THE USE OF SHORT LIVED FLUORESCENT DYES TO CORRECT FOR ARTIFACTS IN THE MEASUREMENTS OF FLUORESCENCE LIFETIMES

Abstract— The ultrafast emission for fluorophores can be used as the effective excitation pulse for prompt response measurements by photo detectors in the same spectral region as that of unknown samples. This method corrects for such artifacts as wavelength and spatial dependence of the response function of the photodetector. It is shown that the emission from triphenylmethane dyes is an excellent effective pulse with relaxation time 2 ps in the red region of the spectrum. A microchannel plate photomultiplier has only a 35 ps increase in response or lag time between excitation at 420 nm and emission at 680 nm.     

三元若丹明激光染料荧光寿命研究

利用单光子计数技术测试了新型三元若丹明激光染料在不同溶剂中的荧光寿命、荧光光谱及其寿命的实验数据.实验表明,所研究的三元若丹明染料存在着有效的分子内能量传递过程,这些过程使得激光染料的荧光量子效率及光稳定性明显改善.     

ZnO纳米粒子的光物理性质──微量水对荧光的影响

ZnO不仅是极其重要的半导体材料,而且还广泛用做化学反应的催化剂,光催化剂和光电转换材料。近年来,ZnO纳米粒子的制备和应用得到了广泛的研究,但许多问题并不十分清楚。譬如,对ZnO颗粒的可见荧光的发生机制就众说纷纭。Henglein等认为ZnO可见荧光由ZnO颗粒表面的阴离子空位引起,而Bahnemann等却认为可见荧光由被捕获的光生电子向被捕获的空穴越迁而引起。本文通过微量水对ZnO超微粒的荧光的影响的研究,进一步阐明ZnO荧光的产生机理。结果支持了Bahnemann的观点。     

新型双发色团染料荧光光谱及其寿命的研究

有效的染料激光操作需要较高的荧光量子效率,若丹明是在500~700 nm光谱区中一类最重要的激光染料.然而,染料的基态分子和三线态对辐射能量的吸收将会大大降低激光输出效率,再者,由于若丹明类染料在紫外区的吸收系数较小,为了有效吸收泵浦能量(如用XeCI准分子激光,308 nm),就必须使用高浓度染料溶液,在这种情况下,若丹明类染料较小的Stokes位移就势必造成基态分子更大的重复吸收,即造成更大的谐振腔损耗^[1].     

PROBING THE STRUCTURE OF HPD BY FLUORESCENCE SPECTROSCOPY

Fluorescence emission spectra indicate that oligomers containing both hematoporphyrin and its dehydration products (vinyl porphyrins) comprise the tumor-localizing fraction of HPD. In the relatively polar solvent methanol, the vinyl porphyrins exhibit reduced fluorescence yields while the hematoporphyrin residues are relatively resistant to fluorescence quenching by Fe+3. In the less polar solvent tetrahydrofuran, fluorescence from oligomeric vinyl porphyrins was enhanced, and Fe+3-induced quenching of oligomeric hematoporphyrin promoted. These, together with other studies in biological systems, suggest a substantial degree of interaction among the porphyrin units contained in these oligomers, as a function of the polarity of the environment.     

PHOTOPHYSICAL PROPERTIES OF 3,3'-DIALKYLTHIACARBOCYANINE DYES IN HOMOGENEOUS SOLUTION

The photophysical properties of 3,3′-dialkylthiacarbocyanine iodides and chlorides were measured in various solvents. It was found that photoisomerization and fluorescence are the major contributors to the deactivation of the excited singlet state; intersystem crossing occurs with only a very low efficiency. In ethanol, a triplet yield of 0.004 and a singlet oxygen quantum yield of 0.002 were determined. The photophysical parameters of these dyes are not substantially influenced by the length of the alkyl chain or the size of the halide counterion. The substitution of an ethyl with an octadecyl-chain only slightly hinders photoisomerization, and the replacement of the chloride with an iodide reduces only marginally the fluorescence lifetimes and fluorescence quantum yields in chloroform. A significant external heavy-atom effect is observed using dibromoethane as a solvent: triplet and singlet oxygen yields increase7–10-fold, and the triplet lifetime decreases from 55 μs to 15 mUs.     

