熒光共振能量轉(zhuǎn)移內(nèi)容

FluorescenceResonanceEnergyFluorescenceResonanceEnergyTransfer-Dr.XiankeCarlZeissMicroscopyWhyweneed為何需要做FRETWhyweneed為何需要做FRETallowsthedetectionofmolecule-moleculeinteractionsintheNanometerwithlightAdaptedfrom:Terjung&Zimmermann,EAMNETteachingmodile,Dr.XiankeSHI,ZeissSlideWHATISWHATISDr.XiankeSHI,ZeissSlideWhatis什么是Whatis什么是FluorescenceResonanceEnergyF?rsterResonanceEnergyTomeasuremolecularDr.XiankeSHI,ZeissSlideWhatis什么是Whatis什么是fromadonorfluorophoretoanacceptorfluorophore.Dr.XiankeSHI,Zeiss5NoPrerequesitesforTheAcceptor′sPrerequesitesforTheAcceptor′sexcitationspectrummustoverlapwiththeDonor′semmissionspectrumFRETisanelectron-basedenergytransfer.(Don′tthinkthat-inthisexample-CFPproducescyanlightandthenexcitesYFP,soYFPthenfluoresces…that′swrong!)PossibleFRETDr.XiankeSHI,Zeiss7PrerequesitesforDonorandacceptormustbeinclose>PrerequesitesforDonorandacceptormustbeinclose>FRETissometimesalsocalledF?rsterResonanceenergytransfer,asF?rsterwasthefirstwhopublishedquantitativetheoryofmolecularresonanceenergytransfer(F?rster1946,1948)FRET~1/rMoreprecise:高低Dr.XiankeSHI,ZeissSlidePossibleApplicationsforConformationalchangesofDue---ChanginginPossibleApplicationsforConformationalchangesofDue---ChanginginIonconcentrationsLigandbindingPhosphorylation,Myristylation….)…-DNAFRETPair:CFP+ProteinFRETPair:Quantum(fluorescentsemiconductorFRETPair:CFP+Adaptedfrom:Dubertret,NatureMaterials4,797–798(2005)&Zaccolo,Circ.Res.94,866-73Dr.XiankeSHI,ZeissSlideFRETBiosensorsandtheirFRETBiosensorsandtheirDr.XiankeSHI,ZeissSlideHOWTODOFRETHOWTODOFRETWITHDr.XiankeSHI,ZeissSlideFRETMicroscopywithLSMFRETMicroscopywithLSMFRETDetection FluorescenceLifetime Dr.XiankeSHI,ZeissFRETWorkflowinImageAcquisitioninTimeFRETWorkflowinImageAcquisitioninTimeSerieswithbleachevent(s)FRETDr.XiankeSHI,ZeissSlidePHOTOBLEACHINGANALYSISFRET實(shí)PHOTOBLEACHINGANALYSISFRET實(shí)驗(yàn)方法一：受體漂白Dr.XiankeSHI,ZeissSlideFRETMicroscopywithLSMFRETFRETMicroscopywithLSMFRETDetection:AcceptorsimpleFRETdetectionDe-quenchingofthedonorafterselectivephotobleachingoftheacceptorcausesanincreaseindonoremissionthatcanbereadilyDr.XiankeSHI,ZeissAcceptorData1---Two2--Track1DonorExcitationandEmissionTrack2AcceptorData1---Two2--Track1DonorExcitationandEmissionTrack2AcceptorExcitationandEmissionSwitch--Line:thesamplemightmoveFrame:thesampleisnotmovingFastacquisition:worseSlowacquisition(longpixeldwell,averaging):goodSNRdirectinfluenceontheFRETcalculation!Dr.XiankeSHI,ZeissSlideNotwellsuitedforlivingsamplesduetoEnsurethatonlytheAcceptorisbleached!Pre-experimentwitha“Donoronly”AcceptorData3AlwaysacquiresomeimagesbeforethebleachAcceptorData3AlwaysacquiresomeimagesbeforethebleachforSingleorRepetitive--Singlebleachisfast,buthardtoanalyze.RepetitiveBleachtakeslongerbutbetteroptionsfor4FortherepetitiveBleach,chooselaserpoweranditerationstogenerateseveraldataDr.XiankeSHI,ZeissSlideAcceptorData???VeryeasysetupReliableanswerto“FRET”orAcceptorData???VeryeasysetupReliableanswerto“FRET”or“noonlytwodatapointsareo-bleachingofthesampleisnotDonorintensityisnotrisingduetobleachingandotherfactorstheAcceptorcannotbebleachedcompletelyaway-->hencetheFRETEff.isalwaystoolowbydefault.