荧光蛋白MiCy、Citrine作为FRET传能对的研究

探索了2种新型荧光蛋白MiCy、Citrine作为荧光共振能量转移技术(FRET)传能对的可能性.首先通过基因工程方法表达纯化了这2种蛋白,并对其进行了光谱学研究,由于MiCy具有高的量子产率,且MiCy与Citrine的光谱重叠积分J较大,计算表明MiCy与Citrine的Forster距离为R0=5.81 nm,比CFP-YFP的大(R0=5 nm).进一步以Ni2+-NTA-agarose为FRET模型,结果表明:无论在荧光分光光度计还是在激光共聚焦显微镜上,都可以明显地检测到MiCy与Citrine的能量转移.由于二者较文献报道的其它FRET对具有更大的Forster距离,而且更适合以激光作为激发光进行测量,因此该FRET对应有更加广泛的应用前景.     

一种耐光漂白的碳菁型pH荧光探针的合成与细胞成像

设计合成了一种引入多个氯原子取代的耐光漂白的碳菁型pH荧光探针(CyCl4), 在500 nm波长光的激发下, 该探针具有530和596 nm两个波长的荧光峰, 其中的596 nm荧光峰在pH 8.5环境中具有最高的荧光信号值, 在pH<8.1和>9.8的条件下则荧光很弱, 通过密度泛函方法计算这三种荧光状态分别对应的三种构象结构A, B和C, 而结构B中的两个磺酸基构成分子内氢键而具有共面性几何构型, 共轭程度最好, 因而其荧光发射能力最强. 测量了该探针的荧光量子产率、瞬态荧光光谱和耐光漂白性能, 其耐光漂白性能强于一般染料型荧光探针. 最后将该探针应用于前列腺癌活细胞荧光成像, 并发现其在细胞膜表面的聚集现象.     

CdS量子点与曙红Y间的荧光共振能量转移研究

在水溶液中,量子点与有机荧光染料之间可能发生荧光共振能量转移(FRET)。本文以发射波长470nm的Cd S量子点为供体,曙红Y为受体,建立了Cd S量子点-曙红Y的FRET体系,研究了该体系的FRET参数。该体系受体供体数目比为8,猝灭效率为45.6%,增强效率为20.1%;供体-受体间的距离为4.4nm;临界能量转移距离为2.4nm。     

2-(2-羟基-5-氨苯基)苯并咪唑与氨基卟啉能量转移的研究

采用紫外光谱法和荧光光谱法研究了二氯甲烷溶液中2-(2-羟基-5-氨基苯基)苯并咪唑(NH_2-HBI)和5-(4-氨基)苯基-10,15,20-三苯基卟啉(NH_2-TPP)两分子之间的能量转移作用,测定了NH_2-HBI与NH_2-TPP作用的结合比及能量转移参数.结果表明,在光的激发作用下,形成了以NH_2-HBI为能量供体、NH_2-TPP为能量受体的荧光共振能量转移(FRET)体系,同时发生了NH_2-HBI激发态分子内质子转移(ESIPT)与FRET的耦合作用, NH_2-HBI分子的醇式构型与酮式构型的荧光都被部分的猝灭,而NH_2-TPP的荧光却明显的增强.两分子形成了1∶1的供体-受体作用分子对,体系的FRET能量转移效率(E_(FRET))为0.114,临界能量转移距离(R_0)为2.293nm,供-受体之间的距离(r)为3.227 nm.     

量子点:FRET的新发展

荧光共振能量转移(FRET)技术作为一种高效的光学“分子尺“,在生物大分子相互作用、免疫分析、核酸检测等方面有广泛的应用.但是许多有机染料吸收光谱较窄而发射光谱较宽,并且光漂白现象比较严重,使得FRET的应用受到了限制,因此迫切需要寻找新的能量供-受体对.由于量子点(QDs)相对于有机染料有很多优点,可以较好地应用于FRET,可能成为FRET领域发展的一个有意义的新方向,近来已引起了人们的关注.本文就FRET的原理以及量子点应用于FRET的最新进展情况做了评述.     

