番红花红T与表面活性剂的作用及其在标记DNA中的应用

对阳离子染料番红花红Ｔ（ＳＴ）在阴离子表面活性剂存在时的溶液状态的吸收光谱和荧光光谱进行了研究。结果表明，低浓度阴离子表面活性剂与ＳＴ形成缔合物，导致ＳＴ的吸收与荧光强度降低；增大表面活性剂的浓度，其分子胶束前预聚集促使染料形成非荧光二聚体，导致荧光急剧猝灭，吸收光谱出现新的特征吸收峰；当表面活性剂浓度大于临界胶束浓度（ＣＭＣ）时，染料二聚体离解，ＳＴ单体增溶于胶团中形成新的高量子产率荧光体。本文     

Mechanisms and advancement of antifading agents for fluorescence microscopy and single-molecule spectroscopy

Modern fluorescence microscopy applications go along with increasing demands for the employed fluorescent dyes. In this work, we compared antifading formulae utilizing a recently developed reducing and oxidizing system (ROXS) with commercial antifading agents. To systematically test fluorophore performance in fluorescence imaging of biological samples, we carried out photobleaching experiments using fixed cells labeled with various commonly used organic dyes, such as Alexa 488, Alexa 594, Alexa 647, Cy3B, ATTO 550, and ATTO 647N. Quantitative evaluation of (i) photostability, (ii) brightness, and (iii) storage stability of fluorophores in samples mounted in different antifades (AFs) reveal optimal combinations of dyes and AFs. Based on these results we provide guidance on which AF should preferably be used with a specific dye. Finally, we studied the antifading mechanisms of the commercial AFs using single-molecule spectroscopy and reveal that these empirically selected AFs exhibit similar properties to ROXS AFs.     

Synthesis and solvent-dependent photophysics of a novel fluorescent triazole-coumarin-based dye

Synthesis of a new coumarin-triazole-based dye and its photophysical parameters such as absorption, fluorescence emission, and fluorescence quantum yield were investigated. Studies have shown that the present dye has symmetry with a mirror image, especially in the ethanol solvent, with respect to the absorption and fluorescence spectra. As a result of the UV-vis and fluorescence spectroscopy techniques used, it was determined that absorption and emission spectra were shifted to the red with increasing solvent polarity. In addition, the spectral data of the synthesized compound exhibited that the stokes shifts are small, usually less than 50 nm, and the quantum yields are significantly high. In accordance with the results obtained, it can be stated that this novel dye synthesized here can offer an insight into application in sensor applications as analytical or biosensors, optoelectronic devices, and medicine industry.     

LASER FLASH SPECTROSCOPY OF METHYLENE BLUE WITH NUCLEIC ACIDS

Absorption spectra, fluorescence spectra and quantum yields, triplet state absorption spectra, yields and rates of decay have been obtained for methylene blue (MB+), MB+/CT-DNA complexes and complexes of MB+ with the synthetic polynucleotides poly[d(G-C)] and poly[d(A-T)]. Strong fluorescence quenching is observed in the complexes of the dye with CT-DNA and poly[d(G-C)] concomitant with a decrease in the triplet yield. The fluorescence and triplet yield of MB+ with poly[d(A-T)] are similar to those of the free dye. The triplet decay times are increased in all three polymer/dye complexes and show a decreased sensitivity to oxygen quenching. These results are interpreted in terms of the binding of the dye to these polymers and the implications of polymer binding on the photosensitizing properties of the dye are discussed.     

Spectroscopic investigations of a novel tricyanofuran dye for nonlinear optics

A novel tricyanofuran dye was synthesized and the dye-in-polymer films were fabricated by spin-coating process. The spectroscopic properties of the dye in the solutions and polymer films were investigated by the absorption spectra and fluorescence emission spectra. It is found that the absorption and fluorescence maxima are largely red-shifted along with the increase of the solvent polarity. And the low values of fluorescence quantum yield in higher polarity solvents suggest the presence of twisted intramolecular charge transfer states of the dye. Moreover, the second order polarizability value of the novel dye was estimated based on the quantum-mechanical two-level model.     