--Dr.XiankeSHI,ZeissSlide!AcceptorDataActivatethebleachedregionAcceptorDataActivatethebleachedregionasanObject.Drawaregionintothebackground,andactivateitas12Dr.XiankeSHI,ZeissSlideAcceptorData!ThegreyvaluesareautomaticallyloadedintoAcceptorData!ThegreyvaluesareautomaticallyloadedintotheThresholdstabwhenaBackgroundregionisWhenchangedaremadeintheThresholdstab,thebackgroundregionisautomaticallyDr.XiankeSHI,ZeissSlideAcceptorDataDefineDonorandAcceptorchannelSetthenumberofframesbeforethebleach(Dpre)DefineifitwasAcceptorDataDefineDonorandAcceptorchannelSetthenumberofframesbeforethebleach(Dpre)DefineifitwasarepetitivebleachIfitwasarepetitivebleach,definewhichframeshallbeusedasthe1234!NorepetitiveistheframerightbeforethebleachistheframerightafterthebleachRepetitiveDpreistheframerightbeforetheischosenbyDr.XiankeSHI,ZeissSlideAcceptorDataPressAnalyzetogeneratetheFRETimageandFRETtableActivatetheOntheAcceptorDataPressAnalyzetogeneratetheFRETimageandFRETtableActivatetheOntheflycheckbox–analysisisupdatedatonce12Region0isthewholeRegion1etc.theDr.XiankeSHI,ZeissSlideAcceptorDataFRET(p)EffAcceptorDataFRET(p)Eff%=TheFRETEff.foreachpixeliscalculatedandthemeanisFRETEff%=ThemeanforDpostandDpreisformed,theFRETEff.iscalculatedDr.XiankeSHI,ZeissSlideAcceptorDataBleachExcelSheet?AcceptorDataBleachExcelSheet?FRET???ExcelReadFRET??ExampleheretwoDr.XiankeSHI,ZeissSlideFRET實(shí)驗(yàn)FRET實(shí)驗(yàn)方法二：受體發(fā)射測定法Dr.XiankeSHI,ZeissSlideFRET:SensitizedemissionEmployingFRETasFRET:SensitizedemissionEmployingFRETasarulerinFRETisanon-radiativetransferofanexcitedstatefromonefluorophore(donor)toanotherFREToccursifdonorandareincloseproximity(1-10FRETpermitsmicroscopicproximityassaysatthemolecularDr.XiankeSHI,ZeissFRET–SensitizedIλNeedscorrectionbasedFRET–SensitizedIλNeedscorrectionbasedonintensityvalues–thedifferentanalysistoolsusedifferentoptions.Dr.XiankeSHI,ZeissDonorEmissionCrosstalkFRET–SensitizedImagingTrack1FRETTrack2FRET–SensitizedImagingTrack1FRETTrack2IIλλDr.XiankeSHI,ZeissFRET–SensitizedExperimentdata33Dr.XiankeFRET–SensitizedExperimentdata33Dr.XiankeSHI,Zeiss!Youneedthreeimageswith3-channelsFRET–SensitizedNamingInconsistentintheInZEN(blackDDonorFRETchannelSample(smalldonoronlyacceptoronlyFRETFcFRET–SensitizedNamingInconsistentintheInZEN(blackDDonorFRETchannelSample(smalldonoronlyacceptoronlyFRETFcasexampleDr.XiankeSHI,ZeissSlideFaAf FdDf FRET–SensitizedExperimentdataDfFaAfFc FRET–SensitizedExperimentdataDfFaAfFc