The Mechanism of Benzothiazole Styrylcyanine Dyes Binding with dsDNA: Studies by Spectral-Luminescent Methods

In the presented work studies of the interaction mode of monomer and two homodimer benzothiazole styryl dyes containing spermine-like linkage/tail group with the double stranded (ds) DNA are reported. For these dyes, equilibrium constant of dye binding to DNA (K _b), as well as the number of dsDNA base pairs occupied by one bound dye molecule (n) were determined. The data obtained show that the presence of spermine-like group containing quaternary nitrogen (Bos-5) results in increase of K _b value as compared to this of unsubstituted analogue (Sbt). Besides, for the dimer dyes containing benzothiazole styryl chromophores, the K _b value is either five times higher (DBos-13) or almost the same (DBsu-10) as compared to this of corresponding monomer Sbt, depending on the position in the benzothiazole ring where the linker is attached. Moreover, the n values for both dimers are significantly different as well, pointing to the bis-intercalative binding mechanism for DBos-13 and for the groove-binding one for DBsu-10. The conclusion about the dimer dyes-dsDNA binding mechanisms is also supported by the study of the fluorescent response of these dyes on the presence of AT- and GC-containing polynucleotides.     

Fluorescence of Styryl Dyes-DNA Complexes Induced by Single- and Two-Photon Excitation

The series of novel monomer and homodimer styryl dyes based on (p-dimethylaminostyryl) benzothiazolium residues were synthesized and studied as possible fluorescent probes for nucleic acids detection. Spectral-luminescent and spectral-photometric properties of obtained dyes in the unbound state and in DNA presence were studied. Fluorescence emission induced by two-photon excitation of dye-DNA complexes in aqueous buffer solution was registered. Two-photon absorption cross section values of the studied dyes in DNA presence were evaluated.     

以萘为π-中心的双芪类衍生物双光子上转换荧光性能研究

研究了2个新的双光子上转换荧光分子—1,4-双-(9-乙基咔唑基)萘(简称为BECVN)和1,4-双-(4’-N,N-二甲氨基苯乙烯基)萘(简称为BMABN)的单光子、双光子光谱性质。在～375 nm Xe灯光源激发下,两样品的DMF溶液发出很强的蓝、绿色荧光(峰位492～541 nm),视感效果非常明显。BMABN分子的线性吸收/发射光谱的峰位与BECVN分子相比,均发生红移;相对荧光量子产率(Φ_f)比BECVN降低了7.4倍。在飞秒钛宝石激光器泵浦下(760 nm),两样品的DMF溶液发出强的双光子上转换荧光发射,峰位与单光子荧光峰位相比发生红移(500～556 nm)。BMABN的双光子荧光强度和双光子吸收截面分别是BECVN的3倍和30.4倍。     

Studies on the Spectral-Luminescent Properties of the Novel Homodimer Styryl Dyes in Complexes with DNA

Series of homodimer styryls containing on (p-dimethylaminostyryl) pyridinium residues that are connected with aliphatic linkage group was synthesized. Spectral luminescent properties of obtained dyes in free state and in nucleic acids presence were studied. It was shown that DNA binding affinity of the novel homodimers exceeds that of parent monomer (p-dimethylaminostyryl)pyridine iodide. For homodimers with the linkage 4–10 carbon atoms preference in binding to DNA than to RNA was observed. It could be concluded that parent monomer has different mechanisms of binding to nucleic acids than corresponding homodimer dye.     

Two-photon induced optical-power limiting and upconverted superradiance properties of a new organic dye HEASPI

A new organic dye trans-4-[p-(N-ethyl-N-hydroxyethylamino)styryl]-N-methylpyridinium iodide (abbreviated as HEASPI hereafter) with large two-photon absorption (TPA) cross section and excellent upconverted superradiance properties was synthesized in our group recently. The TPA cross section was measured to be σ₂′=7.0×10⁻⁴⁸ cm⁴ s/photon by using an open aperture Z-scan system. Linear absorption, single-photon induced fluorescence, two-photon induced fluorescence and two-photon pumped (TPP) upconverted superradiance properties were systematically studied. The highest net upconversion efficiency from the absorbed pump energy to the output upconverted superradiance energy is as high as 19.6% at the pump energy of 2.07 mJ from a mode-locked Nd : YAG picosecond laser. The dye solution also shows a clear optical power limiting effect.     

