大型仪器设备使用与维护讲座(第三讲)

欢迎各位老师参加！---激光共聚焦扫描显微镜应用---奥林巴斯（北京）销售服务有限公司上海分公司光学显微镜技术的进展生物用激光共焦扫描显微镜主要是对______显微观察方法的改进？荧光主要应用：生物荧光相关研究领域几乎含盖整个生物领域的定位、定量、定时、定性相关的研究WHATCANDOBYCONFOCALApplicationsofFluorescence生物检测Bioassays细胞生物学CellBiology核酸/蛋白定量DNA,RNA,andProteinQuantitationSystems分子生物学GenomicsandMolecularBiology离子检测IonIndicators微生物学Microbiology微球/纳米粒子MicrospheresandNanospheres蛋白组学ProteomicsandProteinBiochemistry活性染料ReactiveDyesandChemicals二级检测SecondaryDetection信号传导通路SignalTransduction动物模型SmallAnimalInVivoImagingInvitrogenProprietary&ConfidentialToolboxofLabelsAlexaFluor®DyeSeriesUVtonearIRSulfonatedforincreasedH20solubilityBright,photostable&HighQYBODIPY®DyeSeriesVisibletonearIRZwitterionicHighextinctioncoefficients&QYOregonGreen®,Pacific®dyes,TexasRed®andothergenericdyes(FITC,TRITC)Includespositive,zwitterions,andnegativelychargedmoleculesUVtonearIRIncludingenvironmentallysensitivedyes(pH,polarity)OrganicDyesQuantumDotNanotechnologiesQdot®nanocrystalsAbilitytoarchivesamplesSingleexcitationUniformsizeHighextinctioncoefficientHaptensBiotinandDesthiobiotin(DSB)CompactTightbindingtostreptavidinDSB,facilereversiblebindingwithexcessbiotinAlexaFluor®488BODIPY®FLBiotinDesthiobiotinColorSelection♦Brightness♦PhotostabilityAlexaFluor®DyesacrossthespectrumQuantumDots（量子点）655605585565525nm25nmSizeofthenanocrystaldeterminesthecolorSizeistunablefrom~2-10nm(±3%)SizedistributiondeterminesthespectralwidthHighlyfluorescent,nanometer-size,singlecrystalsofsemiconductormaterials•Mostreliableandconsistentlabelingmethod•Yieldsthehighestleveloflabeling:1dyefor12-20bases•Sameprotocolregardlessofthedyeused•NoworriesaboutdyeinterferencewithenzymeIndirectLabelingGivesHighestandMostConsistentLevelofLabeling“Indirect”Labeling核酸标记CovalentProteinLabelingChemistryDYEDYEDYEProteinconjugate蛋白标记DyeDyeDyeDyeIndicatorResponseTypesCa2+Ca2+Ion-dependentspectralshiftIon-dependentfluorescenceincrease,nospectralshiftExamples•Fura-2•Indo-1•PremoCameleonExamples•Fluo-3•Rhod-2•CalciumGreen•OregonGreen488BAPTA离子指示剂InvitrogenProprietary&ConfidentialTheIlluminatedCell—ProductGuideforFluorescenceImaging•荧光蛋白（B/C/G/Y/RFP）•非破坏性融合表达：共转染示踪活体中蛋白表达及运输等•低/无光毒性•优秀的衍生产品：PA-GFP/Keade/FRET等绿色荧光蛋白家族共聚焦专用器材可以与电动载物台共用，不是普通的多孔板共聚焦专用器材IBIDI培养皿WHATISCONFOCALMICROSCOPYOlympusFV1000configuration1、活细胞或组织切片观察，进行细胞精细结构观察和三维重建2、多维图象获得，X、Y、Z、T、λ（光谱波长）、θ（旋转角度）I（光强度）等多维组合观察4、细胞或组织内离子荧光标记，细胞内Ph值和钙、镁离子浓度比例和动态变化测定3、荧光探针标记的细胞或组织，膜标记、免疫物质、受体或配体，核酸观察，多重荧光标记观察5、利用电生理技术检测细胞或组织的离子通道信号,同时结合图象判断6、蛋白质动力学、生物化学等等ApplicationWHATCANDOBYCONFOCAL多重标记、免疫