asexampleDonotchangelaserpowerwhenswitchingDonotchangeMastergainwhenswitchingDonotchangeMasterforFRETchannelandAcceptorDr.XiankeSHI,Zeiss!StarttheExperimentwiththesettingsoftheFRETsample.Save/ReusethesefortheDonoronlyandAcceptoronlysample.AllthesecoefficientsareFRET–SensitizedExperimentdataFRET–SensitizedExperimentdata???TakeimagesforallthreeImageDonorandFRETchannelinonetracktoreduceerror.Useline-wiseswitch,toreduceerror!Dr.XiankeSHI,ZeissFRET–SensitizedAcceptorLoadtheimageFRET–SensitizedAcceptorLoadtheimageoftheAcceptoronlysampleOpentheFRETtabDrawregionsintotheimage2The2ActivatethemasObjectsor1234Dr.XiankeSHI,ZeissFRET–SensitizedAcceptorandDonor2FRET–SensitizedAcceptorandDonor2OpenParameterstabAssignchannelsRatiovaluesaredisplayedSavetheacceptorvaluesFilewithacceptorvaluessavedinthesamefolderasthe345AcceptorOnly6!RepeatthesestepsfortheDonoronlyDr.XiankeSHI,ZeissFRET–SensitizedOpenFRETtabDrawregionsFRET–SensitizedOpenFRETtabDrawregionsintotheimage22ActivatethemasBackground1234Dr.XiankeSHI,ZeissFRET–Sensitized1OpenParameterstabAssignchannelsLoadDonorFRET–Sensitized1OpenParameterstabAssignchannelsLoadDonorcoefficient25?ChoosetheDonoronly.fret34?fromtheexplorerDonorandAcceptorcoefficientsareDr.XiankeSHI,ZeissFRET–Sensitized3FRET–Sensitized3OpentheFRETtabPressAnalyzebuttonFRETImageisdisplayedFRETtableisdisplayedActivate?onthefly“changesshouldbedisplayedChoosetheanalysisoptiontheFRETimagetobedisplayed(tablewillalwaysshowall)41526Dr.XiankeSHI,ZeissFRET–SensitizedMethodFcbyFRET–SensitizedMethodFcbyD.C.Youvanetal.ThismethodcalculatestheabsoluteintensityofsensitizedemissionintheFRETchannelasFc.Therefore,highFcvaluesoccurathighmolecularFcFfDonorcorr.Acc.Dr.XiankeSHI,ZeissFRET–SensitizedMethodFRETNbyG.W.FRET–SensitizedMethodFRETNbyG.W.Gordonetal.ThismethodemphasizesoccurringatlowconcentrationsofFRETpairs.FfDonorcorr.Acc.GFdG=Gordonaconstantvalueforadonor-acceptorcombinationononesystemDr.XiankeSHI,ZeissFRET–SensitizedMethodN-FRETbyX.FRET–SensitizedMethodN-FRETbyX.Xiaetal.NFprovidesFRETvaluesFRETpairs.NFFfDonorGFdG=Gordonaconstantvalueforadonor-acceptorcombinationononesystemDr.XiankeSHI,ZeissSensitizedEmission–3(main)Dr.XiankeSHI,ZeissNFSensitizedEmission–3(main)Dr.XiankeSHI,ZeissNFFfDonorcorr.GFdMethod3:NF(normalizedFRET)X.Xiaetal.2001IndependentofDonorundAcceptorconcentrationFfDonorcorr.Acc.FnGFdMethod2:Fn(FRETnet)G.W.Gordonetal.FacilitatesgettingFRETSignalsalsoifDonor/AcceptorsignalsareMethod1:Fc(FRETcorrected)D.C.Youvanetal.DependendonDonor/Acceptorconcentration.Ifitsless→noFRETFurtherJares-Erijman,E.A.,FurtherJares-Erijman,E.A.,andJovin,T.M.FRETImage.NatureBiotechnology21:1387-1395Berney,C.andDanuser,G.FRETornoFRET:aquantitativecomparison.BiophysicalJournal84:3992-4010(2003).Piston,D.W.andKremers,G.J.FluorescentproteinFRET:thegood,thebadandtheugly.TrendsinBiochemicalSciences32:407-414(2007).Gordon,G.W.,Berry,G.,Liang,X.H.,Levine,B.andHerman,B.Quantitativefluorescenceresonanceenergytransfermeasurementsusingfluorescencemicroscopy.BiophysicalJournal74:270