Optical Properties of a New Two-photon Absorption Dye DEASPI

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Optical Properties of a New Two-photon Absorption Dye DEASPI

The two-photon absorption (TPA), TPA-induced frequency up-conversion emission, and two-photon pumped (TPP) frequency up-conversion lasing properties of a new synthesized dye Trans-4-［p-(N,N-diethylamino)styryl］-N-methylpyridinium iodide (DEASPI) were experimentally studied. This new dye has a moderate TPA cross-section of σ2=6.9×10-48 cm4*s/photon at 1064 nm, but exhibits a high lasing efficiency. The overall superradiant lasing conversion efficiency is as high as 10.7% at the pump energy of 2.14 mJ.     

Two-photon absorption-induced optical properties of a new lasing dye in two solvents

Pumped by an infrared lasing and its frequency-doubling source, the fluorescence and superradiance emission properties of a new lasing dye trans-4-[4^′-(N,N-diethylamino)styryl]-N-methyl pyridinium methyl sulfate (abbreviated DEASPS), have been studied in benzyl alcohol and chloroform. The two-photon absorption (TPA) and emission properties of DEASPSare influenced by the solvents used. The emission wavelength of the dye in benzyl alcohol is redshifted relative to that in chloroform. The lifetime of two-photon absorption-induced fluorescence is about 529 ps in benzyl alcohol, whereas it is 340 ps in chloroform. Correspondingly, the one-photon-excited fluorescence lifetimes in the two solvents are also given. The upconversion efficiency of DEASPS in chloroform is higher than in benzyl alcohol. Finally, the effective molecular two-photon absorption cross-sections were measured by the nonlinear transmittance method. It is found that the optical limiting effect of the dye in benzyl alcohol is stronger than that in chloroform. Received: 3 May 2001 / Revised version: 6 July 2001 / Published online: 19 September 2001     

Temporal and spectral properties of picosecond two-photon pumped cavity lasing of an organic dye HEASPS

The operation of cavity lasing of a two-photon absorption (TPA) organic dye, trans-4-[p-(N-hydroxyethyl-N-ethylamino)styryl]-N-methylpyridinium p-toluene sulfonate (abbreviated as HEASPS), by using a picosecond infrared laser as the pump source, is reported. The lifetimes of TPA fluorescence of this dye in different solvents were measured. Temporal profiles of cavity lasing show obvious oscillations and magnification of the feedback light. By using the difference of re-absorption coefficients at different conditions, we explained the blue shift for cavity lasing and superradiance compared with TPA-induced fluorescence. The long lifetime of fluorescence is helpful to the generation of cavity lasing. Received: 18 June 2001 / Revised version: 5 November 2001 / Published online: 17 January 2002     

Fluorescence Study of DNA–Dye Complexes Using One-Photon and Two-Photon Picosecond Excitation

Preliminary results of investigation of one-photon- and two-photon-induced fluorescence of acridine orange (AO), epirubicin (ER), hypericin (HYP), and ethidium bromide (EB) in complexes with DNA are presented. A spectrofluorometer based on a picosecond Nd:YAG laser was used for investigations of two-photon (1064-nm, 1-mJ, 40-ps) and one-photon (532- and 355-nm) dye excitation. The spectra of two-photon-induced fluorescence of dyes and their complexes with DNA as well as the kinetics of dyes' fluorescence intensification during their interactions with DNA in dependence on the biomacromolecule concentration were obtained. The intensities of AO, HYP, and EB fluorescence were increased 2.4, 3.2, and 8 times, respectively, after binding with DNA at two-photon excitation, while at one-photon excitation the corresponding values were 2.5, 3.7, and 10 times. The difference in fluorescence enhancement during DNA–dye complex formation at linear and nonlinear excitation may possibly be associated with the fact that the cross sections of one-photon and two-photon absorption, in general, change unequally during the binding of dyes to organic molecules and bathocromic shift of the electronic transitions. It was shown that the peak of AO fluorescence shifted to a longer wavelength on 10 nm after two-photon excitation at 1064 nm in comparison with one-photon excitation at 532 nm.     

Spectroscopic study of interaction of styrylcyanine dye Sbt and its derivatives with bovine serum albumin

The spectral-fluorescent characteristics of styrylcyanine dye Sbt ((E)-2-(4-(dimethylamino) styryl)-3-methylbenzo[d]thiazol-3-ium iodide) and homodimers, dyes conjugated with two chromophores in aqueous solutions without and in the presence of bovine serum albumin (BSA), are studied. It is established that in the presence of BSA for dyes Dbt-5 and Dbt-10, an increase of the absorptivity, a slight broadening and the emergence of new band on the short wavelength range with λ_max=410 nm is observed; also hypsochromic shift of the absorption and fluorescence at 30 nm and 7 nm, respectively for the dye D-183 is observed. The intensity of the fluorescence emission fundamental band in all the studied dyes in the presence of BSA increases by 3.5 to 55 times. The binding constant (K) and number of binding sites (N) of studied dyes with BSA are determined. The dependence of the binding constants with BSA from the dipole moment of dye molecules is identified, which shows that in addition to the electrostatic attraction forces between molecules of styrylcyanine dyes with BSA, hydrophobic interactions are essential. It is shown that the aggregation of dye affects the processes of interaction of the dyes with the BSA.     

