Improved Fluorescent Proteins for Single-Molecule Research in Molecular Tracking and Co-Localization

Three promising variants of autofluorescent proteins have been analyzed photophysically for their proposed use in single-molecule microscopy studies in living cells to compare their superiority to other fluorescent proteins previously reported regarding the number of photons emitted. The first variant under investigation the F46L mutant of eYFP has a 10% greater photon emission rate and > 50% slower photobleaching rate on average than the standard eYFP fluorophore. The monomeric red fluorescent protein (mRFP) has a fivefold lower photon emission rate, likely due to the monomeric content, and also a tenfold faster photobleaching rate than the DsRed fluorescent protein. In contrast, the previously reported eqfp611 has a 50% lower emission rate yet photobleaches more than a factor 2 slowly. We conclude that the F46L YFP and the eqfp611 are superior new options for single molecule imaging and tracking studies in living cells. Studies were also performed on the effects of forced quenching of multiple fluorescent proteins in sub-micrometer regions that would show the effects of dimerization at low concentration levels of fluorescent proteins and also indicate corrections to stoichiometry patterns with fluorescent proteins previously in print. We also introduce properties at the single molecule level of new FRET pairs with combinations of fluorescent proteins and artificial fluorophores. Authors contributed equally to this article.     

Practical Use of Corrected Fluorescence Excitation and Emission Spectra of Fluorescent Proteins in Förster Resonance Energy Transfer (FRET) Studies

Corrected fluorescence excitation and emission spectra have been obtained from several enhanced variants of the green fluorescent protein (EGFP) isolated from the jellyfish Aequorea victoria, blue fluorescence protein (EBFP), cyan fluorescent protein (ECFP), EGFP and yellow fluorescent protein (EYFP–citrine) and from the red fluorescent protein (DsRed) isolated from the coral species Discosoma. The spectra are stored in a database. This report describes how the spectra can be used as templates to derive the critical transfer distance for any pair of fluorescent proteins.     

Fluorescence resonance energy transfer between cerium ion(III) and levofloxacin in micellar solution and its analytical application to the determination of levofloxacin

Fluorescence resonance energy transfer between cerium ion(III) and levofloxacin is studied in a micellar solution of cetyltrimethyl ammonium bromide. A non-fluorescent 1:2 complex was formed between excited cerium ion(III) and ground state levofloxacin. The fluorescence of cerium ion(III) is quenched by levofloxacin with the quenching in accordance with the Stern–Volmer relation. The analytical relationship was established between the ratio of the fluorescence of levofloxacin present and absent cerium ion (III) and the concentration of levofloxacin, which helped to estimate the content of levofloxacin directly.     

消癌平诱导人肺腺癌(ASTC-a-1)细胞内caspase-3活化的荧光光谱分析

采用CCK-8技术检测发现传统中药消癌平（XAP）对人肺腺癌（ASTC-a-1）细胞的增殖活性具有显著的抑制作用。利用共聚焦扫描荧光显微成像、荧光共振能量转移（FRET）及其受体光漂白技术证实了基于FRET技术构建的SCAT3质粒在ASTC-a-1细胞中的稳定表达。将消癌平加入细胞培养液中培育细胞,并在不同的时间检测活细胞内SCAT3的荧光发射光谱,从而监测细胞中caspase-3的活化状态。实验结果表明：(1)消癌平可以有效抑制人肺腺癌（ASTC-a-1）细胞的增殖活性并诱导细胞的死亡,消癌平对细胞的抑制作用具有剂量依赖性。(2)消癌平处理细胞72 h后,细胞内大量的SCAT3被切割,表明细胞内caspase-3的活化水平明显升高。(3)将含消癌平的细胞培养液与细胞共同培养24 h,然后采用没有消癌平的细胞培养液培养细胞48和72 h后,细胞内SCAT3的光谱没有明显改变,表明消癌平作用细胞24 h内没有显著激活细胞内的caspase-3。     

Spectroscopic studies of lanthanide(III) ion complexes with diethyl(phthalimidomethyl) phosphonate

