur according to an exponential law in the absence of random scatter in the deactivation rate constants for the triplet excitations. The results of an experimental study of the DAF decay kinetics for 1,12-benzoperylene in n-hexane at 77 K, where triplet–triplet annihilation occurs in isolated pairs, confirm the validity of the theoretical conclusions.     

Luminescence diagnostics of malignant tumors in the IR spectral range using Yb-porphyrin metallocomplexes

The creation and application of new low-toxic photosensitizers for the luminescence diagnostics of cancer are considered. The new photosensitizers weakly generate singlet oxygen, exhibit developed luminescence, and retain the tumor-tropic properties of the therapeutic photosensitizers. Twenty one ytterbium complexes of porphyrin compounds that differ by the substituents at the periphery of the porphyrin ring are synthesized. The absorption and luminescence spectra and the luminescence decay curves of these substances are studied. The primary toxicological and pharmacokinetic investigations are performed for the most promising compounds in the organisms of experimental animals. The experimental data prove that the Yb-porphyrin complexes are promising as low-toxic markers for the luminescence diagnostics of malignant tumors in the IR spectral range (975–985 nm) that are free of the phototoxicity typical of the conventional porphyrins at a relatively high luminescence contrast and the selective accumulation in tissue.     

Combined depth- and time-resolved autofluorescence spectroscopy of epithelial tissue

A time-resolved confocal fluorescence spectroscopy system is built to measure the fine structure and localized biochemistry of epithelial tissue. It is found that the autofluorescence excited at 405 nm is sensitive to the cellular metabolism and can be used to sense the metabolic status of epithelial tissue. The decay of autofluorescence excited at 405 nm can be accurately fitted with a dual-exponential function consisting of short lifetime (0.4-0.6 ns) and long lifetime (3-4 ns) components. The ratio of the amplitudes of the two components provides information on the fine structure of epithelial tissue. We demonstrate that the combined depth- and time-resolved measurements with single excitation can potentially provide accurate information for the diagnosis of tissue pathology.     

Development of Low-Cost Instrumentation for Single Point Autofluorescence Lifetime Measurements

Autofluorescence lifetime measurements, which can provide label-free readouts in biological tissues, contrasting e.g. different types and states of tissue matrix components and different cellular metabolites, may have significant clinical potential for diagnosis and to provide surgical guidance. However, the cost of the instrumentation typically used currently presents a barrier to wider implementation. We describe a low-cost single point time-resolved autofluorescence instrument, exploiting modulated laser diodes for excitation and FPGA-based circuitry for detection, together with a custom constant fraction discriminator. Its temporal accuracy is compared against a “gold-standard” instrument incorporating commercial TCSPC circuitry by resolving the fluorescence decays of reference fluorophores presenting single and double exponential decay profiles. To illustrate the potential to read out intrinsic contrast in tissue, we present preliminary measurements of autofluorescence lifetime measurements of biological tissues ex vivo. We believe that the lower cost of this instrument could enhance the potential of autofluorescence lifetime metrology for clinical deployment and commercial development.     

Label-Free Spatial Analysis of Free and Enzyme-Bound NAD(P)H in the Presence of High Concentrations of Melanin

The analysis of autofluorescence, often regarded as undesired noise during the imaging of biological samples, allows label free, unbiased detection of NAD(P)H and melanin in native samples. Because both the emission and absorption spectra of these fluorophores overlap and they can hence not be differentiated using emission filters or with different excitation wavelengths, fluorescence lifetime imaging microscopy (FLIM) is used to differentiate between them. In the present paper the application of two-photon excitation microscopy is presented to investigate the autofluorescence of fungal spores. The model organism which was examined is Aspergillus ochraceus. Furthermore a strategy is developed which allows to quantitatively analyze the fluorescence lifetimes of melanin, free NAD(P)H and protein-bound NAD(P)H using forward convolution of a multiexponential decay function with the instrument response function (IRF) and subsequent fitting to the experimental fluorescence data. As a consequence proteins, which are able to bind NAD(P)H, are located with sub-cellular resolution. Furthermore a spatial differentiation of the fluorophores NAD(P)H and melanin inside the spores, is revealed.     

Fluorescence lifetime tomography of live cells expressing enhanced green fluorescent protein embedded in a scattering medium exhibiting background autofluorescence

We present a novel fluorescence lifetime tomography system applied to a highly scattering autofluorescent phantom containing live cells expressing the fluorophore enhanced green fluorescent protein (EGFP). The fluorescence signal was excited using a fiber-laser-pumped supercontinuum source and detected using wide-field time gating imaging. To facilitate rapid 3D reconstruction of the fluorescence lifetime distribution, the time-resolved data were Fourier-transformed in time to give complex functions that formed a data set for the Fourier domain reconstruction. Initially the presence of an unspecified background autofluorescence signal impeded reconstruction of the lifetime distribution, but we show that this problem can be addressed using a simple iterative technique.     