Visualisation of mitochondria in living neurons with single- and two-photon fluorescence laser microscopy

In this work we investigated the relative merits of conventional single-photon confocal laser scanning fluorescence microscopy (CLSM) and two-photon laser scanning fluorescence microscopy (2p-LSM) for the study of mitochondria in living neurons. Dorsal root ganglion neurons were loaded with the mitochondrion-specific fluorescent dye JC-1, the ratio between red (J-aggregates) and green (monomer) fluorescence of which reflects mitochondrial membrane potential. Cells were illuminated at 488 nm for single-photon excitation or at 870 nm for two-photon excitation. In both modalities we found that mitochondria showed: (i) similar appearance; (ii) similar fluorescence ratio values over both whole cell bodies and individual mitochondria; and (iii) similar responses to mitochondrial uncoupler, which dropped the ratio values by 50%. However, 2p-LSM exhibited several advantages over CLSM: (i) better signal/noise ratio in the green emission channel; (ii) less phototoxicity upon repetitive scanning in the focal plane; and (iii) no significant loss of image quality upon repetitive scans in the z direction. We conclude that, while both techniques enable visualisation of individual mitochondria in living cells, 2p-LSM has significant advantages for physiological work requiring time-lapse experiments or four-dimensional reconstructions of mitochondria.     

Molecular Geometry Optimization, Two-Photon Absorption and Electrochemistry of New Diphenylethylene Derivatives Linking with Benzophenone Moiety Through Ether Covalent Bond

This paper presents the molecular geometry optimization, two-photon absorption and electrochemistry of new dyes containing benzophenone part, including 4-(p-benzoyl-benzyloxy)yl-4'-nitro-diphenylethylene (C1), 4-[N-methyl-N-(2-(p-benzoyl-benzyloxy)yl-ethyl]-4'-nitro-diphenylethylene (C2), 4-[N-ethyl-N-(2-(p-benzoyl-benzyloxy)yl-ethyl]-4'-nitro-diphenylethylene (C3), and 4-N, N-bis[(2-(p-benzoyl-benzyloxy)yl-ethyl]-4'-nitro-diphenyl ethylene (C4). The molecular structural parameters show that the coplanarity of diphenylethylene moiety is diminished in the excited state for C1, while it is enhanced for C2, C3 and C4. The electron density distribution of frontier orbital suggests that the derivatives exhibit (π, π) transition with internal charge transfer character, and the extent of charge transfer of C2, C3 and C4 is larger than that of C1. The derivatives display remarkable two-photon absorption (TPA) induced up-converted emission under 800 nm Ti: Sapphire femtosecond laser excitation. The maximal TPA emission wavelength of C2, C3 and C4 is red-shifted with respect to that of C1. TPA cross sections of C2, C3 and C4 are larger than those of C1. The cyclic voltammograms and the fluorescence lifetimes of the derivatives were determined and discussed.     

Measurement of two-photon excitation cross-section of a new symmetric-type fluorene derivative

A new dye, 2,7-bis(4-methoxystyryl)-9,9-bis(2-ethylhexyl)-9H-fluorene, has been synthesized, which is a d-π-d symmetrical-type fluorene derivative. The two-photon absorption (TPA) of this new dye has been experimentally studied by comparable two-photon-induced fluorescence method. This new dye has a TPA cross-section of at 790 nm/13 fs.     

Two-photon absorption and nonlinear optical properties of a new organic dye PSPI

Trans-4-[p-(pyrrolidinyl)styryl]-N-methylpyridinium iodide (abbreviated as PSPI thereafter) is a two-photon absorption (TPA) dye newly synthesized by our research group. It possesses much larger TPA cross-section and much stronger upconversion fluorescence emission than those of common organic dye (such as rhodamine) when excited with near infrared (IR) radiation. TPA spectrum and upconversion efficiency spectrum of HEASPI solution at various wavelengths were measured. There is 34 nm blue shift for the central wavelength of the TPA induced absorption peak compared with two times of the linear absorption peak. The biggest molecular TPA cross-section σ₂^′ is 2.85×10⁻⁴⁷ cm⁴ s/photon at 930 nm. At 1064 nm, σ₂^′ is 3.12×10⁻⁴⁸ cm⁴ s/photon. The highest efficiency is 3.9% at 1010 nm, whereas 2.9% at 1064 nm. Its optical power limiting properties at 930 nm have also been illustrated.     