Molecular and Spectroscopic Characterization of Green and Red Cyanine Fluorophores from the Alexa Fluor and AF Series**

The use of fluorescence techniques has an enormous impact on various research fields including imaging, biochemical assays, DNA-sequencing and medical technologies. This has been facilitated by the development of numerous commercial dyes with optimized photophysical and chemical properties. Often, however, information about the chemical structures of dyes and the attached linkers used for bioconjugation remain a well-kept secret. This can lead to problems for research applications where knowledge of the dye structure is necessary to predict or understand (unwanted) dye-target interactions, or to establish structural models of the dye-target complex. Using a combination of optical spectroscopy, mass spectrometry, NMR spectroscopy and molecular dynamics simulations, we here investigate the molecular structures and spectroscopic properties of dyes from the Alexa Fluor (Alexa Fluor 555 and 647) and AF series (AF555, AF647, AFD647). Based on available data and published structures of the AF and Cy dyes, we propose a structure for Alexa Fluor 555 and refine that of AF555. We also resolve conflicting reports on the linker composition of Alexa Fluor 647 maleimide. We also conducted a comprehensive comparison between Alexa Fluor and AF dyes by continuous-wave absorption and emission spectroscopy, quantum yield determination, fluorescence lifetime and anisotropy spectroscopy of free and protein-attached dyes. All these data support the idea that Alexa Fluor and AF dyes have a cyanine core and are a derivative of Cy3 and Cy5. In addition, we compared Alexa Fluor 555 and Alexa Fluor 647 to their structural homologs AF555 and AF(D)647 in single-molecule FRET applications. Both pairs showed excellent performance in solution-based smFRET experiments using alternating laser excitation. Minor differences in apparent dye-protein interactions were investigated by molecular dynamics simulations. Our findings clearly demonstrate that the AF-fluorophores are an attractive alternative to Alexa- and Cy-dyes in smFRET studies or other fluorescence applications.     

Ratiometric, fluorescent BODIPY dye with aza crown ether functionality: synthesis, solvatochromism, and metal ion complex formation

A new pH and metal ion-responsive BODIPY-based fluorescent probe with an aza crown ether subunit has been synthesized via condensation of 4-(1,4,7,10-tetraoxa-13-aza-cyclopentadec-13-yl)-benzaldehyde with the appropriate 1,3,5,7-tetramethyl substituted boron dipyrromethene moiety. Steady-state and time-resolved fluorometries have been used to study the spectroscopic and photophysical characteristics of this probe in various solvents. The fluorescence properties of the dye are strongly solvent dependent: increasing the solvent polarity leads to lower fluorescence quantum yields and lifetimes, and the wavelength of maximum fluorescence emission shifts to the red. The Catalan solvent scales are found to be the most suitable for describing the solvatochromic shifts of the fluorescence emission. Fluorescence decay profiles of the dye can be described by a single-exponential fit in nonprotic solvents, whereas two decay times are found in alcohols. Protonation as well as complex formation with several metal ions are investigated in acetonitrile as solvent via fluorometric titrations. The aza crown ether dye undergoes a reversible (de)protonation reaction (pKa = 0.09) and shows a approximately 50 nm blue shift in the excitation spectra and a 10-fold fluorescence increase upon protonation. The compound also forms 1:1 complexes with several metal ions (Li(+), Na(+), Mg(2+), Ca(2+), Ba(2+), Zn(2+)), producing large blue shifts in the excitation spectra and significant cation-induced fluorescence amplifications.     

Solvent Polarity and pH Effects on the Spectroscopic Properties of Neutral Red: Application to Lysosomal Microenvironment Probing in Living Cells

Neutral red is a lysosomal probe and a biological pH indicator. In aqueous solutions, the protonated (NRW) and neutral (NR) forms of monomeric neutral red exhibit distinct absorption maxima (535 and 450 nm, respectively) but have the same fluorescence with a maximum at 637 nm and a quantum yield of 0.02. The similarity of the fluorescence spectra at acidic and basic pH suggests deprotonation of cationic species in the first singlet excited state. The NR fluorescence strongly depends on the solvent polarity as shown by addition of increasing amounts of water to pure dioxane, which gradually shifts the fluorescence maximum from 540 nm in pure dioxane to 637 nm in water. The fluorescence quantum yield increases from 0.17 in dioxane to 0.3 upon addition of 7% water and then decreases, reaching 0.02 in pure water. Immediately after incubation of human skin fibroblasts with neutral red, excitation with 435 nm light produces a fluorescence whose maximum is recorded at 575 nm. This fluorescence is located in the perinuclear region and originates from large fluorescent intracytoplasmic spots, suggesting staining of the endoplasmic reticulum-Golgi complex. At longer times, this fluorescence is shifted to 606 nm, suggesting slow diffusion of the lysosomotropic dye toward the more hydrated and acidic interior of ly-sosomes. Addition of a lysosomotropic detergent to cells previously incubated with neutral red shifts the fluorescence to the blue. Thus, in complex biological systems, this probe cannot be a good pH indicator but is a very sensitive probe of lysosomal rnicroenvironrnents.     