Study of ProtoPorphyrin IX Elimination by Body Excreta: A new Noninvasive Cancer Diagnostic Method?

This paper describes the elimination of porphyrins by feces. It was demonstrated that porphyrin accumulates substantially more in tumors than in normal tissues, and consequently more PPIX reaches the blood of patients and animals with tumors, and then, it needs to be eliminated. The fluorescence of feces revealed that there are large amounts of PPIX in the excreta of animals with cancer comparing with healthy animals. The autofluorescence of feces porphyrin extracted with acetone was analyzed using fluorescence spectroscopy of animals inoculated with DU145 cells into the prostate and healthy animals to monitor the PPIX concentration. Emission spectra were obtained by exciting the samples at 405 nm. Significant differences were observed in autofluorescence intensities measured in the 575–725 nm spectral regions for the studied groups. The results showed a noninvasive, simple, rapid and sensitive method to detect cancer by feces analysis.     

Phosphorescence of pi-conjugated oligomers and polymers

We observed phosphorescence from a ladder-type poly-(para-phenylene) and an analogous oligomer containing five phenylene rings. The spectra are similar to the intrinsic fluorescence spectra and bear out a singlet-triplet splitting of 5000 cm(-1) (polymer) and 6800 cm(-1) (oligomer). Phosphorescence decay of the polymer occurs on a 10-100-micros scale obeying a power law and suggestive of nonradiative quenching, while that of the oligomer is asymptotically exponential with an intrinsic decay time of approximately 250 ms. The polymer also exhibits delayed fluorescence. It originates from delayed recombination of geminate electron-hole pairs rather than from triplet-triplet annihilation.     

Kinetics of the Decay of Prolonged Luminescence of Disordered Chrysene under the Conditions of Nonequilibrium Transfer of Energy

We present the results obtained in investigation of the decay of the prolonged luminescence of disordered chrysene in a microsecond range. At the temperature of liquid nitrogen, a highly nonexponential decay of both phosphorescence and annihilation of delayed fluorescence is observed. The observed character of decay is due to relaxation of the energy of electronic excitation in a system of energydisordered centers, when the value of kT is much smaller than the width of the excitedstate energy distribution. At the same time, in the system investigated there is a classical relationship between phosphorescence and delayed fluorescence, i.e., the rate coefficient for the reaction of triplettriplet annihilation is a constant.     

A General Method for Constrained Analysis of Fluorescence Anisotropy Decay: Application of the Steady-State Anisotropy

Time-resolved fluorescence anisotropy is an invaluable method for investigating the internal and rotational dynamics of biomolecules. The range of rotational motions detectable by anisotropy decay is limited by the fluorescence lifetime; typically, a depolarizing motion may be resolved if the associated correlation time is between 0.1 and 10 times the intensity decay lifetime. To extend that range and to improve the recovery of anisotropy decay parameters, a general analytical method has been developed. This procedure utilizes a modification of Lagrange multiplier methods to constrain the values of the iterated kinetic parameters during nonlinear least-squares analysis of anisotropy decay data. The form of the constraint equation is derived from the classic relationship between the decay parameters and the steady-state anisotropy, which can be simply and accurately measured. Application of the constraint to analyses of synthetic data sets increased the accuracy of recovery by decreasing the uncertainty in the iterated parameters. The constraint also enabled the accurate recovery of correlation times that were a factor of 30 greater than the fluorescence lifetime, although it did not improve recovery of correlation times that were much shorter than the lifetime. Using this technique, it should now be possible to characterize the dynamics of larger macromolecules and assemblies than those that can currently be studied by fluorescence anisotropy decay.     

Kinetics of Triplet-Triplet Annihilation in Organic Glasses

In the present paper, the results of the investigation of the decay kinetics of delayed luminescence of organic glasses are presented. A strong deviation of the decay of both phosphorescence and annihilation delayed fluorescence from the exponential law is observed. This effect is shown to be due to the relaxation process of electronic excitation energy in the system with large energetic disorder. At the same time, the presence of two time intervals in which the rate coefficient for triplet-triplet annihilation (TTA) reaction shows different dependence on time is observed. On a short time scale the classical behavior is observed, i.e., the reaction is well described by the second-order equation with a time-independent rate coefficient. At the limit of long times, we have strong dependence of rate coefficient on time, i.e., the electronic excitation energy transport is dispersive. It is shown that behavior observed for the rate coefficient for TTA reaction is due to the relaxation process (on short time scale) and the equilibrium energy migration (in long time limit).