荧光增白剂28的光漂白特性研究

荧光增白剂28(F-28)为欧盟标准EN 648-2006《与食品接触的纸和纸板-荧光增白剂牢度的测定》中判断食品接触材料所含荧光增白剂是否向食品中迁移的标样。我们发现,在规定的波长365nm紫外光激发下,F-28所制成的标样会出现光漂白现象。因此,本文对F-28的光漂白特性进行研究。结果表明,紫外辐照时间为60s时,四级(3mg/L)、三级(8mg/L)、二级(31mg/L)、一级(125mg/L)滤纸和玻纤的最大荧光值衰减率分别为0.16%、0.29%、0.92%、0.44%和2.59%、1.99%、0.49%、3.14%;当辐照时间延长到1800 s时,最大荧光值衰减率分别增加为4.67%、4.22%、5.51%、2.75%和35.37%、39.78%、28.09%、39.38%。因此,在对食品接触材料是否含有荧光增白剂以及是否迁移的测试中,必须考虑光漂白因素。同时,滤纸作为迁移实验载体,在降低光漂白和稳定性方面比玻纤更具优势。     

新型SARS-CoⅤ 3CL蛋白酶荧光多肽底物的设计、制备及其酶动力学研究

以邻氨基苯甲酸(Abz)为荧光发射基团、2,4-二硝基苯基乙二胺(Eddnp)为荧光猝灭基团,设计合成了SARS-CoⅤ3CL蛋白酶的新型荧光多肽底物:H2N-E(Eddnp)STLQSGLK(Abz)-CONH2.用液相色谱-质谱(LC-MS)联用技术进行了表征,表明该多肽底物能被SARS-CoⅤ3CL蛋白酶识别,并在QS之间被专一性酶解.另外,利用该多肽底物的荧光共振能量转移(FRET)特性,对SARS-CoⅤ3CL蛋白酶的酶解动力学性质进行了研究,结果表明,此荧光多肽底物可以作为荧光探针,应用于SARS-CoⅤ3CL蛋白酶活性的测定及其抑制剂的筛选.     

量子点:FRET的新发展

荧光共振能量转移(FRET)技术作为一种高效的光学“分子尺”,在生物大分子相互作用、免疫分析、核酸检测等方面有广泛的应用。但是许多有机染料吸收光谱较窄而发射光谱较宽,并且光漂白现象比较严重,使得FRET的应用受到了限制,因此迫切需要寻找新的能量供-受体对。由于量子点(QDs)相对于有机染料有很多优点,可以较好地应用于FRET,可能成为FRET领域发展的一个有意义的新方向,近来已引起了人们的关注。本文就FRET的原理以及量子点应用于FRET的最新进展情况做了评述。     

CdTe量子点与罗丹明6G荧光共振能量转移体系的构建及机理研究

以巯基乙酸为修饰剂,水相合成不同尺寸的CdTe量子点(QDs),研究不同发射波长的CdTe QDs与罗丹明6G(R6G)之间的荧光共振能量转移(FRET)规律。结果发现,以发射波长为515nm的CdTe QDs为供体与R6G能量转移效率为58.5%,以R6G为供体与发射波长为605nm的CdTe QDs能量转移效率为49.4%,即在CdTe QDs-R6G的FRET体系中,量子点既可作为供体也可作为受体。在此基础上,构建了CdTe QDs(供体)-R6G(第一受体)-CdTe QDs(第二受体)的三元FRET体系,并研究了其双荧光共振能量转移的机理。     

量子点:FRET的新发展

荧光共振能量转移（FRET）技术作为一种高效的光学“分子尺”，在生物大分子相互作用、免疫分析、核酸检测等方面有广泛的应用。但是许多有机染料吸收光谱较窄而发射光谱较宽，并且光漂白现象比较严重，使得FRET的应用受到了限制，因此迫切需要寻找新的能量供-受体对。由于量子点（QDs）相对于有机染料有很多优点，可以较好地应用于FRET，可能成为FRET领域发展的一个有意义的新方向，近来已引起了人们的关注。本文就FRET的原理以及量子点应用于FRET的最新进展情况做了评述。     

Fluorescence energy transfer-sensitized photobleaching of a fluorescent label as a tool to study donor-acceptor distance distributions and dynamics in protein assemblies: studies of a complex of biotinylated IgM with streptavidin and aggregates of concanavalin A

A photokinetic method of detection of fluorescence resonance energy transfer (FRET) between special fluorescent labels is applied to study time-averaged spatial distribution of labeled proteins in protein assemblies. Prolonged irradiation of a sample at the absorption maximum of the energy donor initiates FRET-sensitized fluorescence photobleaching of the energy acceptor label, which was monitored by steady-state fluorimetric measurements. Kinetics of the acceptor photobleaching and kinetics of decreasing the efficiency of FRET from donors to unbleached acceptors were determined. The FRET efficiency was found from measuring sensitization of acceptor fluorescence. Analysis of the photokinetic data permits to estimate the time-averaged distribution of acceptors on donor-acceptor distances in the range of characteristic distances of FRET. Dynamic processes influencing donor-acceptor distances can be also investigated by the method. Application of the method is demonstrated by the studies of a complex of biotinylated IgM with streptavidin and aggregates composed of concanavalin A and sodium dodecyl sulphate. A new thiadicarbocyanine dye was used as the acceptor label. R-phycoerythrin and tetramethylrhodamine isothiocyanate were the donor labels. In the IgM-streptavidin complex, 16% of acceptors most contributed to FRET provided 90% of FRET efficiency, whereas acceptors made about the same time-averaged contribution to FRET in the concanavalin A aggregates.     