The photochemical characteristics of aromatic enediyne compounds substituted with electron donating and electron withdrawing groups

trans- and cis-1-(4-Dimethylaminophenyl)-6-(4-nitrophenyl)hex-3-ene-1,5-diynes (trans- and cis-DANE) were synthesized and their photochemical properties were studied. The absorption spectra of trans-DANE red-shifted compared with the parent compound bisphenylethynylethene (BEE) due to intramolecular charge transfer. The fluorescence spectra, Stokes shift, fluorescence lifetime, fluorescence quantum yield, and quantum yield of trans-to-cis photoisomerization of trans-DANE showed strong dependence upon the solvent polarity in the less-polar region. No fluorescence emission from trans-DANE was observed in medium-polar and polar solvents. The quantum yield of cis-to-trans isomerization was almost solvent independent. The donor-acceptor substituents shifted the equilibrium between the trans perpendicular triplet state and the trans planar triplet state to the trans triplet state, and resulted in an increase in the triplet lifetime. Comparison of the photochemical properties of trans-DANE with trans-4-dimethylamino-4'-nitrostilbene (DANS) suggests that trans-DANE is a possible fluorescent probe in the non-polar region.     

Photophysical characterization of a highly conjugated bipyridyl-based dye synthesized by a unique two-step approach

In this article, we present a new and simple, yet efficient, two-step approach to synthesize 4,4'-bis(dibutylaminostyrylstyryl)-2,2'-bipyridine with high yield, as well as its linear and nonlinear optical characterizations in THF and toluene solutions. We show that its one- and two-photon absorption spectra are similar in both solvents. Nevertheless, the relaxation processes of this compound exhibit dependence on the solvent polarity. The one- and two-photon induced fluorescence signal of this molecule in solution reveals that its excited state is highly stabilized in THF solution rather than in toluene. Analysis of the fluorescence quantum yield, lifetime, and radiative and nonradiative decay rates are in agreement with Lippert's model for solute-solvent interactions. The optical measurements demonstrate that this dye is a promising candidate for multiphoton fluorescence imaging, optical limiting, and dye lasers.     

THE PHOTOPHYSICS OF MEROCYANINE 540. A COMPARATIVE STUDY IN ETHANOL AND IN LIPOSOMES

Abstract— Absorption and fluorescence emission spectra, fluorescence lifetimes, fluorescence quantum yields, photoisomerization quantum yields and triplet quantum yields were measured for Merocyanine 540 (MC540) in ethanol and in large unilamellar dimyristoyl phosphatidylcholine vesicles. The major differences in the photophysics between the two media are the increase of the fluorescence quantum yield from 0.15 in ethanol to 0.6 in vesicles at 25° C, and the appearance of a second fluorescence decay with a lifetime of 1.87 ns in the latter medium. Upper and lower limits for the photoisomerization quantum yields were determined by combining the data from laser flash photolysis and optoacoustic spectroscopy. The decrease in photoisomerization quantum yield upon incorporation of the dye into the lipid bilayer by a factor 2 suggests that this process competes directly with fluorescence. The temperature dependence of the fluorescence and photoisomerization quantum yields in solution supports this model. In both media MC540 has a very low triplet quantum yield with values 0.002 > (> ø_T > 0.02 in ethanol and 0.01 > ø_T- > 0.09 in liposomes Our data are consistent with the model whereby the dye is incorporated into the lipid bilayer as a monomer with two different orientations and this model is adopted on the basis of the biexponential behaviour of the fluorescence and photoisomer decay.     

A novel FRET approach for in situ investigation of cellulase–cellulose interaction

A novel real-time in situ detection method for the investigation of cellulase–cellulose interactions based on fluorescence resonance energy transfer (FRET) has been developed. FRET has been widely used in biological and biophysical fields for studies related to proteins, nucleic acids, and small biological molecules. Here, we report the efficient labeling of carboxymethyl cellulose (CMC) with donor dye 5-(aminomethyl)fluorescein and its use as a donor in a FRET assay together with an Alexa Fluor 594 (AF594, acceptor)–cellulase conjugate as acceptor. This methodology was successfully employed to investigate the temperature dependency of cellulase binding to cellulose at a molecular level by monitoring the fluorescence emission change of donor (or acceptor) in a homogeneous liquid environment. It also provides a sound base for ongoing cellulase–cellulose study using cellulosic fiber.     