Complexation and photophysical properties of complexes of lanthanide ions, Ln(III), with diethyl(phthalimidomethyl)phosphonate ligand, DPIP, were studied. Interactions between Ln(III) and DPIP were investigated using Nd(III) absorption and Eu(III) and Tb(III) luminescence (emission and excitation) spectra, recorded in acetonitrile solution containing different counter ions (NO₃^-, Cl^- and ClO₄^-). Results of the absorption spectroscopy have shown that counter ions play a significant role in the complexation of Ln(III)/DPIP complexes. Studies of luminescence spectra of Eu(III) and Tb(III) ions proved that the formation of Ln(III)/DPIP complexes of stoichiometry Ln:L=1:3 is preferred in solution. Based on the results of elemental analysis, Nd(III) absorption spectra and IR and NMR data, it was shown that the DPIP ligand binds Ln(III) ions via oxygen from phosphoryl group, forming complexes of a general formula Ln(DPIP)₃(NO₃)₃·H₂O, in which the NO₃^- ions are coordinated with the metal ion as bidentate ligands. Luminescent properties and energy transfer, from the ligand to Ln(III) ions in the complexes formed, were studied based on the emission and excitation spectra of Eu(III) and Tb(III). Their luminescent lifetimes and emission quantum yields were also measured.     

Ln(III)-cored complexes based on 2-thenoyltrifluoroacetone ligand for near infrared emission: Energy transfer pathway and transient absorption behavior

Lanthanide(III)-cored complexes based on 2-thenoyltrifluoroacetone (TTA) ligand for near infrared (NIR) emission have been developed to investigate the energy transfer pathway from the antenna ligand to the Ln³⁺ ion. Their photophysical studies indicate the sensitization of Ln³⁺ luminescence by energy transfer through the excited triplet state of β-diketone ligand. Nanosecond (ns) transient absorption behavior of Ln³⁺-[TTA]₃(terpy) complexes at room temperature is explored. The triplet-triplet absorption spectrum for Gd³⁺-[TTA]₃(terpy) is observed under degassed condition, whereas it is hardly observed in Er³⁺-[TTA]₃(terpy) complex. The sensitizing process in Er³⁺-[TTA]₃(terpy), through the triplet state of TTA ligand to Er³⁺ ion, is also independent on the presence of oxygen. It indicates that the energy transfer rate through the excited triplet state of β-diketone ligand to Er³⁺ ion occurs approximately faster than that of the oxygen quenching rate.     

荧光共振能量转移-荧光寿命显微成像(FRET-FLIM)技术在生命科学研究中的应用进展

细胞是动植物结构和生命活动的基本单位.细胞过程的一个重要特点就是其生化组分在时空调控上的相互作用关系.然而,利用传统的生化方法(如酵母双杂交系统、pull-down系统等)很难在空间上评估活细胞内分子间的相互作用.光学技术的快速发展,为研究活细胞中生物分子的时空动态提供了新的遗传研究工具,其中荧光共振能量转移-荧光寿命...     

Fluorimetric Determination of Thyroxine Hormone with Eu(III)-(Pyridine-2,6-Dicarboxylate) Tris Complex

We describe a method for the determination of thyroxine (Thy) using its quenching effect on Eu(PDA)₃ ^3- tris complex fluorescence. The relative fluorescence intensities are measured at fixed _exc = 282 nm, _em = 615 nm by titrating the metal complex with Thy in piperazine buffer solution at pH 6.5. Data indicated an associative type of reaction of two molecules valid between 0.0 < R < 1.0, R being the mole ratio of Eu(PDA)₃ ^3- to Thy. Over this ratio and up to (R 1.0) collisional quenching of Eu(PDA)₃ ^3- complex ion emission is seen as a result of heavy atom effect, intermolecular energy transfer playing the main role. This is also confirmed by the Stern-Volmer equation. In optimized experimental conditions, the L- form of Thy is determined in a range of 15.5–551.6 g/ml (2.0 × 10^–5 – 7.1 × 10^–4 M) with relative error of ±1.17%.     