新型以若丹明染料为母体的三发色团化合物的荧光性能

若丹明染料作为一种重要的激光染料,它们具有较高的荧光量子效率。这类染料在紫外区的吸收是相当小的,于是,当它们被使用在横向泵浦激光装置上时,就需要相当高的染料浓度,这样才能有效地吸收紫外泵浦光(如XeCI激光器,308 nm)。问题是,这类染料的发射光谱与其吸收光谱的低能量部分有重叠,亦即它门的Stokes位移较小,并且由于染料的荧光量子效率通常小于100,以致使一部分染料的激发辐射被基态染料分子再吸收,从而导致激发辐射的损失。与此同时,为了有效吸收泵浦光能量,染料的浓度通常很高,这样,激发辐射损失就会相当大。     

以吖啶染料荧光猝灭为基础的卤素阴离子检测研究

合成了两种吖啶及吖啶橙盐类小分子化合物和带有吖啶盐的三元共聚物.研究了它们在溶液中的荧光被卤素离子猝灭的问题.发现它们能强烈地被碘离子所猝灭而不易被氯离子猝灭,表明这类化合物有可能用于在有氯离子存在条件下对碘、溴离子的检测.对荧光猝灭机理进行的研究发现,卤素离子的猝灭能力并不和它们对发光化合物的系间窜越和三重态的生成等有关,而是和阴离子对极化分子的作用减弱了分子内的电荷转移能力相关.     

PHOTOPHYSICAL and PHOTOCHEMICAL PROPERTIES OF COUMARIN DYES IN AMPHIPHILIC MEDIA

Abstract— Photophysical properties of coumarin dyes solubilized in aqueous detergent solutions have been investigated including measurement of absorption and fluorescence emission maxima, and fluorescence quantum yields. Use of coumarin 4 as a fluorescence probe of sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide (CTAB) solutions led to the conclusion that the sites for dye incorporation in micelles are significantly hydrogen-bonded (hydrated). The inhibition of photochemical decomposition for detergent-solubilizcd dyes has also been observed. Electron transfer from micelle-bound dye to a water soluble acceptor, methyl viologen, has been investigated by flash photolysis.     

PHOTOPHYSICAL STUDIES ON THE BINDING OF TETRASULFONATOPHENYLPORPHYRIN TO LENS PROTEINS

Previous studies have shown that mesotetra(p-sulfonatophenyl)porphine (TPPS) binds to lens proteins. This characteristic should increase the residence time of the sensitizer in the lens and therefore enhance the probability of inducing photooxidative damage to that tissue in vivo. Subsequent in vivo studies have verified that contention. The present studies were performed to determine the effect of such binding on the spectroscopy and photophysics of the porphyrins. It was found that the binding of TPPS (1) quenches the fluorescence of lens proteins, (2) causes a shift in the ground state absorption spectra, fluorescence excitation spectra and the triplet excited state spectrum of TPPS to longer wavelengths and (3) results in an increase in the triplet state lifetime of TPPS. In the presence of the isolated crystallins the average triplet lifetime increases in the following order: gamma less than beta less than alpha.     

皖南尖吻蝮蛇毒糖甙水解酶(NADase)的荧光光谱研究

本文用荧光光谱法研究了皖南尖吻蝮蛇毒糖苷水解酶(NADase)的性质。在pH<6时，pH对NADase的荧光强度影响较大，而在pH>6，pH对NADase的荧光强度几乎没有什么影响，Cu²⁺的加入可引起NADase的内源荧光强度的降低，通过荧光滴定测得Cu²⁺与NADase结合常数K_EM为5.3×10³(mol/L)^-1。I^-对NADase发光的淬灭作用很小，I     

ARTIFACTS IN FLUORESCENCE EMISSION SPECTROSCOPY RELATED TO WOOD''S ANOMALY

Fluorescence emission spectra of porphyrins acquired with a monochromator using a 1500 line/mm holographic grating were affected by polarization-induced effects (Wood's anomaly) which affected the transmission of different wavelengths of light. This effect resulted in a blue-shift in the emission optimum, and distortions in the fluorescence spectrum at longer wavelengths. These anomalies could be eliminated by insertion of polarizers, orientation = 0 degrees, in emission and excitation paths. A monochromator with a 1200 line/mm holographic grating was essentially free from these distortions.     