荧光3channelacquisition:Nuclei(TO-PRO-3),Microtubules(FITC),Mitochondria(Mitotracker),3channelacquisition:Green(Alexfluo488)andBlue(DRAQ5)Red(Alexfluo568)视丘下部大脑皮层第三脑室丘脑厚（脑片）组织皮肤里层•Measurement–Intensity•(accumulated,mean,max,min)–Areaandperimetermeasurement–Time-lapsemeasurement–2Ddatahistogramdisplay–Colocalization多重标记、免疫荧光LocalizationandCo-localizationActivity-InducedRapidSynapticMaturationMediatedbyPresynapticCdc42Signaling中科院神经所段树民老师Neuron50,401–414,May4,2006Exampleoftheimage.Mousebrainslice）Dye:YFPOB：ＵｐｌａｎSAPO10xNumber：5X5=25imagesEachpoint:XYZimage512x512、15Z-Slices大视野多Z轴拼图Sampleprovidedby:Ms.MikakoSakurai,Mr.MasayukiSekiguchi(SectionChief)DepartmentofDegenerativeNeurologicalDiseases,NationalInstituteofNeuroscience,NationalCenterofNeurologyandPsychiatry三维重建、光谱扫描EGFPEYFP三维重建分析模块强大的3D功能3D反卷积运算功能灵活方便的3D控制软件1、鼠标任意拖动,任意切面、多种内嵌式切法2、3D与2D的任意转换3、XYZ三维任意投射控制FromYasuhiroKamei光谱拆分（Unmixing）GFP（细胞质内）FITC（细胞膜）GFP（细胞质内）FITC（细胞膜）TheIntensity:ROI1(GFP)andROI2(CFP)IntensityNormalized■XYlambdaacquisitionconditionLaserPower40%(457nm),HVabout500-600,C.A.150,andDM457/515,ScanSpeed:125kHzResolution10nmof,Step2nm,46images(465to565nm)CFP(Nucleus)/GFP(Microtubules)InfluenceofDM457/515.TheMargeimageafterUn-Mixing自动化光谱扫描技术GettingfluorescencespectraldataCh1Lambdascanningbytiltingofgrating---2nmλ高精度光谱扫描技术离子浓度比例和动态测量Fluo3/Ca2+wave~50mS快速钙信号采集——线扫描功能Ca2+Sparks•XYt•4000line/sec(512size)•16images/sec(256x256pixelimagesize)•16frames/secondat(256x256pixels)•Xt•XYZt•upto4DtemporaldetectionproceduresXYt/andXYZtimagestacks(4D)•cultureneuroncellareinjenctionwithIP3andcalusumelevelisstainedbyFlow3钙离子动态分析–Controloftheexperiments–Precision•MicrosecondorderHightemporalprecision–Flexibility•Interactivelychangingconditionswhilescanning–Programmable•Flowcontrol•Specialprogramlanguage–MultipointtimelapsMultichannelacquicision活体长时间的观察共聚焦在长时间动态观察中的应用：CalciumTomographicviewofthespecimen(microscopicCT)3-Dreconstructionfromz-sectionseriesThickspecimenCross-talkcorrectionFarredemissionwhichcannotbedetectedbyyoureyesandCCD.GreenFluorescentProtein(GFP)MultiphotonExcitationLaserScanningMicroscopyMoreApplicationFRETFRAPFLIPFLIMUNCAGINGphotoactivedFRETbackgroudHowFRETworks:FREToccurswhen1.Iftheemissionspectrumofa“donor”moleculeoverlapstheexcitationspectrumofan“acceptor”molecule2.Thedistancebetweenthesetwomoleculesarewithin10-100Å,3.thedonorandacceptorfluoro-phoresmustbeinfavorablemutualorientation.thentheexciteddonormoleculewilltransferenergynon-radiatively(withoutemittingofphoton)totheacceptor.Thatis,when