Green Fluorescent Protein (GFP) as a Marker for Cell Viability After UV Irradiation

Generation of Chinese Hamster Ovary (CHO) cell lines stably expressing green fluorescent protein (GFP) was achieved using a plasmid vector that encoded the red-shifted pCX-xGFP under the control of a strong hybrid promoter composed of a CMV enhancer and a -actin/-globin gene promoter. Cotransfection of the promoter-less pSV2-Neo helper plasmid transmitting neomycin resistance was followed by selection with the antibiotic G418. Constitutive GFP expression could be visualized in living and fixed cells using fluorescence spectroscopy, fluorescence microscopy, and flow cytometry. DNA repair-proficient (AA8) and deficient (UV5) CHO strains were used for survival tests after UVC irradiation. Cells carrying the GFP construct (AA8-pGFP, UV5-pGFP) show the same response to UV irradiation (colony forming ability) as their nontransformed parental cell lines (AA8, UV5). Using GFP as a marker for cell viability, cells were harvested after certain postirradiation growth periods and the numbers of GFP expressing cells and fluorescence intensities were determined by FACS analysis. Generally, GFP fluorescence in irradiated cells is not seen when cell membranes are damaged (leak-out of the soluble GFP). Irradiated cells without membrane damage express GFP continuously (leading to a dose-dependent increase in GFP contents).     

The use of the isotropic orientation factor in fluorescence resonance energy transfer (FRET) studies of the actin filament

A Dale-Eisinger style analysis (R. E. Daleet al., Biophys. J. 26, 161, 1979) is used to produce three-dimensional plots that display the limits on the average orientation factor k ² that is required to calculate molecular distances in F-actin from fluorescence resonance energy transfer measurements. Maxima and minima plots are generated for the transfer of energy from a donor to a single acceptor and for transfer to multiple acceptors that are related by F-actin helical symmetry. The analysis is performed in terms of dipole cone half-angles rather than depolarization factors, in order to facilitate the modeling of the multiple acceptor problem. Calculations are carried out under the restrictive condition of a single electric dipole moment per fluorophore. In addition, both surface and volume averaging of the donor and acceptor dipoles are considered. Comparisons between the plots show that for the multiple acceptor cases with F-actin symmetry, there is a great reduction in the range for maxima and minima limits on k ². The calculations also suggest guidelines for the choice of fluorescence label that will result in an average orientation factor occurring within acceptable limits, i.e., inside the limits for which k ²=2/3 may be employed. Thus, without having detailed knowledge of the mean donor or acceptor dipole relative orientations, the use of k ²=2/3 in radial coordinate studies of F-actin is more than reasonable and is fairly assured of being correct.     

A sensitive fluorimetric method for determination of trace amounts of nitrite based on luminescence energy transfer

An efficient luminescence energy transfer (LET) system based on terbium(III)-sodium hexametaphosphate (Tb/SHMP) chelates as donor and 4-((4-(2-aminoethylamino)naphthalen-1-yl)diazenyl)benzenesulfonic acid dihydrochloride (ANDBS) as acceptor was developed for sensitive determination of trace nitrite. Stable and strong fluorescence Tb/SHMP chelates were prepared in aqueous solution. Based on Griess Reaction, ANDBS was generated by the quantitative reaction of nitrite, sulfanilamide and N-(1-naphtyl)-ethylenediamine dihydrochloride (N1NED). The degree of the overlap was effective for LET between the emission spectrum of Tb/SHMP chelates and the absorption spectrum of ANDBS. Based on the luminescence intensity quenching of Tb/SHMP chelates in proportion to the trace amounts of nitrite, a new assay for the selective and sensitive determination of nitrite was developed. Under the optimum conditions, the linear calibration graph was obtained with a linear range of 0.00040-0.20 μg mL⁻¹ (R=0.99657). The detection limit of was 0.00010 μg mL⁻¹ (R=0.99657). The method was applied successfully to the determination of nitrite for synthetic samples.     

