荧光探针和分子传感器复习进程.ppt

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1、XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY荧光探针和分子传感器XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFluorescent ProbesuIntroductionuFluorescent 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光 uApplication 1.Radical probes 2.Ion probes 3.NIRF Fluorescence image 4.DNA sequence5.Protein p

2、robes6.DNA probes7.Membrane probes8.pH probes200507052XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYThankyouThank you 200507053XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFluorescence research uKey word:Fluorescen*uPeriod:Jan/2005-Jun/2005uData 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

3、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200507054XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYMolecular ProbeMolecular reporterMolecular reporterFrom J.Fluoresce

4、nceFrom J.Fluorescence200507055XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYMolecular probes Molecular probes 200507056Jablonski diagramJablonski diagramT1h Ah AAbsorption10-15 sVibrationalVibrational relaxrelax,VR,10VR,10-12-12 1010-14-14 s sInternal conversion,IC,10-1110-13 sIntersystem crossing,ISC,

5、10-210-6 sFluorescencePhosphorescence ICISCS0S1S23 2 1 010-8 s10-3 100 sh Fh PHOMOLUMO200507057XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYCharacteristics of FluorophoreExcitation wavelength 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

6、lifetime 荧光寿命光寿命 Fluorescence quantum yield 荧光量子产率荧光量子产率 Fluorescence Anisotropy 各各项异性异性 r Fluorescence Polarization 荧光偏振光偏振 p200507058XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYExcitation and emission spectra 1Excitation spectrumEmission spectraF emF ex em ex200507059Fluorescence lifetime relaxation

7、(10relaxation(10-12-12 s)s)S S0 0S S1 1S S1 1hvhvA AhvhvF F k knrnrSimplified Jablonski diagram Simplified Jablonski diagram :Emission rate :Emission rate K Knrnr:nonradiative:nonradiative decay ratedecay ratelDefinitionDefinitionAverage lifetimeAverage lifetime:the average the average time the mole

8、cule spends in time the molecule spends in the excited state prior to the excited state prior to return to the ground state.return to the ground state.l lExpressionExpression2005070510Fluorescence Quantum YieldlDefinition The fluorescence quantum yield is the ratio The fluorescence quantum yield is

9、the ratio of the number of photons emitted to the of the number of photons emitted to the number absorbed number absorbed lExpression 2005070511Sensor (1)+S RTS S S S 信号单元，信号单元，信号单元，信号单元，R R R R 识别单元，识别单元，识别单元，识别单元，T T T T 靶分子靶分子靶分子靶分子hvhvhv自由基传感自由基传感Z.-Y.Bian,X.-Q.Guo*,Analytical Science,21 553-559

10、,20052005070512XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYSensor(2)DDhue-hvhvhvPET Photoinduced Electron TransferhvHg2+E.M.Nolan,et al.,J.Am.Chem.Soc.,2003,125:14270-14271 SRT2005070513Sensor(3)RShvhv FRET F D,ex D,em A,ex A,em F D,ex D,em A,ex A,emT+hvhvFluorescence Resonance Energy Transfer 2005070

11、514XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYProbing DNAMolecular beacons Molecular beacons 分子信标分子信标分子信标分子信标 ADQFTarget DNAssDNA binding proteinDenaturing reagent QFFQ2005070515XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYSensor(4)+S-RThvhvAl3+hv2005070516XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYSenso

12、r(5)+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2005070517XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYThe Fusion of Integal Membrane ProteinThe Fusion of Integal Membrane Protein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2005070518Cell tracerA 30

13、mthick section of 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 fi

14、xed,embedded,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 th

15、en 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.C7000C7000C C6868HH105105ClCl2 2NN3 3O O 荧光染色的斑马鱼胚胎荧光染色的斑马鱼胚胎荧光染

16、色的斑马鱼胚胎荧光染色的斑马鱼胚胎2005070519Tracer Tracer AgAgF-AgF-AgAb Ab 抗体抗体抗体抗体在在免免疫疫反反应应前前后后荧荧光光探探针针自自身身的的荧荧光光量量子子产产率率不不发发生生变变化化的的情情况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，荧光反比抗原量F F标记抗原标记抗原待测抗原待测抗原 FF FF荧光分子荧光分子F F FF FF2005070520FRET FRET Fluorescence Resonance Ener

