1、XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY200507051XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY200507052u全国高校化学教育研究中心第四届学术委员会副主任，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，中国化学会理事学会理事u 先后获得国家级奖先后获得国家级奖2项，省部级奖项，省部级奖4项，发表科研与项，发表科研与教学研究论文教学研究论文100100余篇。余篇。u研究兴趣：荧光光谱分析的原理与技术，在研项目涉研
2、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。术研究，食品安全等。u联系方式联系方式:xqguojingxian.xmu.edu 0592-2181637(L),2187288(O),2182355(H)XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY200507053uIntroductionuFluorescent Sensor 荧光传感荧光传感 uFluorescent Tracer 荧光示踪荧光示踪 uFRET 共振能量转移荧光共振能量转移荧
3、光 uApplication 1.Radical probes 2.Ion probes 3.NIRF Fluorescence image 4.DNA sequence5.Protein probes6.DNA probes7.Membrane probes8.pH probesXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY200507054Thank you Thank you XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY200507055uKey word:Fluorescen*uPeriod:Jan/2019
4、-Jun/2019uData base:美国化学会美国化学会 (ACS)(ACS)数据库数据库 3566 3566 篇篇 WPSWPS电子期刊全文数据库电子期刊全文数据库 206 206 篇篇 SDOSSDOS全文数据库（上交全文数据库（上交 镜像）镜像）1890 1890 篇篇 英国皇家化学会（英国皇家化学会（RSC)RSC)数据库数据库 120 120 篇篇研究与应用领域涉及：生命研究与应用领域涉及：生命 环境环境 材料材料 等等讲座要求了解：讲座要求了解：荧光荧光 探针探针 表征表征 应用应用 研究意义研究意义XIAMEN UNIVERSITYFLUORESCENCE SPECTROSC
5、OPY200507056XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY200507057 T1h Ah AAbsorption10-15 sInternal conversion,IC,10-1110-13 sIntersystem crossing,ISC,10-210-6 sFluorescencePhosphorescence ICISCS0S1S23 2 1 010-8 s10-3 100 sh Fh PHOMOLUMOXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY200507059Excitation wav
6、elength 激发波长激发波长 exEmission wavelength 发射波长发射波长 emExtinction(absorption)coefficient 吸光系数吸光系数 Stokes shift Fluorescence lifetime 荧光寿命荧光寿命 Fluorescence quantum yield 荧光量子产率荧光量子产率 Fluorescence Anisotropy 各项异性各项异性 r Fluorescence Polarization 荧光偏振荧光偏振 pXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070510
7、 1Excitation spectrumEmission spectraF emF ex em exnrk 1nrk+S RThvhvhv自由基传感自由基传感Z.-Y.Bian,X.-Q.Guo*,Analytical Science,21 553-559,2019XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070514DDhue-hvhvhvPET Photoinduced Electron TransferhvHg2+E.M.Nolan,et al.,J.Am.Chem.Soc.,2019,125:14270-14271 SRTRShvhv
8、 FRET F D,ex D,em A,ex A,em F D,ex D,em A,ex A,emT+hvhvFluorescence Resonance Energy Transfer XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070516ADQFTarget DNAssDNA binding proteinDenaturing reagent QFFQXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070517+S-RThvhvAl3+hvXIAMEN UNIVERSITYFLUORESCENCE
9、 SPECTROSCOPY2005070518+S-RT+竞争型竞争型+S-RT催化剂中间体催化剂中间体+hvhv催化型催化型XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070519荧光素标记鼠蛋白质罗丹明标记人蛋白质鼠细胞人细胞37C15C融合融合异核体异核体From Instant notes,Biochemistry,E2TracerTracer膜膜内内蛋蛋白白侧侧向向运运动动A 30 mthick section of a zebrafish embryo stained with CellTracker CM-DiI(C7000,C70
10、01)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,embedded,frozen in liquid nitrogen and sectioned on a cryostat.After block
11、ing 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 then viewed sequentially through optical filter sets appropriate for rhodamine a
12、nd fluorescein,and the resulting images were superimposed.The image was contributed by William Trevarrow,Beckman Institute,California Institute of Technology.在免疫反应前后荧光探针自身的在免疫反应前后荧光探针自身的荧光量子产率不发生变化的情况荧光量子产率不发生变化的情况测反应剩余的标记抗原，荧光正比抗原量测反应剩余的标记抗原，荧光正比抗原量测固相荧光，荧光反比抗原量测固相荧光，荧光反比抗原量测固相洗脱，荧光反比抗原量测固相洗脱，荧光反比抗
13、原量标记抗原标记抗原待测抗原待测抗原荧光分子荧光分子XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070523JNrnkKDDT6422128)10(ln9000Rate of energy transfer D quantum yield of D D lifetime of Dn refrcative indexN Avogadros number F D,ex D,em A,ex A,emXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070524k=2/3randomize by rotation
14、al diffusion prior to energy transferXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070525 K.Kikuchi,et al.,TrAC,2019,23(6),407-413XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070526环腺苷酸依赖蛋白激环腺苷酸依赖蛋白激酶支架结构四聚体酶支架结构四聚体表征表征cAMPBefore hydrolysisAfter hydrolysis K.Kikuchi,et al.,TrAC,2019,23(6),407-413Be
15、foreAfter 香豆素phosphodiesterasehvhvhvXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070528PDEXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070529 F D,ex D,em A,ex A,em F D,ex D,em A,ex A,emLF absQF absDonor EmiLF(内酯）QF(醌型）K.Kikuchi,et al.,TrAC,2019,23(6),407-413PTP 蛋白质酪氨酸磷酸酶XIAMEN UNIVERSITYFLUORESCEN