Hydrophobicity effect on interactions between organic molecules in nanocages of surfactant micelle

Interactions between the cationic polymethine dyes 3,3′-diethyloxacarbocyanaine iodide (DiOC₂), 3,3′-dioctadecyloxacarbocyanine iodide (DiOC₁₈), and 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) in the nano-scale volume of anionic sodium dodecylsulfate (SDS) micelles have been studied using optical spectroscopy techniques. The solubilization of pairs of dye molecules (DiOC₁₈/DiI or DiOC₂/DiI) within SDS micelles was monitored by Forster resonance energy transfer (FRET) between the dyes in each pair. The influence of the hydrophobicity of the dyes DiOC₂ and DiOC₁₈ on the efficiency of their binding to SDS micelles and, consequently, on the efficiency of FRET between DiOC₁₈ and DiI or DiOC₂ and DiI fluorophors has been analyzed. It has been shown that hydrophobic interactions in addition to electrostatic interactions are of key importance for the binding between the positively charged dyes and negatively charged surfactant micelles. Translated from Zhurnal Prikladnoi Spektroskopii, Vol. 75, No. 5, pp. 646–652, September–October, 2008.     

Use of Terbium(III) as a Probe for the Ion-Binding Properties of Tactic Polyacids and Triacidic Model Compounds

The binding properties of Tb(III) ions in configurationally different environments were investigated using luminescence intensity and lifetime measurements. The emission intensity of Tb(III) (⁵D₄–⁷F₅ transition) is directly dependent upon the number of coordinated water molecules (quenchers) bound in the inner coordination sphere. The more efficiently a ligand coordinates to Tb(III) ion, the more water molecules are expelled from the coordination sphere, thereby enhancing the luminescence intensity and lifetime of the ion. Isotactic and syndiotactic poly(methacrylic acids) (PMAs) were neutralized and complexed with Tb(III) ions in aqueous solutions. The luminescence intensities and lifetimes were monitored with _exc = 265, the hypersensitive excitation band at 286 and 370 nm. The isotactic PMA/Tb(III) complex exhibited a six times greater luminescence intensity than the syndiotactic PMA complex. Lifetime measurements showed 2.4 water molecules coordinated to Tb(III) ion in the isotactic PMA complex, while 3.4 water molecules were found to remain in the syndiotactic complex. Similar studies were also conducted on small organic model compounds such as Kemp's triacid and its configurational isomer. These data supported the polymeric results where the isotactic model, Kemp's triacid, exhibited a higher luminescence intensity and a longer lifetime than the Kemp's isomer. Lifetime results showed the Kemp's molecule retained approximately 2.7 water molecules, compared to four water molecules for the isomer. The validity of using the Kemp's molecules as polymeric models is also discussed.     

Fluorescent polystyrene microspheres with large Stokes shift by fluorescence resonance energy transfer

We prepared fluorescent microspheres with notably large Stokes shift and long-wavelength fluorescence by applying fluorescence resonance energy transfer (FRET) between two common julolidine dyes. Short distance between dye molecules caused by high dye concentration results in efficient FRET in microspheres. However, adequate dye concentration and moderate molar ratio of the donor and acceptor should be chosen to avoid aggregation of dye molecules, which leads to the decrease of fluorescent intensity. Microrspheres with average distance between dye molecules of 1.94 nm and molar ratio of 3.08:1 realize highly efficient FRET with no fluorescence of donor and intense long-wavelength emission of acceptor. In addition, the applied solvent evaporation method for preparing microspheres provided better protection of dyes from ambient medium than traditional surface-labeled method. These results demonstrate the feasibility of applying FRET in microspheres to expand useful fluorescent probes, and reveal their potential application in bioassays field.     