Effect of TiO2 colloids on the fluorescence behavior of two cyanine dyes

The photophysical properties of two typical cyanine dyes [3,3'-diethyl-9-methyl-thiacarbocyanine iodide (dye A) and anhydro-3,3'-disulfopropyl-5,5'-diphenyl-9-ethyloxacarbocyanine hydroxide (dye B)] in the absence and presence of TiO(2) colloids have been investigated by UV-visible spectroscopy, (1)H-NMR spectroscopy, fluorescence spectroscopy, fluorescence lifetime measurements, and ESR measurements. It was found from the absorption spectra and NMR results that there are two isomers in the ground state of these dyes. Steady-state fluorescence spectra show that the fluorescence intensities of dye A and dye B are enhanced and quenched by TiO(2) colloids, respectively. Time-resolved fluorescence lifetime measurements indicate that the lifetimes of dye A and dye B in the presence of TiO(2) colloids are longer and shorter than those obtained in the absence of TiO(2) colloids, respectively. ESR measurements demonstrate that the electron transfer efficiency from (1)dye B* to the conduction band of TiO(2) is much larger than that from (1)dye A* to the conduction band of TiO(2). The different fluorescence behavior of dye A and dye B can be intepreted in terms of whether phi(Tr,nr)(0)-phi(Tr,nr) (the reduction of the quantum yield for radiationless transition in the excited singlet state (1)dye* caused by the TiO(2) colloids) is larger or smaller than phi(ET) (the quantum yield of electron transfer from (1)dye* to the conduction band of TiO(2) colloids).     

The Fluorescence Properties of Hypocrellin B and its Amino‐substituted Derivative: Photinduced Intramolecular Proton Transfer and Photoinduced Intramolecular Electron Transfer¶

We report on the emission spectra and emission quantum yields of a newly synthesized hypocrellin dye, Z‐demethoxy‐ 2,3‐ethylenediamino hypocrellin B (EDAHB), and its parent HB in different solvents of varying polarity. Our results demonstrate that EDAHB is one of the few dyes that exhibit highly solvent polarity‐dependent fluorescence in the useful region (680–730 nm). Therefore, it offers some applications in the biomedical field as a fluorescent probe molecule. The solvatochromic effect of EDAHB is proposed to be due to a distinct change in the dipole moment of the dye on excitation. A photoinduced intramolecular proton transfer and a photoinduced intramolecular electron transfer process are considered relevant for the fluorescence properties of HB and EDAHB, respectively.     

一种具有双重功能的可视化pH荧光探针的合成及光谱研究

以二烯单酮结构为荧光团,酚羟基为脱质子基团,合成了一种具有双重功能的可视化pH荧光分子探针.pH滴定实验表明,探针的紫外吸收和荧光光谱均对溶液的pH值有很强的依赖性,当体系溶液由酸性变为碱性时,探针的紫外吸收光谱发生明显的红移,并伴有溶液颜色的显著变化;荧光光谱强度在酸性条件下随pH值的变化不大,而在碱性条件下随pH值...     

Photophysical behavior of a dimeric cyanine dye (BOBO-1) within cationic liposomes

This study is aimed at establishing optimal conditions for the use of 2,2'-[1,3-propanediylbis[(dimethyliminio)-3,1-propanediyl-1(4H)-pyridinyl-4-ylidenemethy-lidyne]]bis[3-methyl]-tetraiodide (BOBO-1) as a fluorescent probe in the characterization of lipid/DNA complexes (lipoplexes). The fluorescence spectra, anisotropy, fluorescence lifetimes and fluorescence quantum yields of this dimeric cyanine dye in plasmid DNA (2694 base pairs) with and without cationic liposomes (1,2-dioleoyl-3-trimethylammonium-propane [DOTAP]), are reported. The photophysical behavior of the dye in the absence of lipid was studied for several dye/DNA ratios using both supercoiled and relaxed plasmid. At dye/DNA ratios (d/b) below 0.01 the fluorescence intensity increases linearly, whereas lifetime and anisotropy values of the dye are constant (tau approximately 2.5 ns and = 0.20). By agarose gel electrophoresis it was verified that up to d/b = 0.01 DNA conformation is not considerably modified, whereas for d/b = 0.05-0.06 a single heavy band appears on the gel. For these and higher dye/DNA ratios the fluorescence intensity, anisotropy and average lifetime values decrease with an increase in BOBO-1 concentration. When cationic liposomes are added to the BOBO-1/DNA complex, an additional effect is noticed: The difference in the environment probed by BOBO-1 bound to DNA leads to a decrease in quantum yield and average lifetime values, and a redshift is apparent in the emission spectrum. For fluorescence measurements including energy transfer (FRET), a d/b ratio of 0.01 seems to be adequate because no considerable change on DNA conformation is detected, a considerable fluorescent signal is still measured after lipoplex formation, and energy migration is not efficient.     