Study on the Synthesis and Spectra of a Novel Kind of Carbozole Benzothiazole Indole Styryl Cyanine Dye with a Carbazole Bridged Chain

Based on the frequently-used cyanine dye probe thiazole orange (TO) and Cy3, a novel kind of styryl cyanine dye was designed and synthesized. Carbazole was inserted into the structures of two cyanine dyes, TO and Cy3, to act as a bridge to link the benzothiazole and indole. This modification resulted in a novel kind of carbozole benzothiazole indole cyanine dye with a carbazole-bridged chain. The dyes were characterized by HNMR and MS. The spectra of the novel dyes were also studied and the results showed that the fluorescence wavelength of novel carbazole benzothiazole indole cyanine dye shifted red, the Stokes shift and Fluorescence quantum yields increased and the fluorescence intensity was enhanced compared to that of TO. These results indicated that the novel dye could be used as an excellent fluorescent probe in biological labeling.     

Synthesis and Spectra of a Kind of Novel Longer-Wavelength Benzoxazole Indole Styryl Cyanine Dye with a Carbazole-Bridged Chain

Based on cyanine dye probe oxazole yellow (YO) and Cy₃, a series of novel styryl cyanine dyes were designed and synthesized. Carbazole was inserted into the structures of YO and Cy₃ to act as a bridge to link the benzoxazole and indole group. This modification resulted in a novel kind of benzoxazole indole styryl cyanine dye with a carbazole-bridged chain. The dyes were characterized by ¹HNMR and MS. The spectra of the novel dyes were also performed and the results showed that the maximum emission wavelength of the carbazole styryl cyanine dye was shifted red, the Stokes shift increased and the fluorescence intensity enhanced compared with those of YO and Cy₃. These results indicated that the novel dye could be used as an excellent fluorescent probe in biological labeling.     

A Series of Carbazole Cationic Compounds with Large Two-Photon Absorption Cross Sections for Imaging Mitochondria in Living Cells with Two-Photon Fluorescence Microscopy

A series of carbazole cationic compounds based on donor- Π—acceptor (D-Π-A) structure were synthesized and characterized. They exhibit large two-photon absorption cross sections when excited by a 810 nm a laser beam, and their photophysical properties show that the intramolecular charge transfer (ICT) character is predominant. Moreover these compounds can easily pass though the intact cell membrane of living cells, amongst, 3-(1-hydroxyethyl -4-vinylpyridium iodine)-N-butyl carbazole (9B-HVC) has been proven to be capable of accumulating within the mitochondria possessing large membrane potential and imaging this organelle in living cells by means of two-photon fluorescence microscopy. At the same time usable fluorescent photos can be obtained at lower incident excitation power (5 mW) and low-micromolar concentrations (2 μM), which does not result in significant reduction in cell viability over a period of at least 24 h.     

有机染料HEASPS在不同波长时的激发态吸收和双光子吸收性质研究

提出了有机染料反式 4 [4′ (N 羟乙基 N 乙基胺基 )苯乙烯基 ] N 甲基吡啶对甲苯磺酸盐 (简称HEASPS)非线性吸收的三态模型。认为在较短波长 (<1 0 0 0nm)时 ,双光子吸收和激发态吸收同时存在。成功地解释了该染料双光子吸收峰相对线性吸收峰的两倍的明显蓝移 ,以及最高上转换效率波长相对最强吸收波长的明显红移。提出了一种新的计算方法 ,利用不同波长的非线性透过率和不同波长的上转换效率 ,求出了该染料的双光子吸收截面和激发态吸收系数。     

取代效应对脂滴检测NAPBr染料分子的双光子吸收和激发态性质影响的理论探究

双光子荧光染料分子在生物医学成像中具有广阔的应用前景,但取代效应对分子结构以及光物理性质影响的探求相对匮乏. 本文设计并研究了一系列脂滴检测染料分子,分析了分子的光学性质以及无辐射跃迁等. 通过分子内弱相互作用和电子- 空穴布居分析,阐述了其内在机理. 结果表明,所研究的分子均具有优良的光物理性能、高效荧光量子产量、大的斯托克斯位移以及显著的双光子吸收截面等. 本工作合理地解释了实验现象并阐述了取代效应对脂滴检测NAPBr染料分子的双光子吸收和激发态性质的影响,这为设计新型的高效有机分子提供了理论指导.