WO2019207004A1迭代荧光成像[专利] - 图文

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(51)InternationalPatentClassification:

G01N33/58(2006.01)G01N33/50(2006.01)(21)InternationalApplicationNumber:

PCT/EP2019/060532

(22)InternationalFilingDate:

24April2019(24.04.2019)

(25)FilingLanguage:(26)PublicationLanguage:(30)PriorityData:18169077.7

24April2018(24.04.2018)

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patent(Rule4.17(H))Published:

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EnglishEnglish

EP

(71)Applicant:UNIVERSITATZURICH[CH/CH];Ramistr.

71,8006Zurich(CH).(72)Inventors:GUT,Gabriele;UniversityofZurichWin-terthurerstrasse190,8057Zurich(CH).PELKMANS,Lu?cas;UniversityofZurichWinterthurerstrasse190,8057Zurich(CH).HERRMANN,Markus;MGH&BWHCen-terforClinicalDataScience100CambridgeSt,Boston,MA02114(US).(74)Agent:SCHULZJUNGHANSPATENTANWALTE

PARTGMBB;Grohbeerenstr.71,10963Berlin(DE).(81)DesignatedStates(unlessotherwiseindicated,forevery

kindofnationalprotectionavailable):AE,AG,AL,AM,AO,AT,AU,AZ,BA,BB,BG,BH,BN,BR,BW,BY,BZ,CA,CH,CL,CN,CO,CR,CU,CZ,DE,DJ,DK,DM,DO,DZ,EC,EE,EG,ES,FI,GB,GD,GE,GH,GM,GT,HN,HR,HU,ID,IL,IN,IR,IS,JO,JP,KE,KG,KH,KN,KP,KR,KW,KZ,LA,LC,LK,LR,LS,LU,LY,MA,MD,ME,MG,MK,MN,MW,MX,MY,MZ,NA,NG,NI,NO,NZ,OM,PA,PE,PG,PH,PL,PT,QA,RO,RS,RU,RW,SA,SC,SD,SE,SG,SK,SL,SM,ST,SV,SY,TH,TJ,TM,TN,TR,TT,TZ,UA,UG,US,UZ,VC,VN,ZA,ZM,ZW.(84)DesignatedStates(unlessotherwiseindicated,forevery

kindofregionalprotectionavailable):ARIPO(BW,GH,GM,KE,LR,LS,MW,MZ,NA,RW,SD,SL,ST,SZ,TZ,UG,ZM,ZW),Eurasian(AM,AZ,BY,KG,KZ,RU,TJ,TM),European(AL,AT,BE,BG,CH,CY,CZ,DE,DK,EE,ES,FI,FR,GB,GR,HR,HU,IE,IS,IT,LT,LU,LV,MC,MK,MT,NL,NO,PL,PT,RO,RS,SE,SI,SK,SM,TR),OAPI(BF,BJ,CF,CG,Cl,CM,GA,GN,GQ,GW,KM,ML,MR,NE,SN,TD,TG).

(54)Title:ITERATIVEFLUORESCENCEIMAGING

(57)Abstract:Theinventionrelatestoamethodformultiplexstainingofabiologicalsampleinvolvingtheuseofabuffercombinationofblockingbuffer,imagingbufferandelutionbufferthatallowsformultiplestainingroundsofbiologicalsamples.Theblockingbuffercomprisesacompoundthatiscapableofbindingtohydrophobicbindingsitesnon-specificallyandasulfhydryl-reactivecompound.TheimagingbufferisatneutralpHandcomprisesaradicalscavenger,andtheelutionbufferisatpHlowerthan4andcomprisesabufferingcomponent,areducingagentandatleastonecompounddisruptinghydrogenbonds.Theinventionfurtherrelatestobuffersusedinthepracticeofthemethodoftheinvention,andtoakitcontainingthesebuffers.

IterativeFluorescenceImaging

Thepresentinventionrelatestoamethodthatallowsformultiplexantibody-stainingofbiologicalsamplesforimagingincludingbuffersforblocking,imagingandelution.

Background

Variousmethodshaverevolutionizedourabilitytoobtainmultiplexedmeasurementsoftheabundanceofhundredsorthousandsofdifferentmolecularspeciesfromsinglecells.Thesehavebroughtthepromisethatthroughlarge-scaleefforts,allfunctionallyrelevantcelltypesofahumanbodywill,inanunbiasedmanner,emergefromsuchdata.Sincesomeofthesemethodscanbeappliedinsitu,theidentifiedcelltypescanthenbeplacedwithinthecontextofacellpopulationortissue.However,itiswellknownthattheabundanceofaproteinorproteinstate,orofanRNAtranscript,isnotdirectlyinformativeaboutitsinvolvementincellular

function.Thisdependsonthespecificintracellularlocationandinteractionwithothermoleculesandintracellularstructures,whichmayonlyinvolveasmallfractionofthetotalcellularpool.Moreover,thephenotypeofanindividualcellisdeterminedbythefunctionalstate,abundance,morphology,andturnoverofitsintracellularorganellesandcytoskeletal

structures.Therefore,toobtainfunctionallyrelevantinformation,theseunbiasedlarge-scalemethodsneedtoextendthelengthscaleofmolecularmultiplexingintotheintracellulardomain,andultimatelyacquiretemporalinformation.Recently,atour-de-forcestudyachievedhigh-

resolutionintracellularimmunofluorescenceimagingof12,000proteins,fromwhichan

averagesubcellularmapofthehumanproteomewascreated.However,tounderstandhowthesubcellulardistributionoftheproteomeisfunctionallylinkedtothephenotypicstateofacellanditsmicroenvironmentandhowitrespondstovaryingconditions,suchmapsmustbe

directlymeasuredinthesamesinglecellandacrossmanycellsinsitu.Whilevariouspowerfulmethodsexistthatcanachievespatiallyresolvedantibodymultiplexingontissuesorsinglecells,nonemeetallrequirementstosimultaneouslycoverthetissue,singlecell,andhighly

resolvedintracellularlengthscalewhilstpreservingsamplequalityinahigh-throughputmannerformultipleconditionsandbecombinedwithlarge-scaleimageprocessingandmultivariatestatisticalapproachestoextracttherichamountofbiologicalinformationpresent

insuchdata.

Themainissuewithrecordingsuchmultiplexeddataistoallowformultipleantibodystainingroundswithoutcrosslinkingtheantibodytothesample.

Theobjectiveofthisinventionistoprovidemeansandmethodsthatallowformultipleantibodystainingroundswithoutcrosslinkingtheantibodytothesample.