TIME-GATED FLUORESCENCE SPECTROSCOPY OF THE TUMOR LOCALIZING FRACTION OF HpD IN THE PRESENCE OF CATIONIC SURFACTANT

Time-gated fluorescence spectroscopy was performed on the tumor localizing fraction (TLF) of HpD in buffer at different concentrations of cationic surfactant. This technique obtains emission spectra with programmable delay relative to the excitation pulse. According to the measured fluorescence decay-time constants (approximately 0.7, approximately 3 and approximately 15 ns) three gates were considered, delayed by 0, 5 and 18 ns, respectively, to evaluate the contribution of the emitting molecular species to the spectra. Simultaneous to these measurements, fluorescence decay waveforms and time-integrated spectra were also detected. In buffer and in detergent micelles the fluorescence spectra are given by the superposition of the emission of the different molecular species present in the solution, and no appreciable interaction among the chromophores is observed. On the contrary, in the pre-micellar range of the surfactant, evidence for the existence of an energy transfer mechanism was found. This effect seems to be related to the configurational state of the TLF polymeric chains and depends on the relative TLF/surfactant concentration.     

AGE-RELATED CHANGES IN THE FLUORESCENCE OF MELANIN and LIPOFUSCIN GRANULES OF THE RETINAL PIGMENT EPITHELIUM: A TIME-RESOLVED FLUORESCENCE SPECTROSCOPY STUDY

The photophysical properties of purified populations of melanin and lipofuscin granules from human retinal pigment epithelium, and their changes with donor age, have been investigated using high-sensitivity time-resolved fluorescence spectroscopy techniques with picosecond gating capabilities. The overall fluorescence intensity of both melanin and lipofuscin granules clearly increased with increasing donor age, the increase being most marked for melanin. In all granule populations the fluorescence decays were multiexponential with subnanosecond and nanosecond decay components. The resultant time-integrated and time-gated spectra also exhibited marked age-variations for each type of granule. Young melanin showed spectral patterns similar to those of bovine melanin, while a yellow-orange fluorescence band appeared in melanin samples from older age groups. Lipofuscin granules exhibited a blue, a yellow and an orange band whose relative amounts were age-related. The results demonstrate the potential of time-resolved techniques for discriminating fluorophores in vitro and in situ, and have confirmed results previously obtained using extraction techniques. Furthermore, the ability of this technique to identify and quantify individual fluorophores within granules may provide an important insight into the origin and development of lipofuscin within the retinal pigment epithelium and ultimately into the mechanisms of age-related retinal diseases.     

FLUORESCENCE LINE NARROWING SPECTROSCOPY OF Zn PORPHYRINS*

Fluorescence line narrowing (FLN) spectroscopy was used to study the vibrational features of Zn porphyrins, including Zn octaethylporphine, Zn mesoporphyrin, Zn deuteroporphyrin and Zn coproporphyrin, and the interaction of these porphyrins with their solvent matrices. A number of ground-state vibrational levels were identified for each of the Zn porphyrins. Six of these levels persistent throughout almost all spectra were analyzed in terms ofcontributions of specific internal vibrational modes of the porphyrins. A number of other vibrational levels were identified as characteristic of the peripheral substituents of Zn porphyrin. Phonon wing contribution in the FLN spectra increased with increased polarity of the solvent matrix, and the Stokes shift calculated from the conventional spectra was correlated with the strength of phonon coupling. Specific vibrational modes were found to couple differently with the solvent and to have different phonon wings. The conventional and energy-selected spectra of Zn coproporphyrin in 5 M guanidine HCI suggest that guanidine ligates to the Zn atom.     

CHARACTERIZATION OF THE PHOTOREACTION BETWEEN DNA AND AMINOMETHYL-TRIMETHYLPSORALEN USING ABSORPTION AND FLUORESCENCE SPECTROSCOPY

–The use of absorption and fluorescence spectroscopy for following the progress of the photo-reaction between DNA and 4'-aminomethyl-4,5',8-trimethylpsoralen (AMT) has been investigated. Absorption at long wavelengths and fluorescence both decline upon intercalation of AMT into the DNA helix. The loss of fluorescence from AMT and the accompanying appearance of monoadduct fluorescence upon irradiation by UV light can be easily followed by using the excitation beam of a spectro-fluorometer as the source of irradiation and monitoring the changing emission spectrum. Where cross-link formation is possible, the subsequent decline of monoadduct fluorescence is seen as well. This suggests that the 4,5-tnonoadduct is a precursor of cross-links. Both monoaddition and cross-linking are more rapid with poly d(A·T) than with calf thymus DNA or poly d(A·T). Excitation spectra can be helpful in resolving the levels of AMT and 4',5'-monoadduct when both are contributing to the emission spectrum. Some changes are observed in the emission spectrum of AMT–poly d(A·T) monoadducts after prolonged irradiation which indicate further photoreaction.     