Competition Between Energy Transfer Quenching and Chelation Enhanced Fluorescence in a Cu (II) Coordinated Conjugated Polymer System

An investigation of the mechanism of the fluorescence quenching by Cu²⁺ for a conjugated polymer system initially designed as a fluorescence “turn-on” chemosensor based on chelation enhanced fluorescence (CHEF) is described in this paper. Unlike all other metal cations tested, the polymer/Cu²⁺ hybrid system with a 1:1 ratio between the receptor and Cu²⁺ has only weak fluorescence with λ_max = 490 nm and a quantum yield of 0.004 in THF at room temperature. In solvent glasses at 77 K the fluorescence remained quenched suggesting that the quenching mechanism was due to energy transfer between the Cu²⁺ and the conjugated polymer backbone. The energy transfer quenching competes effectively with the electron transfer involved in the CHEF resulting in a more selective chemosensory system. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

半导体聚合物量子点-亚甲基蓝荧光能量转移体系测定水中Bi~(3+)

基于半导体聚合物量子点和亚甲基蓝间的荧光共振能量转移构筑了一种铋离子荧光探针。亚甲蓝通过静电作用吸附到半导体聚合物量子点表面使其荧光发生猝灭。铋离子存在时,由于它与半导体聚合物量子点的结合力强于亚甲蓝,使体系的能量转移得到抑制,荧光恢复。在最优化条件下,线性范围为0~0.5%mol/L(R=0.995 5),最低检出限为16 nmol/L。该方法成功用于湖水中的Bi3+测定,结果令人满意。     

估算供体-受体对荧光共振能量传递效率的研究

在有机白光LED的研究中,能量传递效率计算方法的研究是一项很有意义的工作。作者在总结前人工作的基础上,提出了一种利用有机分子发射谱和激发谱估算有机分子荧光共振能量传递(FRET)效率相对值的新方法,这种方法的优点是数据处理过程简单,对实验仪器设备要求不高,并且不需要通常计算方法所需的荧光量子产率等参数,缺点是只能计算能量传递的相对值。利用这种方法计算了甲萘酚、溴甲酚紫、萤光素钠盐三种能量受体和能量供体核黄素在不同浓度下能量传递效率的相对值,计算结果与实验结果基本符合,验证了此方法的合理性。     

Fluorescence anisotropy of cyanobacterial phycobilisomes oriented in polyvinyl alcohol (PVA) films

Polarized absorption (at 296 and 85 K), fluorescence, and photoacoustic (at 296 and 85 K) spectra of antenna complexes—phycobilisomes isolated from cyanobacteriaTolypothrix tenuis andOscillatoria and embedded in isotropic and anisotropic polyvinyl alcohol films—were measured. From the sets of polarized components of emission, the anisotropy of fluorescence for the pools of differently oriented molecules was calculated. On the basis of polarized photoacoustic and emission spectra, the competition between the process of thermal deactivation of excitation and excitation energy transfer in a chain of excitation donor and acceptor chromophores of phycobilisomes is discussed.     

Spectroscopic studies on the molecular interaction between salicylic acid and riboflavin (B2) in micellar solution

The interaction between salicylic acid (SA) and riboflavin (RF) was studied by Fluorescence Resonance Energy Transfer (FRET) in micellar solution. The riboflavin strongly quenches the intrinsic fluorescence of SA by radiative energy transfer. The extent of energy transfer in sodium dodecyl sulphate (SDS) micellar solution of different concentration is quantified from the energy transfer efficiency data. It is seen that the energy transfer is more efficient in the micellar solution. The critical energy transfer distance (R₀) was determined from which the mean distance between SA and RF molecules was calculated. The quenching was found to fit into Stern-Volmer relation. The results on variation of Stern-Volmer constant (K_sv) with quencher concentration obtained at different temperatures suggested the formation of complex between SA and RF. The association constant of complex formation was estimated and found to decrease with temperature. The values of thermodynamic parameters ΔH, ΔG and ΔS at different temperatures were estimated and the results indicated that the molecular interaction between SA and RF is electrostatic in nature.     

Phosphorescence of Poly(Methylphenyl)Silane Films

In the 5–100 K temperature range, the photoluminescence spectra of poly(methylphenyl)silane (PMPS) and polystyrene films doped with acetophenone, propiophenone, benzophenone, diphenyl, and naphthalene, as well as the thermostimulated luminescence spectra of neat PMPS films, have been investigated. It has been shown that the PMPS film phosphorescence spectrum consists of a structureless band with a maximum near 450 nm, and the literature structural phosphorescence spectrum of this polymer may belong to an uncontrolled impurity or a defect with chromophore groups of acetophenone.