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1、Fluorescent Probes,荧光探针,厦门大学化学系 郭祥群,20050705,2,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),20050705,3,Fluorescent Probes,IntroductionFluorescent S
2、ensor 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 probes6.DNA probes7.Membrane probes8.pH probes,20050705,4,20050705,5,Fluorescence research,Key word:Fluorescen*Period:Jan/2005-Jun/2005Data base:美国化学会(ACS)数据库
3、 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,20050705,6,Molecular Probe,Molecular reporter,From J.Fluorescence,20050705,7,homepage,Molecular probes,Jablonski diagram,20050705,9,Characteristics of Fluorophore,Excitation wavelength 激发
4、波长 exEmission wavelength 发射波长 emExtinction(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,20050705,10,Excitation and emission spectra,1,Excitation spectrum,Emission spectra,F,em,em,
5、ex,Fluorescence lifetime,Simplified Jablonski diagram,:Emission rate,Knr:nonradiative decay rate,DefinitionAverage lifetime:the average time the molecule spends in the excited state prior to return to the ground state.,Expression,Fluorescence Quantum Yield,Definition,The fluorescence quantum yield i
6、s 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,21 553-559,2005,20050705,14,Sensor(2),E.M.Nolan,et al.,J.Am.Chem.Soc.,2003,125:14270-14271,S,R,T,Sensor(3),Fluorescence Resonance Energy Transfer,20050705,16,Probing
7、DNA,Molecular beacons 分子信标,20050705,17,Sensor(4),Al3+,hv,20050705,18,Sensor(5),竞争型,S-R,T,催化型,20050705,19,The Fusion of Integal Membrane Protein,From Instant notes,Biochemistry,E2,Tracer,膜内蛋白侧向运动,Cell tracer,A 30 mthick section of a zebrafish embryo stained with CellTracker CM-DiI(C7000,C7001)prior t
8、o 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,embedded,frozen in liquid nitrogen and sectioned on a cryostat.After blocking with 0
9、.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 then viewed sequentially through optical filter sets appropriate for rhodamine and fluores
10、cein,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 Resonance Energy Transfer,20050705,23,Principle of FRET,Rate of energy transfer,D quantum yield
11、 of D,D lifetime of D,r distance between A and D,n refrcative index,N Avogadros number,k orientation factor,J overlap integral,20050705,24,Orientation factor,k=2/3,randomize by rotational diffusion prior to energy transfer,20050705,25,FRET sensor design based on donor and acceptor distance changes,K
12、.Kikuchi,et al.,TrAC,2004,23(6),407-413,20050705,26,FRET probing cAMP,K.Kikuchi,et al.,TrAC,2004,23(6),407-413,FRET probing PDE 磷酸二酯酶,Before hydrolysis,After hydrolysis,K.Kikuchi,et al.,TrAC,2004,23(6),407-413,Before,After,香豆素,phosphodiesterase,hv,hv,hv,20050705,28,Emission spectra,20050705,29,FRET
13、sensor design based on modulation of spectral overlap,有利于能量转移,不利于能量转移,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,香豆素,荧光素,20050705,31,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
14、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。,20050705,32,OH抽氢导致DNA断裂,20050705,33,硫中心自由基引起细胞损伤,20050705,34,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、组织的自由基作用研究,20050705,35,比率型荧光分子-Probing NO,Soh,N.et al.,Chem.Commun.,2002,2250-2251,荧胺,香豆素,OH,比率型荧光探针-Prob
15、ing 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,20050705,37,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,Emission,20050705,38,Ca2+indicator Indo-1,Indo-1-Ca2+,20050705,39,ICT principle,20050705,40,Image of Fura-2-Ca2+,False-c
16、olor 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 excitation at 340 nm and 380 nm,reveals the mobilization of internal Ca2+stores without contribution from Ca2+influx.,Fura-