Quantitative FRET analysis with the EGFP-mCherry fluorescent protein pair

Fluorescence resonance energy transfer (FRET) between fluorescent proteins (FPs) is a powerful tool to investigate protein–protein interaction and even protein modifications in living cells. Here, we analyze the E⁰GFP-mCherry pair and show that it can yield a reproducible quantitative determination of the energy transfer efficiency both in vivo and in vitro . The photophysics of the two proteins is reported and shows good spectral overlap (Förster radius R ₀ = 51 Å), low crosstalk between acceptor and donor channels, and independence of the emission spectra from pH and halide ion concentration. Acceptor photobleaching (APB) and one- and two-photon fluorescence lifetime imaging microscopy (FLIM) are used to quantitatively determine FRET efficiency values. A FRET standard is introduced based on a tandem construct comprising donor and acceptor together with a 20 amino acid long cleavable peptidic linker. Reference values are obtained via enzymatic cleavage of the linker and are used as benchmarks for APB and FLIM data. E⁰GFP-mCherry shows ideal properties for FLIM detection of FRET and yields high accuracy both in vitro and in vivo . Furthermore, the recently introduced phasor approach to FLIM is shown to yield straightforward and accurate two-photon FRET efficiency data even in suboptimal experimental conditions. The consistence of these results with the reference method (both in vitro and in vivo ) reveals that this new pair can be used for very effective quantitative FRET imaging.     

Optimization of pairings and detection conditions for measurement of FRET between cyan and yellow fluorescent proteins.

Detection of F?rster resonance energy transfer (FRET) between cyan and yellow fluorescent proteins is a key method for quantifying dynamic processes inside living cells. To compare the different cyan and yellow fluorescent proteins, FRET efficiencies were measured for a set of the possible donor:acceptor pairs. FRET between monomeric Cerulean and Venus is more efficient than the ECFP:EYFP pair and has a 10% greater F?rster distance. We also compared several live cell microscopy methods for measuring FRET. The greatest contrast for changes in intramolecular FRET is obtained using a combination of ratiometric and spectral imaging. However, this method is not appropriate for establishing the presence of FRET without extra controls. Accurate FRET efficiencies are obtained by fluorescence lifetime imaging microscopy, but these measurements are difficult to collect and analyze. Acceptor photobleaching is a common and simple method for measuring FRET efficiencies. However, when applied to cyan to yellow fluorescent protein FRET, this method becomes prone to an artifact that leads to overestimation of FRET efficiency and false positive signals. FRET was also detected by measuring the acceptor fluorescence anisotropy. Although difficult to quantify, this method is exceptional for screening purposes, because it provides high contrast for discriminating FRET.     

A PHOTOCHEMICAL TECHNIQUE TO ENHANCE SENSITIVITY OF DETECTION OF FLUORESCENCE RESONANCE ENERGY TRANSFER

Abstract –A photochemical kinetic method of measuring small values of efficiency of fluorescence resonance energy transfer (FRET) between special probes is proposed. The FRET efficiency ( Ω ) is determined from kinetics of the photochemical reaction of the energy acceptor sensitized by FRET from the energy donor. The choice of an appropriate donor-acceptor pair permits the minimization of background reactions. Application of the method is demonstrated by the detection of FRET from 2,5-W.s(5- tert -butyl-2-benzoxasolyl)thiophen (BBOT) to acridine orange (AO) in phospholipid vesicles. Photobleaching of AO in the presence of CBr₄ was applied as a photochemical reaction of the acceptor. The reaction was monitored by steady-state fluorescence measurements. The FRET measurements were carried out by the proposed technique when the probe/lipid ratio and Ω were as small as 1.1 times 10^-5 M/M and 0.0017, respectively. Under these conditions, the rate constant of AO photobleaching was increased by 26% as compared with that of the reference sample without BBOT. The results suggest that applications of the technique may be useful in the study of the membrane topography.     