17、gy Transfer Fluorescence Resonance Energy Transfer 2005070521XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYPrinciple of FRETRate of energy transfer D quantum yield of D D lifetime of Dr r distance between A and Ddistance between A and Dn refrcative indexN Avogadros numberk k orientation factororientatio

18、n factorJ J overlap integraloverlap integral F D,ex D,em A,ex A,em2005070522XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYOrientation factork=2/3randomize by rotational diffusion prior to energy transfer2005070523XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFRET sensor design based on donor and FRET sensor

19、 design based on donor and acceptor distance changes acceptor distance changes K.Kikuchi,et al.,TrAC,2004,23(6),407-4132005070524XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFRET probing cAMP K.Kikuchi,et al.,TrAC,2004,23(6),407-413 K.Kikuchi,et al.,TrAC,2004,23(6),407-413cAMP cyclic adenosine monophos

20、phate cAMP cyclic adenosine monophosphate 环腺苷酸环腺苷酸环腺苷酸环腺苷酸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cAMP2005070525FRET probing PDE 磷酸二酯酶磷酸二酯酶Before hydrolysisAfter hydrolysis K.Kikuchi,et al.,TrAC,2004,23(6),407-413BeforeAfter 香豆素phosphodiesterasehvhvhv2005070526XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOP

21、YEmission spectraPDE2005070527XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYFRET sensor design based on modulation of spectral overlap F D,ex D,em A,ex A,em F D,ex D,em A,ex A,em有利于能量转移有利于能量转移有利于能量转移有利于能量转移不利于能量转移不利于能量转移不利于能量转移不利于能量转移2005070528Probing phosphatase 磷酸酶磷酸酶LF absQF absDonor EmiLF(内酯）QF(醌型）K

22、.Kikuchi,et al.,TrAC,2004,23(6),407-413PTP 蛋白质酪氨酸磷酸酶香豆素香豆素香豆素香豆素荧光素荧光素荧光素荧光素2005070529XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY生物活性自由基研究的重要性生物活性自由基研究的重要性OH OOH O2 2-RO ROO RO ROO NO NO 活性氧自由基活性氧自由基活性氧自由基活性氧自由基其他其他其他其他 R GS R GS 为维持生命所必须为维持生命所必须为维持生命所必须为维持生命所必须过量产生会导致生物大分子损伤过量产生会导致生物大分子损伤过量产生会导致生物大分

23、子损伤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命过程中所产生的自由基，是行使生理功能及介导其生命过程中所产生的自由基，是行使生理功能及介导其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生化反应所必需的。如在线粒体呼吸链上电子传递过程生化反应所必需的。如在线粒体呼吸链上电子传递过程生化反应所必需的。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。中产生的自由基在线粒体能量代谢中起着重要的作用。中产生的自由基在线粒体能量代谢中起着重要的作用。中产生的自由基在线粒体能量代谢中起着

24、重要的作用。2005070530XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYOH抽氢导致抽氢导致DNA断裂断裂2005070531XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY硫中心自由基引起细胞损伤硫中心自由基引起细胞损伤 2005070532XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY顺磁标记荧光探针顺磁标记荧光探针顺磁标记荧光探针顺磁标记荧光探针-Probing free radical-Probing free radicalParamagnetic(Low fluor

25、escence)Diamagnetic(High fluorescence)产物抗磁性，稳定产物抗磁性，稳定产物抗磁性，稳定产物抗磁性，稳定自由基之间的反应快速自由基之间的反应快速自由基之间的反应快速自由基之间的反应快速多功能探针：多功能探针：多功能探针：多功能探针：ESRESR；荧光（强度、偏振、寿命等）；荧光（强度、偏振、寿命等）；荧光（强度、偏振、寿命等）；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荧光显微技术应用于细胞、组织的自由基作用研究荧光显微技术应用于细胞、组织的自由基作用研究荧光显微技术应用于细胞、组织的自由基作用研究2005070533XIAMEN

