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1、Fluorescent Probes,荧光探针,厦门大学化学系 郭祥群,郭祥群博士，博士生导师,全国高校化学教育研究中心第四届学术委员会副主任，教育部高等学校本科教学工作水平评估专家，中国化学会理事 先后获得国家级奖2项，省部级奖4项，发表科研与教学研究论文100余篇。 研究兴趣：荧光光谱分析的原理与技术，在研项目涉及生物活性自由基的荧光表征，海洋污染生物监测技术研究，食品安全等。 联系方式: 0592-2181637(L), 2187288(O), 2182355(H),20050705,2,Fluorescent Probes,Introduction Fluorescent Senso
2、r 荧光传感 Fluorescent Tracer 荧光示踪 FRET 共振能量转移荧光 Application 1. Radical probes 2. Ion probes 3. NIRF Fluorescence image 4. DNA sequence,5. Protein probes 6. DNA probes 7. Membrane probes 8. pH probes,20050705,3,Fluorescence research,Key word: Fluorescen* Period: Jan/2005-Jun/ 2005 Data base: 美国化学会 ( ACS
3、 )数据库 3566 篇 WPS电子期刊全文数据库 206 篇 SDOS全文数据库（上交 镜像） 1890 篇 英国皇家化学会（RSC) 数据库 120 篇 研究与应用领域涉及：生命 环境 材料 等 讲座要求了解： 荧光 探针 表征 应用 研究意义,20050705,4,Molecular Probe,Molecular reporter,From J. Fluorescence,20050705,5,homepage,www. probes. com,Molecular probes,20050705,6,Jablonski diagram,Characteristics of Fluoro
4、phore,Excitation wavelength 激发波长 ex Emission wavelength 发射波长 em Extinction (absorption) coefficient 吸光系数 Stokes shift Fluorescence lifetime 荧光寿命 Fluorescence quantum yield 荧光量子产率 Fluorescence Anisotropy 各项异性 r Fluorescence Polarization 荧光偏振 p,20050705,8,Excitation and emission spectra,1,Excitation s
5、pectrum,Emission spectra,F,em,em,ex,20050705,9,Fluorescence lifetime,Simplified Jablonski diagram, : Emission rate,Knr: nonradiative decay rate,Definition Average lifetime: the average time the molecule spends in the excited state prior to return to the ground state.,Expression,Fluorescence Quantum
6、Yield,Definition,The fluorescence quantum yield is the ratio of the number of photons emitted to the number absorbed,Expression,Sensor (1),自由基传感,Z.-Y. Bian, X.-Q. Guo*, Analytical Science, 21 553-559, 2005,Sensor (2),E. M. Nolan, et al., J. Am. Chem. Soc., 2003, 125: 14270-14271,S,R,T,20050705,13,Se
7、nsor (3),Fluorescence Resonance Energy Transfer,Probing DNA,Molecular beacons 分子信标,20050705,15,Sensor (4),Al3+,hv,20050705,16,Sensor (5),竞争型,S-R,T,催化型,20050705,17,The Fusion of Integal Membrane Protein,From Instant notes, Biochemistry, E2,Tracer,膜内蛋白侧向运动,20050705,18,Cell tracer,A 30 mthick section o
8、f a zebrafish embryo stained with CellTracker CM-DiI (C7000, C7001) prior to immunohistochemical analysis. A one-day-old zebrafish embryo was immobilized and impaled in the hindbrain with a microelectrode filled with 1 mg/mL CellTracker CM-DiI in 95% ethanol. One day later, the brain was fixed, embe
9、dded, frozen in liquid nitrogen and sectioned on a cryostat. After blocking with 0.1% Triton X-100 in PBS containing 2% BSA and 2% normal goat serum, the 30 mthick section was incubated with primary anti-glial antibody in conjunction with fluorescein goat antimouse IgG antibody. This section was the
10、n viewed sequentially through optical filter sets appropriate for rhodamine and fluorescein, and the resulting images were superimposed. The image was contributed by William Trevarrow, Beckman Institute, California Institute of Technology.,C7000,C68H105Cl2N3O,,荧光染色的斑马鱼胚胎,Tracer,FRET Fluorescence Res
11、onance Energy Transfer,Principle of FRET,Rate of energy transfer,D quantum yield of D,D lifetime of D,r distance between A and D,n refrcative index,N Avogadros number,k orientation factor,J overlap integral,20050705,22,Orientation factor,k = 2/3,randomize by rotational diffusion prior to energy tran
12、sfer,20050705,23,FRET sensor design based on donor and acceptor distance changes,K. Kikuchi, et al., TrAC, 2004,23(6),407-413,20050705,24,FRET probing cAMP,K. Kikuchi, et al., TrAC, 2004,23(6),407-413,20050705,25,FRET probing PDE 磷酸二酯酶,Before hydrolysis,After hydrolysis,K. Kikuchi, et al., TrAC, 200
13、4,23(6),407-413,Before,After,香豆素,phosphodiesterase,hv,hv,hv,Emission spectra,20050705,27,FRET sensor design based on modulation of spectral overlap,有利于能量转移,不利于能量转移,20050705,28,Probing phosphatase 磷酸酶,LF abs,QF abs,Donor Emi,LF (内酯）,QF (醌型）,K. Kikuchi, et al., TrAC, 2004,23(6),407-413,PTP 蛋白质酪氨酸磷酸酶,香
14、豆素,荧光素,生物活性自由基研究的重要性,OH O2- RO ROO NO,活性氧自由基,其他,R GS,为维持生命所必须 过量产生会导致生物大分子损伤,生命过程中所产生的自由基，是行使生理功能及介导其生化反应所必需的。如在线粒体呼吸链上电子传递过程中产生的自由基在线粒体能量代谢中起着重要的作用。,20050705,30,OH抽氢导致DNA断裂,20050705,31,硫中心自由基引起细胞损伤,20050705,32,顺磁标记荧光探针-Probing free radical,产物抗磁性，稳定,自由基之间的反应快速,多功能探针：ESR；荧光（强度、偏振、寿命等）,荧光显微技术应用于细胞、组织的
15、自由基作用研究,20050705,33,比率型荧光分子-Probing NO,Soh, N. et al., Chem. Commun.,2002, 2250-2251,荧胺,香豆素,20050705,34,OH,比率型荧光探针-Probing OH,Soh, N. et al., Chem. Commun.,2004, 496 - 497,以与自由基作用的特征靶分子或官能团作为识别基团,荧光素,罗丹明,脱氧胸腺嘧啶核苷二聚体,Ca2+ indicator Fura-2,钙具有使原生质胶体稳定以及调节细胞膜的结构和通透性的作用。对各种细胞的兴奋性具有正负效应。,Excitation,Emiss
16、ion,20050705,36,Ca2+ indicator Indo-1,Indo-1-Ca2+,,20050705,37,ICT principle,20050705,38,Image of Fura-2-Ca2+,False-color image（假色成像） of free Ca2+ concentration in a Purkinje neuron （梨状神经元）from embryonic mouse cerebellum（胚鼠小脑） The composite image, which represents the ratio of images obtained with e