A PHOTOCHEMICAL TECHNIQUE TO ENHANCE SENSITIVITY OF DETECTION OF FLUORESCENCE RESONANCE ENERGY TRANSFER

Abstract: A photochemical kinetic method of measuring small values of efficiency of fluorescence resonance energy transfer (FRET) between special probes is proposed. The FRET efficiency (ω) is determined from kinetics of the photochemical reaction of the energy acceptor sensitized by FRET from the energy donor. The choice of an appropriate donor-acceptor pair permits the minimization of background reactions. Application of the method is demonstrated by the detection of FRET from 2,5- bis (5- tert -butyl-2-benzoxasolyl)thiophen (BBOT) to acridine orange (AO) in phospholipid vesicles. Photobleaching of AO in the presence of CBr₄ was applied as a photochemical reaction of the acceptor. The reaction was monitored by steady-state fluorescence measurements. The FRET measurements were carried out by the proposed technique when the probe/lipid ratio and ω were as small as 1.1 × 10⁻⁵ M/M and 0.0017, respectively. Under these conditions, the rate constant of AO photobleaching was increased by 26% as compared with that of the reference sample without BBOT. The results suggest that applications of the technique may be useful in the study of the membrane topography.     

Determination of cellulase colocalization on cellulose fiber with quantitative FRET measured by acceptor photobleaching and spectrally unmixing fluorescence microscopy

The determination of cellulase distribution on the surface of cellulose fiber is an important parameter to understand when determining the interaction between cellulase and cellulose and/or the cooperation of different types of cellulases during the enzymatic hydrolysis of cellulose. In this communication, a strategy is presented to quantitatively determine the cellulase colocalization using the fluorescence resonance energy transfer (FRET) methodology, which is based on acceptor photobleaching and spectrally unmixing fluorescence microscopy. FRET monitoring of cellulase colocalization was achieved by labeling cellulases with an appropriate pair of FRET dyes and by adopting an appropriate FRET model. We describe here that the adapted acceptor photobleaching FRET method can be successfully used to quantify cellulase colocalization regarding their binding to a cellulose fiber at a resolution <10 nm. This developed quantitative FRET method is promising for further studying the interactions between cellulase and cellulose and between different types of cellulases.     

Fluorescence lifetime based distance measurement illustrates conformation changes of PYL10-CL2 upon ABA binding in solution state

The fluorescence lifetime based FRET distance measurements using sitespecific incorporated unnatural amino acid HC and Alexa488 as FRET pair revealed the different conformations of PYL10-CL2 upon ABA binding.     

Photobleaching pathways in single-molecule FRET experiments

To acquire accurate structural and dynamical information on complex biomolecular machines using single-molecule fluorescence resonance energy transfer (sm-FRET), a large flux of donor and acceptor photons is needed. To achieve such fluxes, one may use higher laser excitation intensity; however, this induces increased rates of photobleaching. Anti-oxidant additives have been extensively used for reducing acceptor's photobleaching. Here we focus on deciphering the initial step along the photobleaching pathway. Utilizing an array of recently developed single-molecule and ensemble spectroscopies and doubly labeled Acyl-CoA binding protein and double-stranded DNA as model systems, we study these photobleaching pathways, which place fundamental limitations on sm-FRET experiments. We find that: (i) acceptor photobleaching scales with FRET efficiency, (ii) acceptor photobleaching is enhanced under picosecond-pulsed (vs continuous-wave) excitation, and (iii) acceptor photobleaching scales with the intensity of only the short wavelength (donor) excitation laser. We infer from these findings that the main pathway for acceptor's photobleaching is through absorption of a short wavelength photon from the acceptor's first excited singlet state and that donor's photobleaching is usually not a concern. We conclude by suggesting the use of short pulses for donor excitation, among other possible remedies, for reducing acceptor's photobleaching in sm-FRET measurements.     

Separating structural heterogeneities from stochastic variations in fluorescence resonance energy transfer distributions via photon distribution analysis

We establish a probability distribution analysis (PDA) method for the analysis of fluorescence resonance energy transfer (FRET) signals to determine with high precision the originating value of a shot-noise-limited signal distribution. PDA theoretical distributions are calculated explicitly including crosstalk, stochastic variations, and background and represent the minimum width that a FRET distribution must have. In this way an unambiguous distinction is made between shot-noise distributions and distributions broadened by heterogeneities. This method simultaneously and effectively extracts highly resolved information from FRET distributions. The theoretical histograms match the exact profile of histograms generated from constant transfer efficiency experimental data with a chi2 near unity. The chi2 surface suggests an ultimate level of precision with FRET of < 1% of the F?rster radius. Distributions of FRET signals in donor-acceptor-labeled DNA were unambiguously identified as being broader than shot-noise variations could explain. A model describing a Gaussian distribution of distances was tested with the PDA method and demonstrated 5 A inhomogeneities due to dye motions. The capability of this method to recover quantitative information from FRET distributions has potential applications for studying molecular conformations and dynamics. Potential sources for artifacts such as acceptor photobleaching, spectrally different observation volumes, and fluctuations of the F?rster radius are ruled out.