17、2-Ca2+,胚鼠小脑梨状神经元中游离Ca2+浓度荧光成像,20050705,41,Image of spiral Ca2+wave,Fluo-3(F1240)confocal image of a spiral Ca2+wave initiated by injection of a nonhydrolyzable analog of inositol 1,4,5-triphosphate（肌醇三磷酸非水解模拟物）in a Xenopus laevis oocyte（瓜蟾卵母细胞）.The image was contributed by David Clapham,Harvard Univ
18、ersity,and reproduced with permission from Cell 69,283(1992).,瓜蟾卵母细胞中螺旋Ca2+波共焦荧光成像,Fluo-3,20050705,42,Na+indicator,SBFI,SBFI,SBFI-Na+,海马切片CA1锥形神经元Na+成像,20050705,43,Cd2+indicator,Fluorescence emission spectra of a mixture of 1 M BTC-5N(B-6845)and 1 M TCPP in solutions containing zero to 1.0 M Cd2+.,B
19、TC-5N,tetrapotassium salt,20050705,44,Zn2+indicator,APTRA-BTC,tripotassium salt,Fluorescence excitation spectra of APTRA-BTC(A-6895)in solutions containing zero to 25 M Zn2+.,20050705,45,pH indicator,5-sulfofluorescein diacetate,sodium salt(SFDA),DNA-based pH sensor,Tatsuo Ohmishi et al.,Biochemistr
20、y 2005,44 7125-7130,20050705,47,Tatsuo Ohmishi et al.,Biochemistry 2005,44 7125-7130,DNA-based pH indicator,表征MCF-7乳腺癌细胞编程性死亡过程,Apoptosis,MCF-7 乳腺癌细胞,20050705,48,荧光成像,通过静脉注射或腹腔注射到生物体内的近红外荧光探针吸着并聚集于肿瘤等组织中，肿瘤中存在特异的细胞、蛋白质、酶等，这些物质可与荧光探针结合或反应而发出荧光，可根据荧光的位置、强度得知肿瘤的位置、大小，是否转移、扩散及肿瘤的阴性、阳性等一系列信息.,20050705,49
21、,近红外成像 为什么选择近红外？,GFP 绿色荧光蛋白,Endostatin内生抑制素,Cy5.5 花菁荧光染料,NIR1,NIR2,GFP 不同滤光片,CY5.5标记的内生抑制素特异识别肿瘤,20050705,51,20050705,52,20050705,53,Self-quenching probe,Ralph Wissleder,et.al.Nature America 1999,17:375-378,酶活化,酶作用位点,C-PGC,肿瘤伴生溶酶体蛋白酶特异识别自猝灭荧光探针（或细胞成像）,多甲氧基聚乙烯乙二醇-聚赖氨酸-cy5.5(C-PGC),20050705,54,2005070
22、5,55,Phase contrast image 相差成像,NIRF image 肿瘤细胞成像,NIRF,NIRF,Lysosomal compartment 溶酶体分隔,20050705,56,NIRF image,DNA sequence,20050705,58,近红外成像荧光探针,七次甲基花菁素类(图2，a),Protein probes,蛋白质荧光探针RG对牛血清白蛋白（BSA）的响应图,WWW,20050705,60,DNA probe,Absorption and fluorescence emission spectra of ethidium bromde bound to
23、DNA,Ethidium bromide 溴化乙锭,20050705,61,DNA probe,Hoechst 33258(bis-benzimide),Absorption and fluorescence emission spectra of Hoechst 33258 bound to DNA.,20050705,62,Normal and camptothecin-treated bovine pulmonary artery endothelial cells(BPAEC)were stained with C4-BODIPY 500/510 C9(B-3824 and Hoech
24、st 33258(H-1398,H-3569).BODIPY and Hoechst fluorescence were recorded using longpass filters appropriate for fluorescein and DAPI,respectively.An overlay of the images indicates that a red shift in BODIPY fluorescence is significant in normal cells(left panel)compared to that in apoptotic cells(righ
25、t panel,as indicated by the fragmented nuclear staining of the Hoechst dye).Image contributed by Wan-Nan Uang,Molecular Probes,Inc.,正常细胞,编程性死亡细胞,Hoechst 33258 staining,DNA staining,DNA staining,Viable Madin-Darby canine kidney(MDCK)cells sequentially stained with BODIPY FL C5-ceramide(D-3521),LysoTr
26、acker Red DND-99(L-7528)and Hoechst 33258(H-1398,H-3569).Green-fluorescent BODIPY FL C5-ceramide localized to the Golgi apparatus,red-fluorescent LysoTracker Red stain accumulated in the lysosomes and blue-fluorescent Hoechst 33258 dye stained the nuclei.The multiple-exposure image was acquired with
27、 bandpass filters appropriate for fluorescein,Texas Red dye and DAPI.Image contributed by Chii-Shiarng Chen,Molecular Probes,Inc.,Hoechst 33258 stain N 胞核,BODIPY FL C5 stain G 高尔基体,Lysotracker stain L 溶酶体,DNA staining,卵磷脂,Membrane probes,Membrane probe,Emission intensities of DPPG vesicles labeled with of Ru(bpy)2(dppz)2+at various cholesterol concentrations,measured as a function of increasing temperature towards the lipid phase transition temperature,膜探针表征生物膜的流通性,