Synthesis and photophysics of novel 8-hydroxyquinoline aluminum metal dye with hole transfer groups

A novel luminescent dye metal complex, (CZHQ)3Al, with 8-hydroxyquinoline aluminum and hole-transporting carbazole units was designed and synthesized. The (CZHQ)3Al optical properties were carefully investigated by UV-vis absorption and fluorescence spectra in diluent solution. The results showed that the luminescent quantum yield of (CZHQ)3Al was 0.62 in DMSO and it emitted red-light with the band gap of 2.89 eV estimated from the onset absorption. In addition, the light-emission of (CZHQ)3Al can be quenched by electron acceptor (dimethylterephalate), where the process followed the Stern-Volmer equation. However, the fluorescent intensities of (CZHQ)3Al were slowly increased with the addition of electron donor (N,N-dimethylaniline). Furthermore, the molecular interactions of (CZHQ)3Al with fullerene (C60) and carbon nanotubes (CNTs) were also respectively investigated, which indicated the metal dye can be used as new fluorescent probe.     

ENVIRONMENTAL EFFECTS ON THE ELECTRONIC SPECTRAL PROPERTIES OF 1-PYRENECARBOXALDEHYDE AND THEIR APPLICATION IN PROBING BIOLOGICAL STRUCTURES

Abstract— The electronic spectra of 1-pyrenecarboxaldehyde are sensitive to environmental polarity. In the fluorescence spectrum especially, an extensive loss of structure, large red shifts and an increase in the quantum yield is progressively observed on going from aliphatic to polar solvents or even mixtures between the two types of solvent. When 1-pyrenecarboxaldehyde interacts with substances possessing an amino group its fluorescence quantum yield increases without a simultaneous spectral shift or loss of spectral structure. These phenomena are explained in terms of the effect of the solvent on the nπ *- ππ * state coupling. The effect of temperature and the pH on the electronic spectral properties are discussed. The solvent-sensitive photophysical properties of 1-pyrenecarboxaldehyde can be used to probe the interior of lecithin vesicles. These properties, as well as the dynamics of the interaction of the hydrocarbon with proteins, can yield information about mitochondrial membranes.     

荧光量子点及其在生物检测中的应用

量子点(QDs)是一种零维的半导体纳米晶体,与传统的有机染料相比,具有独特的光学特征。由于它们具有激发光谱宽、发射光谱窄、发射波长精确可调、量子产率高和荧光稳定性好等特点,作为新一代的生物荧光探针,已被广泛应用于生物检测。本文介绍了QDs的基本概念和性质,探讨了QDs的制备方法及表面修饰,对其毒性也作了简要分析,提供了QDs在荧光免疫分析、生物芯片、生物传感器及体内成像等方面的应用实例。随着技术发展的不断进展,QDs在生物分析领域有着更为广泛的潜在的应用前景。     

Photophysics on surfaces: determination of absolute fluorescence quantum yields from reflectance spectra

A method for the calculation of absolute fluorescence quantum yields for dyes attached to solid particles based on reflectance measurements is reported. The same procedure allows calculation of true reflectance spectra (free of fluorescence) for highly fluorescent materials as well. Samples ofcresyl violet were immobilized by adsorption on microgranular cellulose in the concentration range 4.5 x 10(-9) to 3.8 x 10(-6) mol g(-1). Diffuse and total reflectance spectra were recorded with and without insertion of an optical absorption filter between the output of the integrating sphere of a reflectance spectrometer and the photodetector in order to block fluorescence partially. From these data, the relative emission spectrum of the dye, the filter transmission spectrum, and the detector sensitivity, true reflectances and absolute fluorescence quantum yields were recovered. Observed fluorescence quantum yields, affected by dye aggregation and inner filter effects, were concentration and wavelength dependent, ranging approximately between 0.1 and 0.6. The analysis of remission function spectra showed that dye aggregation is negligible up to a concentration of 1.41 x 10(-7) mol g(-1). Fluorescence data were corrected for reemission and reabsorption using a suitable model [Lagorio, M. G.; Dicelio, L. E.; Litter, M. I.; San Roman, E. J. Chem. Soc., Faraday Trans. 1998, 94, 419]. Application of this model to samples showing no aggregation yielded a wavelength-independent true fluorescence quantum yield of 0.60 +/- 0.05, similar to values found in solution. The usage of cresyl violet as a reference for the evaluation of fluorescence quantum yields for weakly fluorescing samples in the solid phase is discussed.