解析细胞内部的量化工具-高内涵

解析细胞内部的量化工具——高内涵分析平台刘育林Tel:13910309742Email:[e-mail]2History•1970sD.LansingTalyor•Cellomics,Inc.foundedin1996,HeadquartersPittsburgh,PA•FirstHCSReaderin1997•Strategicpartnerships:CarlZeiss,Spotfire,IBM,andEMC•PartofThermoFisherfamilyin2006.•Number1HCSProvider–GloballeaderinHCSwith300placements–Top20pharmaceuticalcompanies–Leadingacademiccentersglobally3HistoryinChina•2005年，全国第一台由CellomicsInc.生产的高内涵设备进驻国家新药筛选中心•2006年，国内第一篇高内涵综述文章，中国医学科学院药物研究所•直至2010年，ThermoFisherScientificCellomics公司提供的高内涵平台已经占据了60%的国内市场份额。将近20台高内涵设备为各大院所及知名药物研发企业提供服务。4细胞组学时代的到来——解析细胞内涵DNAGenesMessengerRNAGenomicsProteinProteomicsCellomicsWholeCell5高内涵研究细胞的本质6细胞组学的最新手段：高内涵技术对每个细胞进行多通道、多靶点的荧光扫描检测，由成像技术捕获图像信息后，经专用分析系统进行多指标在线分析，最终高效率地获取细胞内及细胞间的综合细胞学信息的检测技术平台。7什么是高内涵？全自动单细胞多靶点定量的精细的细胞分析系统高内涵：8每个细胞逐个动态跟踪分析9多靶点•Multipletargets,multipleprocesses,overtime•Individualcellresponsestelltherealstory!NuclearMorphology细胞核形态CalciumHomeostasis钙平衡MitochondrialTransmembranePotential线粒体膜电位MembranePermeability细胞通透性每个细胞还原真实的生物学现象细胞凋亡过程10从定性观察到定量标准SymbolCompoundEC50(nM)R(-)-Isoproterenol(+)-bitartrate(±)-Isoproterenolhydrochloride(-)-EpinephrinebitartrateS(+)-Isoproterenol(+)-bitartrate(-)-alpha-MethylnorepinephrineAlbuterolhemisulfateXMean12.025.943.738766018872.82.13.878181213SD10-11001011021031041051060102030405060708090100Log(nM)%MaxResponse10-11001011021031041051060102030405060708090100HCS11Samelabelintensity,differentdistribution•Location,location,location…Onelabel,severalpotentialdistributions……andheterogeneity精细的细胞内部分析12HCSAdvantage技术优势•高通量定量•自动,使用多孔板•简单,便于使用酶标仪读板机•灵活,试剂使用广泛•逐个细胞观察•空间结构分析和定位荧光显微镜•单个细胞分析•每个细胞多参数分析•结果可视化，配套分析软件流式细胞仪13酶标仪荧光显微镜流式细胞仪+++++++++++++++++++++自动化操作逐个细胞分析多指标分析附着细胞自动数据处理悬浮细胞空间结构细胞形态高内涵技术技术优势14•CellCountingandProliferation细胞增殖计数•Averagecellcountperfieldperwell每个孔每个视野视野细胞计数•CellularIntensity/Expression细胞内靶标的强度表达•Total,Average,&VariationofIntensityinprimaryobjectandderivedTargetregions靶标激活或表达的整体，平均，偏差水平统计•SpotAnalysis单点分析•TargetAreaandCount目标大小及计数•IntracellularTranslocation胞间迁移•AverageIntensityDifference荧光强度差值检测（细胞质和细胞核间）•CellMorphology细胞形态•ObjectArea,Perimeter,Length,Width,Shape目标大小、周长、长、宽、形状等•CellHealth&Toxicity细胞健康和毒性•ObjectNumber有效细胞计数•Area,Perimeter细胞核大小和周长等DataAnalysis数据分析15ThermoCellomics高内涵生物学应用通用型应用•靶点激活•细胞空间分析•细胞形态分析•逐点扫描毒性检测应用•细胞健康指标分析•彗星检测•细胞毒性分析•微核检测•斑马鱼毒性神经学应用•神经生长分析肿瘤学应用•小管形成•细胞周期•细胞黏附•细胞运动信号通路应用•分子转位•质膜转位•共定位分析受体内化•GPCR信号转导•微点发现16ThermoCellomics高内涵生物学应用ReceptorActivationCellmembranereceptorbindingGPCRinternalizationLabeledLigandinternalizationCellproliferationCellmorphologyCellsurvivalsignalingCellmigrationsignalingToxicityCellviabilityApoptosis/necrosisNuclearcountFluorescentproteinlocalizationTranscriptionfactorsReportergeneexpressionCellcyclestatusDNAreplicationstudiesWoundhealingStemcelldifferentiationColocalizationNuclear-cytoplasmictranslocationPlasmamembranetranslocationNeuriteoutgrowthSynaptogenesisTubeformationMicrotubulearrangementCytoskeletalreorganizationMicronucleiformationGenotoxicityHepatotoxicityOxidativestressPhospholipidosisCholeostasisCalciumhomeostasisNeurotoxicityStressresponseCellclassificationViralclearingGenefunctionModelorganismstissuesRNAiiNOS17细胞形态18细胞骨架F-actin•IncontrolcellstheF-actin(red)patternisfibrous,withamixtureofhigh,medium,andlowintensitiesthroughoutthecellbody•Incytochalasintreatedcellsthereisare-distributionofredfluorescence;Itismostlyneartheedgesofthecellandisbright1920微丝和点的检测21小管形成TubeFormationRawImage&OverlaysfromAutomatedAnalysisEndothelialcells(HUVECs)onMatrigel™formtubes内皮细胞形成小管F-ActinLabelHoechstnodeconnectedtubeunconnectedtuberejectedobjectnuclei©Cellomics,Inc.1999-200622nuclei小管形成TubeFormation23MyotubeFormationAssay肌小管020406080100120ValidobjectcountNumberofnucleipermyotubeMeanmyotubeareaMeanmyotubelengthMeanmyotubeprolatevolumeDrugEffectas%ofControlControlNifedipine-myotubeinhibitorNoTreatment100mMNifedipineBioApplicationUsed:MorphologyExplorerMultiplemeasuresofsize,shape,textureandpositioncanbecombinedtodefine“activity”24对照药物1Channel1Nucleus–DNAcontentChannel2PhosphorylatedRbChannel3TotalRb细胞周期CellCycleBioApplication2505101520253035404550020406080100120140160180AutomaticAssignmentofCellCyclePhasebasedonDNAContent%CellsinDifferentPhases01020304050607080G1G1SG2G2CellCyclePhase%ofCellsUntreatedNocodazoleDNAContent(DAPIIntensity)NumberofCellsDNAContentUntreatedNocodazoleRatioofG1/G2cells=4.64RatioofG1/G2cells=0.05G2G126Serialdilution(2-fold)ofeachcompoundfromrighttoleft.CellCycleProfiles27钙流检测(每个细胞的检测)•Timevs.intensityofcalciumindicator•Stimulatordilutionseries1101000µM0.010.128singlecelltracesE2(10-6M)F2(10-5M)G2(10-4M)B2(10-9M)C2(10-8M)D2(10-7M)WellMEANs29E2(10-6M)F2(10-5M)G2(10-4M)B2(10-9M)C2(10-8M)D2(10-7M)Actualtimepoints(ongraph)~1.4secintervals.So,shownhere:~every5thframe,for½thetotaltraceWellMEANs30分子转位Translocation通道靶标标记物分析区域1（蓝色）细胞核Hoechst33342基础目标2(绿色)NFkB间接荧光标记1°NFkB抗体；2°AlexaFluor488的2抗CircandRingNUCLEUSNFkBCircRegion–blueRingRegion-fromcirctoRed31NFkBTranslocationDoseResponse01002003004005000.0010.010.11101