26、 UNIVERSITYFLUORESCENCE SPECTROSCOPY比率型荧光分子比率型荧光分子-Probing NOSoh,N.et al.,Chem.Commun.,2002,2250-2251Soh,N.et al.,Chem.Commun.,2002,2250-2251NOFRETFRET exex=360nm=360nm emem=470nm=470nm emem=410nm=410nm exex=360nm=360nm荧胺荧胺荧胺荧胺香豆素香豆素2005070534 OHOH比率型荧光探针比率型荧光探针-Probing OHSoh,N.et al.,Chem.Commun.,2

27、004,496-497Soh,N.et al.,Chem.Commun.,2004,496-497以与自由基作用的特征靶分以与自由基作用的特征靶分以与自由基作用的特征靶分以与自由基作用的特征靶分子或官能团作为识别基团子或官能团作为识别基团子或官能团作为识别基团子或官能团作为识别基团荧光素荧光素荧光素荧光素罗丹明罗丹明罗丹明罗丹明脱氧胸腺嘧啶核苷二聚体脱氧胸腺嘧啶核苷二聚体脱氧胸腺嘧啶核苷二聚体脱氧胸腺嘧啶核苷二聚体2005070535XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYCa2+indicator Fura-2Fura-Ca2+Fura钙具有使原生

28、质胶体稳定以及调节细胞膜的结构和通透钙具有使原生质胶体稳定以及调节细胞膜的结构和通透钙具有使原生质胶体稳定以及调节细胞膜的结构和通透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。性的作用。对各种细胞的兴奋性具有正负效应。性的作用。对各种细胞的兴奋性具有正负效应。性的作用。对各种细胞的兴奋性具有正负效应。Excitation Emission2005070536XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYCa2+indicator Indo-1Indo-1-Ca2+2005070537XIAMEN UNIVERS

29、ITYFLUORESCENCE SPECTROSCOPYICT principle2005070538XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYImage of Fura-2-Ca2+False-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 image

30、s obtained with excitation at 340 nm and 380 nm,reveals the mobilization of internal Ca2+stores without contribution from Ca2+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2005070539XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYImage of spiral Ca2+waveFluo-3(F1240)confocal image of a spiral Ca2+wav

31、e 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 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-32005070540X

32、IAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYNa+indicator SBFISBFISBFI-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2005070541XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYCd2+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+.BTC-5N,tetrapota

33、ssium salt2005070542XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYZnZn2+2+indicator indicatorAPTRA-BTC,tripotassium salt Fluorescence excitation spectra of APTRA-BTC(A-6895)in solutions containing zero to 25 M Zn2+.2005070543XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYpH indicator5-sulfofluorescein diacet

34、ate,sodium salt(SFDA)2005070544DNA-based pH sensor Tatsuo Ohmishi et al.,Biochemistry 2005,44 7125-7130 Tatsuo Ohmishi et al.,Biochemistry 2005,44 7125-71302005070545XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY Tatsuo Ohmishi et al.,Biochemistry 2005,44 7125-7130 Tatsuo Ohmishi et al.,Biochemistry 200

35、5,44 7125-7130DNA-based pH indicator表征表征表征表征MCF-7MCF-7乳腺癌细乳腺癌细乳腺癌细乳腺癌细胞编程性死亡过程胞编程性死亡过程胞编程性死亡过程胞编程性死亡过程ApoptosisMCF-7 MCF-7 乳腺癌细胞乳腺癌细胞2005070546XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY荧光成像荧光成像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

36、、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.2005070547XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY近红外成像近红外成像 为什么选择近红外？为什么选择近红外？2005070548GFP 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

37、滤光片CY5.5CY5.5标记的内生抑制素特异识别肿瘤标记的内生抑制素特异识别肿瘤2005070549XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070550XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070551XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYSelf-quenching probeRalph Wissleder,et.al.Nature America 1999,17:375-378Ralph Wissleder,et.al.Nature Am

38、erica 1999,17:375-378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（或细胞成像）自猝灭荧光探针（或细胞成像）多甲氧基聚乙烯乙二醇多甲氧基聚乙烯乙二醇-聚赖氨酸聚赖氨酸-cy5.5(C-PGC),-cy5.5(C-PGC),2005070552XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY2005070553XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYPhase contrast Phase contrast image image

