Adaptive elements for STED microscopy [Elektronische Ressource] / presented by Andreas Engler

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rDissertationAndreassubmittedNaturaltoOraltheDoCombinedbFrnaculties2009fodegreerrthepNaturaDiplom-PhysiklbSciencesFandJunefofortheMathematicsofofctotheofRupScienceserto-CaresentedrolayUniversiteryEnglerofoHeidinerankfurth/M.lexamination:b24th,erg,GermanyKaAW.daptiveDr.elementsProf.foBrennerrStefanSTEDHellmicroscopDr.yrl-HeinzReferees:Prof.stateZusammenfassungPSFMouorescencederneFuoreszenztungMikroskcooofpelseerecsinadpropnicscanninghulatedtthemehrimageduelrcwithh3DBeugung?glicinBleicihrerberreicmicroscophbarenvAu?sungdernbmoescincreasehr?nkt.noDieseyEigenscscanninghaftreduceserh?hcepttwhicihfactors.rentheseEinussofaufbleacdieLebBildgebungAufnvhonwurden.zellbiologiscarehnentheObimag-jekten.ds,DieyvsuboTherlaiegendecaseArbTED)eitthebTefasstrsiceshformitadaptivVosed,erfahren,ediefdieyEigenscyheableacfxictefurthernAdiesermethomoscandernencessesMikroskfrustratedopBeobacehwundesenttlidiecghrascvverbMoessern.microscopExemplariscresolutionhdiraction,anmore.stimyulofafortedThisemissionersdepleticonsiderablyothenensem(STEes,D)ofMikroskmicroscopopieofwirdoptimizedderlighEinuss(SLM)desfScdepletionhaltmyuresolutionstersnoisef?rmicroscopdiewithAu?sungserh?helungroutiundprodiealeddetektierbarenqualitPhotonengenerateddurcahbdieisNutzungheinestispatialsimlighloadtsamplemoimagedulatorsThis(SLM)extendedoptinomcallyimethoereducesrt.ing,MitanddiesemsevprogrammierbareneectsWecerkzeugetterwirdbinationeinevnieenablesdenaforgewdynamicesenedFlexibilit?

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rDissertationAndreassubmittedNaturaltoOraltheDoCombinedbFrnaculties2009fodegreerrthepNaturaDiplom-PhysiklbSciencesFandJunefofortheMathematicsofofctotheofRupScienceserto-CaresentedrolayUniversiteryEnglerofoHeidinerankfurth/M.lexamination:b24th,erg,GermanyKaAW.daptiveDr.elementsProf.foBrennerrStefanSTEDHellmicroscopDr.yrl-HeinzReferees:Prof.stateZusammenfassungPSFMouorescencederneFuoreszenztungMikroskcooofpelseerecsinadpropnicscanninghulatedtthemehrimageduelrcwithh3DBeugung?glicinBleicihrerberreicmicroscophbarenvAu?sungdernbmoescincreasehr?nkt.noDieseyEigenscscanninghaftreduceserh?hcepttwhicihfactors.rentheseEinussofaufbleacdieLebBildgebungAufnvhonwurden.zellbiologiscarehnentheObimag-jekten.ds,DieyvsuboTherlaiegendecaseArbTED)eitthebTefasstrsiceshformitadaptivVosed,erfahren,ediefdieyEigenscyheableacfxictefurthernAdiesermethomoscandernencessesMikroskfrustratedopBeobacehwundesenttlidiecghrascvverbMoessern.microscopExemplariscresolutionhdiraction,anmore.stimyulofafortedThisemissionersdepleticonsiderablyothenensem(STEes,D)ofMikroskmicroscopopieofwirdoptimizedderlighEinuss(SLM)desfScdepletionhaltmyuresolutionstersnoisef?rmicroscopdiewithAu?sungserh?helungroutiundprodiealeddetektierbarenqualitPhotonengenerateddurcahbdieisNutzungheinestispatialsimlighloadtsamplemoimagedulatorsThis(SLM)extendedoptinomcallyimethoereducesrt.ing,MitanddiesemsevprogrammierbareneectsWecerkzeugetterwirdbinationeinevnieenablesdenaforgewdynamicesenedFlexibilit?timagesundyQualit?tdiedeshScschaltmnellerustersensprozesseerreicermhht.3DWahmen,eiter-bislhinnwurdedurczurzuezienhestenhenAbbildungerhindertvAbstractondernlebfar-eldendenesProbnotenlimitedmitySTEDaeiny-scThishnellesertlincreasesoimpactkthesealesadaptivcell-biologicalesing.Strahthesislscanvverfahrenmethoenthattimprowicekqualitelt,ofwbleelcmicroscophesadieclassAbbmoildungszeituorescenceumes.eininuencevielfacthehesisvwitherk?rztspatialundtdabdulatoreiingleicohzeitistimgemissiondie(SProbmicroscopetoscthehandonsignaltratioohnetheane.Bildqualit?togethereinzub??en.aDiesesvKerroonzeptcorrectionwurdeneumSLMeinvidwunriveiexibilitteandresyneuartigesthelokSTED-PSF.alurthermore,adaptivneweseBeleuceamhpatterntungsvproperfahrenwhicerwshortenseitert,acquisitionwmelcandhesultaneouslydasilluminationBleicohentheundwithphototounimpairedxiscqualithe.Wirkungencon-inisdenbProbanotherenvumloeinadaptivvielfacilluminationhesd,reduziert.hDerphotoSchhdarkutztransitionsderphototoProbeectseeralnimmThesetareindteressanreasedterbWresolution.eisecommitofderadaptiAu-e?sungdsdesunpMikroskedopstedzu.timesDieimagingKfastomprobinationanderenablesneuenSTEDadaptivpreviouslyenbVphotoerfahrenhing.erlaubtscanningContents.1.Intro.ductionevtoyfa.r-eld5optical.microscopeyi1and1.1.Ma.jor.aerimendbv.ancemen.ts.indopticaltofar-eldnmicprinciroscop.ySTED.tal.......A.of...mirror1.1.23.4Imageeformation38inSTEDconfo.calandmicroscopby4tecinhniquesreduction.of.49.of.....tation...552.1.3.Breaking.the4.5diraction.limit....ok.I.implemen.b...3.3.shift.........erimen.of.p...A.pattern.y..5.1.43.6Theofadaptiveescconcepts.fordaptiveSTEDphotomicroscopmi-y4.1.the.b.hing.b.The.l...........4.311RESCue2yA.daptive.STED-PSExpF.creation.13.2.1.SLMs.for.STED.microscopandy............and.Bibliography.endix.Exp.tal.tation.the.eam.concept...29.Decoupling.eam.and.rotation..13.2.2.The.error.correction.pro32cedureExp.tal.aluation.th.QuadScanner.erformance.....3.5.daptiv.scanning.in.microscop...........41.Conclusions.limitations15a2.3aptivExppatternerimeneamtalannvngalidation45ofAtilluminationhereduceSLMbleachingerrorSTEDccroscoporr49eThectionof.total.um.er.switc.cycles.its.enet.4.220basic2.4pTheesadvRESCuean.tage.of.an.e.rr.or.corrected.SLM..51.Implemen.of.in.microscop...........4.4.erimen.results..24.3.A.daptive.pattern.b.eam.scanning.STED.microscop.y.26573.1DiscussionBeamSummaryscanning.and.RESOLFT.microscop.y..............66.Conclusion.Outlo.68.78.App.7926I3.2VAbbellreviationsum2Dmicroscoptewuouso-dimensionalaccess3Dythreet-dF?rsteriGSDmensionaleADmirrorAlzheimerrelaxationDiseaseuorescenceAIetimeanalogFPGAinputFRAPAphotoOFWHManalogbrillaryoutputIAaOTFpixelacoustoDMopticaldirecttunabledarklteerFAspOMuorescenceacoustoFopticalofmoprogrammabledulatorarraAPDrecoayvhingalenergyanchalf-mheAPphotootediostatedeIAPPwaminoidvprecursordTproteindwAFMtimeatomicdeformableforceDMAmicroscopmemoryeD-RexAPDstateaEMvlectronalyancCShecorrelationphotoectroscopdioFLIMdelifCLEMimagingconOtrolledeldlighviewteldexpgaosureemicroscopyyuorescenceCPUvcenrtralafterprobleaccessingFRETunitresonancecTtransfercycfull-widthleaximtimeGFCTFglialconprtrastintransfergroundfunctiondepletionCWIcontinlmIOhedinput(uorescenoutputsiLCUVliquidRcrystalsSMLEDemissionlighTTLtersibleemittingrodiinolighdeblThstimlostatewTPSeruThthresholdt)LUTopticlotransitionokmeanupregiontablerTOPSLMopmoerationmoamplierrPCnoiseplersonalT-RexcomputerTFTPBSTi:Sapplateolarizingransistor-transistorbeream-splittervioletPMFrevpsaturableoallarizingt)mainRMStainingotsinglesquaredmoOIdeofbteresterreacPMTtimephotospatialmtultiplierdulatortubsingleedePPephaseSNplategnalPSFSTEDpuoinatedtdepletionspreadtripletfunctionrelaxationQEthinquantransistorstumtitanium-sapphireeciencythinRESCuesplinesreductiontoflogicexcitationuppandthresholdsignalultrasuppression(lighcyclesIVRESOLFTy1toIntrospductionmitoasfatr-eldesopticaltomicroscopZernikycompletely1.1aMstateajoendenrtrastadvancementsdistinctinconceptopticaleryfayr-eldductionmicroscopconybCellwbiologyofaimsshiftedatThisgettingvapdeepber1.1understandingtofoflife.tNewunoinsighntstheinmatoercellularhprooaceFluorescencedouresafterareonessenduetialradiation-lesstohromeunderstandtheandercurethemastronglyjorddiseshiftasesallosuctionhtheasofcanceropticalorSinceAlzheimercellletheadingtrasttoindexaconbasetterenandandlongeraslifebfor[4]helduman.kind.breakthroughSincewcompinonenuorescence,tsvidesoftandhingen-teractionstwithintaneoustheacellacanabsorptionnotobaevobservsolvedandbreyuorothethenakenergyedphotoneyloe,relativtheenergyapplicatioednlossofetoandolsitsisStoknecessaryThe.herebThistheirntheterestsignalinlighthethemicrodiccosmorstriggered(seetheg.devcanelofopusagemmenossibilittduceofendenthoreariations.rstuorophoresopticalafar-eldtmicroscopenhancedeinbofylabHansdiscoanduorescenceSaccohariastrastJansen,ywhicehorrepresendarktsmicroscopthe[5]cornerstoneTheforjorstillwongoinghoresearcevhtheintrotheofeldwhicofpromicroscopaedierendevappreclopmenfort.trastHeration.erebisyhe,ontheemissionfar-eldflighphotontymicroscopuorophoreetheholdsofaphnotabletpwithositionshortercomparedatoelengthothertomicroscopicentecrelaxationhnfastivibrationalqueslaxationsucthehcasinAexcitedtomictheFoforceemittedMicro-isscoptoesw(AFM)energy[1]eortheElecoftabsorbronphoton.Menergyicroscopisesdy(EM)dep[2,t3],callebafterecausediscoopticalerer:far-eldesmicroscop[6].esStokareshiftcapablyewstoseobservaea-structuresofandconprogeneratingcessesandinsideexcitationlivingtcells.yTheuserstsimpleopticalhroicmicroscoprresorwltersereg.hinderedandb1.3).yuorophorestheirstainpstructuresotheortheconoftrast,hewhicoershpwyasprobasedcononindepabsorptiontandabsorptionreection.refractionThisvaThiswofexpaseriencedtrsteadysprogressagenowvgreatlyerbthethedecadesvbtionyimmdevuorescenceelopmenelingtsthesucvhofasproteinsthemark-phaseIt2n1RaInrmationtrobductionofto[15].far-elduorescopticaalwmicroscopyytheerscessing[7].nWithimpthedistinguishaidareasoftuorescenRatTheproteins,isspinecicitsproteinstioninsideTtheproplivingtcelloscctechniquesanOnebtheeoflabofeledtandAimagedob[8].trastIntrastgeneral,arethetimmtheunovideduorescencetolabwelingmicroscopusesexcitationorganicThedyhighesewithandacalprimary-secondaryMianrepresentibvoopticaldy1.2bindingotoationthectproteinkofoscincalterest.sampleThistheprobcedureinhasOthersevperalalsoadvisanitages.terest.Theiflaboeledthesitepast-likwasellbasjectsthedeneddyAlthougherep-canspbconeturescthehangedelemenblighywjustonreplacotingeldthevprimthearerformanceylorPhotontheesecondarydataanthetibe,obdypre-microscopsptheectivtoelythis.limitationAmicdditionallyy,magesevconeralmicroscopsecondaryastanthetibaspoimagediesthcadomainsnmicbindyonofeacThinsideprimaryconanstructurestibjorocommonlydythe.counThismienhancesthetheofsignalhandorthereforeelengththeeconcontrast.alThhighereofotherofmacjorresolvlabgeneratedelingecomestecw.hniquecriterionisumthewhicusetofpuorescobenceresolvproteins.%Asmallestcellwlineocanfulllingbcriterioneresolutiontransystems-termfectedarbitrarilytopropromoreduceecicitaanduorescentrasttstruc-proteinofconnectedteresttremainingoeakthetsproteinfar-eldoftinyterest.ereThisdiractiwlimitedaspyand,wideandetection.imminuno-labenelingofoflaser,thepceldetectioldevicesisiknotthenMultipliereubcessary(PMT)ancomputationalymore.proBymadeusingconfodymicroscopesrstoroseduorescenytnskyproteinsossiblewithThisdierenettsexcitationrsand/orstepemissionospercomeectraremainimoregthanofonefar-eldstructurercanopb.eIdetectedfosiminultaneouslyf.calTherefoyrContregenerinasteractionsmostandortantleoincaliza-formationtionsofcanebeyeofmeasuredencewithrrespopectistoimagingeacbiologi-hstructures.other.oThestructuresattributesaoftheatrastuorescenthesetismoleculemadepattribute.endisstronglygeneratedonyitsdierenceenphotonvironmentst.theSevscellaneouseralofotherimage.tattributeselighcsuchniquesasuseolarizationthiswcvharacteristiccantobcollectused.furtherhigherinformationtrastabequivoutenthetolabspatialelednformationproteinthestructures,jectforinexampleStructuresFluorescenceannotLIfetimeeMicroscoped,ythe(FLIM)con[9],bFluorescencetoReso-lonanceTheEnergyyleighTdenesransferminim(FRET)con[10]at,hFluorescenceoinRecotvoeryoinAfterePhotojectsbleacstillh-ableing25(FRAP)[16].[11,resulting12]distanceandetFluorescenceeenCorrelationwSpobectroscstilloptheyyleigh(FisCS)as[13,of14].imagingAlthough.allthetheseresolutionindenedvitentions