16、CE SPECTROSCOPY2005070531XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070532XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070533GSHROSGSGS-GSSGO2O2蛋白质巯基细胞膜磷酯-XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070534NOFluorophoreNOFluorophore.+R.RIIIParamagnetic(Low fluorescence)Diamagnetic(High fluoresc
17、ence)XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070535NHOCOO-ONON HNN HH NOOO M eF eNOHCOO-ONONONHNNHHNOOOMeFeNOXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070537Fura-Ca2+FuraExcitation EmissionXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070538Indo-1-Ca2+XIAMEN UNIVERSITYFLUORESCENCE SPECTROS
18、COPY2005070539ICT principleXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070540False-color image（假色成像）（假色成像）of free Ca2+concentration in a Purkinje neuron（梨状（梨状神经元）神经元）from embryonic mouse cerebellum（胚鼠小（胚鼠小脑）脑）The composite image,which represents the ratio of images obtained with excitation at 340
19、nm and 380 nm,reveals the mobilization of internal Ca2+stores without contribution from Ca2+influx.Fura-2-Ca2+胚鼠小脑梨状神经元中游离胚鼠小脑梨状神经元中游离Ca2+浓度荧光成像浓度荧光成像XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070541Fluo-3(F1240)confocal image of a spiral Ca2+wave initiated by injection of a nonhydrolyzable analo
20、g 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 University,and reproduced with permission from Cell 69,283(1992).瓜蟾卵母细胞中螺旋瓜蟾卵母细胞中螺旋Ca2+波共焦荧光成像波共焦荧光成像Fluo-3XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070542SBFISBFISB
21、FI-Na+海马切片海马切片CA1锥形锥形神经元神经元Na+成像成像XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070543Fluorescence 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+.BTC-5N,tetrapotassium saltXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070544APTRA-BTC,tripot
22、assium salt Fluorescence excitation spectra of APTRA-BTC(A-6895)in solutions containing zero to 25 M Zn2+.XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY20050705455-sulfofluorescein diacetate,sodium salt(SFDA)XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070547ApoptosisXIAMEN UNIVERSITYFLUORESCENCE SPECT
23、ROSCOPY2005070548通过静脉注射或腹腔注射到生物体内的近红外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、阳性等一系列信息转移、扩散及肿瘤的阴性、阳性等一系列信息.XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY
24、2005070549GFP 绿色绿色荧光蛋白荧光蛋白Endostatin内生抑制素内生抑制素Cy5.5 花菁花菁荧光染料荧光染料NIR1,NIR2,GFP 不同滤光片不同滤光片XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070551XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070552XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070553酶活化酶活化酶作用位点酶作用位点C-PGC肿瘤伴生溶酶体蛋白酶特异识别肿瘤伴生溶酶体蛋白酶特异识别自猝灭荧光探针
25、（或细胞成像）自猝灭荧光探针（或细胞成像）XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070554XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070555Lysosomal compartment 溶酶体分隔溶酶体分隔XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070556Template 3-CCGGTAGCAACT-5Primer 5-GG-3GGCCAGGCCATCGTTGAGGCGGCCGGCCATCGGCCATCGGGCCATCGTTGGGC
26、CATGGCCATCGTGGCCATCGTTdATPdCTPdGTPdTTPddATPdATPdCTPdGTPdTTPddCTPdATPdCTPdGTPdTTPddGTPdATPdCTPdGTPdTTPddTTPF1-AF2-CF3-GF4-TDNA polymerasedATP 脱氧腺苷dCTP 脱氧胞苷dGTP 脱氧鸟苷dTTP 脱氧胸苷ddATP 双脱氧腺苷ddCTP 双脱氧胞苷ddGTP 双脱氧鸟苷ddTTP 双脱氧胸苷FXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070558七次甲基花菁素类七次甲基花菁素类(图图2，a)WWW.prob
27、esXIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070560Absorption and fluorescence emission spectra of ethidium bromde bound to DNA Ethidium bromide 溴化乙锭溴化乙锭XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070561Hoechst 33258(bis-benzimide)Absorption and fluorescence emission spectra of Hoechst 33258 bo
28、und to DNA.XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070562Normal and camptothecin-treated bovine pulmonary artery endothelial cells(BPAEC)were stained with C4-BODIPY 500/510 C9(B-3824 and Hoechst 33258(H-1398,H-3569).BODIPY and Hoechst fluorescence were recorded using longpass filters appropria
29、te 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(right panel,as indicated by the fragmented nuclear staining of the Hoechst dye).Image contributed by Wan-N
30、an Uang,Molecular Probes,Inc.正常细胞正常细胞编程性死亡细胞编程性死亡细胞Hoechst 33258 staining Hoechst 33258 stain N 胞核胞核BODIPY FL C5 stain G 高尔基体高尔基体Lysotracker stain L 溶酶体溶酶体 N CH3CH3H3CCH2CH2OP OOOCH2CHH2CO OC OCO+-卵磷脂卵磷脂0102030405060024641 oC RuDPPG vesicle containingcholesterolEX=463nm,EM=650nm,0%20%33%50%Emission Intensity T oCNNNNRuNNNN2+Ru(bpy)2(dppz)2+