荧光光谱法研究美托拉宗与牛血清白蛋白的相互作用

采用荧光光谱技术研究了在pH7.40的Tris-HCl缓冲介质中美托拉宗与BSA相互作用。实验发现,美托拉宗对BSA有较强的荧光猝灭作用,根据292K和311K时美托拉宗对BSA的荧光猝灭作用,利用Stern-Volmer方程及双倒数方程处理实验数据,表明美托拉宗对BSA的荧光猝灭作用属于动态猝灭过程,根据F rster非辐射能量转移理论计算出了美托拉宗与BSA间的结合距离r=2.77 nm,结合过程的热力学数据表明,二者主要靠疏水作用力结合;进一步采用同步荧光光谱探讨了美托拉宗对BSA构象的影响。     

FLUORESCENCE PROBE STUDIES ON THE SECOND CRITICAL MICELLE CONCENTRATION OF SEVERAL KINDS OF SURFACTANTS

The fluorescence intenstiy ratios (F₂/F₁) of excimer (F₂) to monomer (F₁) of probe (pyrene) were measured as a function of the concentration of the surfactant in several systems, which consist of sodium hexadecyl sulfonate (C₁₆As)-anionic surfactant, cetyltrimethylammonium bromide (CTAB)-cationic surfactant and pentaoxyethylene decyl ether (C₁₀E₅)-nonionic surfactant. The second CMC values were obtained according to F₂/F₁ ~ surfactant concentration curve break. In addition, we also studied the variation of second CMC vs NaCl concentration. It was found that the second CMC values of C₁₆A₅ and CTAB decreased with the increase of NaCl concentration. But the present of NaCl had no influence on the second CMC value of C₁₀E₅. Results showed that sphere-shaped micelles turn into rod-like micelle at the second CMC. The results were interpreted in terms of diffusion of pyrene molecules and the distribution of pyrene among micelles.     

伊文思蓝作荧光探针研究牛血清白蛋白与红霉素之间的竞争反应

用伊文思蓝(EB,Evans blue)作荧光探针研究了红霉素(erythromycin,EM)对牛血清白蛋白(BSA,bo-vine serum albumin)的竞争反应。根据Stern-Volmer方程研究了荧光猝灭的类型及机理,伊文思蓝与牛血清白蛋白作用,使牛血清白蛋白荧光发生静态猝灭,即伊文思蓝和牛血清白蛋白形成了一种稳定的复合物。伊文思蓝与牛血清白蛋白的结合常数KBSA-EB=1.122×106L/mol,结合点数n=0.9935。当加入红霉素后,牛血清白蛋白的相对荧光强度恢复,表明红霉素与牛血清白蛋白发生了竞争反应,探讨了相关机理,得到了伊文思蓝与红霉素的结合常数KEB-EM=1.950×104L/mol。     

ON THE COMPLEX FORMATION OF ACRIDINE DYES WITH DNA-IV. THE EQUILIBRIUM CONSTANTS OF SUBSTITUTED PROFLAVINE AND ACRIDINE ORANGE DERIVATIVES*

Abstract— The binding constants of the complex formation of several acridine dyes with calf thymus DNA are reported. The results indicate, that the binding tendency increases with the basicity of the dyes in the order acridine < 3–amino-acridine < 3.6–bis-monocetamino-acridine < 3.6–diamino-acridine ? 3.6–bis-dimethylamino-acndine < 3.6–diamino-10–methyl-acridine. The ring-N alkylated amino-acridines give in general the highest brinding constants, but there is no significant difference in the extrapolated values, K₀, at diminishing dye concentration if the chain length of the alkyl groups is increased from methyl to amyl. Only substitution with phenyl seems to inhibit the binding. On the other hand, the weaker binding of the acndine itself is sensitive to the ring-N alkylation and the binding tendency decreases in the order acridine > 10–methyl-acridine > 10–propyl-acridine. These findings could be explained, if the electrostatic binding involving the positively charged ring nitrogen, which seems to be more important for the more weakly interacting acridines, is complemented by other binding mechanisms, including possibly non-electrostatic interactions in the case of those amino-acridines having the strong binding effect.