39、 相差成像相差成像相差成像相差成像NIRF image 肿瘤细胞成像肿瘤细胞成像NIRFNIRFNIRFNIRFLysosomal compartment 溶酶体分隔溶酶体分隔2005070554XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYNIRF image2005070555Template 3-CCGGTAGCAACT-5Primer 5-GG-3GGCCAGGCCATCGTTGAGGCGGCCGGCCATCGGCCATCGGGCCATCGTTGGGCCATGGCCATCGTGGCCATCGTTdATPdCTPdGTPdTTPddATPdATPdC

40、TPdGTPdTTPddCTPdATPdCTPdGTPdTTPddGTPdATPdCTPdGTPdTTPddTTPF1-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FDNA sequence2005070556XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY近红外成像荧光探针近红外成像荧光探针七次甲基花菁素类七次甲基花菁素类(图图2，a)2005070557Protein probes蛋白质

41、荧光蛋白质荧光蛋白质荧光蛋白质荧光探针探针探针探针RGRG对牛对牛对牛对牛血清白蛋白血清白蛋白血清白蛋白血清白蛋白（BSABSA）的）的）的）的响应图响应图响应图响应图WWW2005070558XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYDNA probeAbsorption and fluorescence emission spectra of ethidium bromde bound to DNA Ethidium bromide 溴化乙锭溴化乙锭2005070559XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCO

42、PYDNA probeHoechst 33258(bis-benzimide)Absorption and fluorescence emission spectra of Hoechst 33258 bound to DNA.2005070560XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPYNormal and camptothecin-treated bovine pulmonary artery endothelial cells(BPAEC)were stained with C4-BODIPY 500/510 C9(B-3824 and Hoec

43、hst 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(rig

44、ht panel,as indicated by the fragmented nuclear staining of the Hoechst dye).Image contributed by Wan-Nan Uang,Molecular Probes,Inc.正常细胞正常细胞编程性死亡细胞编程性死亡细胞Hoechst 33258 staining DNA staining 2005070561DNA stainingDNA stainingViable Madin-Darby canine Viable Madin-Darby canine kidney(MDCK)cells kidney

45、(MDCK)cells sequentially stained with sequentially stained with BODIPY FL C5-ceramide(BODIPY FL C5-ceramide(D-D-35213521),LysoTracker Red DND-99),LysoTracker Red DND-99(L-7528L-7528)and Hoechst 33258()and Hoechst 33258(H-H-13981398,H-3569H-3569).Green-).Green-fluorescent BODIPY FL C5-fluorescent BOD

46、IPY FL C5-ceramide ceramide localizedlocalized to the Golgi to the Golgi apparatus,red-fluorescent apparatus,red-fluorescent LysoTracker Red stain LysoTracker Red stain accumulated in the lysosomes accumulated in the lysosomes and blue-fluorescent Hoechst and blue-fluorescent Hoechst 33258 dye stain

47、ed the nuclei.33258 dye stained the nuclei.The multiple-exposure image The multiple-exposure image was acquired with bandpass was acquired with bandpass filters appropriate for filters appropriate for fluorescein,Texas Red dye and fluorescein,Texas Red dye and DAPI.Image contributed by DAPI.Image co

48、ntributed by Chii-Shiarng Chen,Molecular Chii-Shiarng Chen,Molecular Probes,Inc.Probes,Inc.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 DNA stainingDNA staining2005070562+-卵磷脂卵磷脂Membrane probesMembrane probes2005070563Membrane probe Emission intensities of DPPG E

49、mission intensities of DPPG vesicles labeled with of vesicles labeled with of Ru(bpy)2(dppz)2+at various Ru(bpy)2(dppz)2+at various cholesterol concentrations,cholesterol concentrations,measured as a function of measured as a function of increasing temperature towards increasing temperature towards the lipid phase transition the lipid phase transition temperature temperature 膜探针表征生物膜的膜探针表征生物膜的膜探针表征生物膜的膜探针表征生物膜的流通性流通性流通性流通性2005070564XIAMEN UNIVERSITYFLUORESCENCE SPECTROSCOPY此课件下载可自行编辑修改，仅供参考！此课件下载可自行编辑修改，仅供参考！感谢您的支持，我们努力做得更好！谢谢感谢您的支持，我们努力做得更好！谢谢