4-Time domain lifetime measurements

Time-resolvedmeasurementsarewidelyusedinfluores-cencespectroscopy,particularlyforstudiesofbiologicalmacromoleculesandincreasinglyforcellularimaging.Time-resolvedmeasurementscontainmoreinformationthanisavailablefromthesteady-statedata.Forinstance,consideraproteinthatcontainstwotryptophanresidues,eachwithadistinctlifetime.Becauseofspectraloverlapoftheabsorptionandemission,itisnotusuallypossibletoresolvetheemissionfromthetworesiduesfromthesteady-statedata.However,thetime-resolveddatamayrevealtwodecaytimes,whichcanbeusedtoresolvetheemissionspectraandrelativeintensitiesofthetwotryptophanresidues.Thetime-resolvedmeasurementscanrevealhoweachofthetryptophanresiduesintheproteinisaffectedbytheinterac-tionswithitssubstrateorothermacromolecules.Isoneofthetryptophanresiduesclosetothebindingsite?Isatryp-tophanresidueinadistaldomainaffectedbysubstratebindingtoanotherdomain?Suchquestionscanbeansweredifonemeasuresthedecaytimesassociatedwitheachofthetryptophanresidues.Therearemanyotherexampleswherethetime-resolveddataprovideinformationnotavailablefromthesteady-statedata.Onecandistinguishstaticanddynamicquenchingusinglifetimemeasurements.Formationofstat-icground-statecomplexesdonotdecreasethedecaytimeoftheuncomplexedfluorophoresbecauseonlytheun-quenchedfluorophoresareobserved.Dynamicquenchingisarateprocessactingontheentireexcited-statepopula-tion,andthusdecreasesthemeandecaytimeoftheentireexcited-statepopulation.Resonanceenergytransferisalsobeststudiedusingtime-resolvedmeasurements.Supposeaproteincontainsadonorandacceptor,andthesteady-statemeasurementsindicatethedonoris50%quenchedbytheacceptor.Theresultof50%donorquenchingcanbedueto100%quenchingforhalfofthedonors,or50%quenchingofallthedonors,orsomecombinationofthesetwolimit-ingpossibilities.Thesteady-statedatacannotdistinguishbetweentheseextremecases.Incontrast,verydifferentdonorintensitydecayswouldbeobservedforeachcase.Ifallthedonorsare50%quenchedbytheacceptors,andtheacceptorsareatasingledistance,thenthedonordecaywillbeasingleexponentialwithalifetimeofhalftheunquenchedlifetime.If50%ofthedonorsarecompletelyquenchedand50%arenotquenched,thenthedonorlife-timewillbethesameastheunquenchedlifetime.Amulti-exponentialdecaywouldbeobservedifthedonorispartial-lyquenchedbytheacceptorandsomeofthedonorsdonothaveanearbyacceptor.Thetime-resolveddonordecaysarehighlyinformativeaboutthepurityofthesampleaswellasthedonor-to-acceptordistance.Therearemanyotherinstanceswherelifetimemeas-urementsareadvantageousoversteady-statemeasure-ments.Oneimportantapplicationiscellularimagingusingfluorescencemicroscopy.Whenlabeledcellsareobservedinafluorescencemicroscope,thelocalconcentrationoftheprobeineachpartofthecellisnotknown.Additionally,theprobeconcentrationcanchangeduringthemeasurementduetowashoutorphotobleaching.Asaresultitisdifficulttomakequantitativeuseofthelocalintensities.Incontrast,iftheprobeemissioniswellabovethebackgroundsignal,fluorescencelifetimesaretypicallyindependentoftheprobeconcentration.Manyfluorescencesensorssuchasthecalciumprobesdisplaychangesinlifetimeinresponsetoanalytes.Also,resonanceenergytransfer(RET)revealstheproximityofdonorsandacceptorsbychangesinthedonorlifetime.Becauseofadvancesintechnologyfortime-resolvedmeasurements,itisnowpossibletocreatelifetimeimages,wheretheimagecontrastisbasedonthelifetimeineachregionofthesample.Fluorescencelifetimeimagingmicroscopy,orFLIM,hasnowbecomeanaccessibleandincreasinglyusedtoolincellbiology(Chapter22).An4Time-DomainLifetimeMeasurements97understandingofFLIMmustbebasedonanunderstandingofthetechnologyusedfortime-resolvedfluorescencemea-surements.Priortodescribingthetechnologyfortime-resolvedmeasurementswepresentanoverviewofthetwodominantmethodsfortime-resolvedmeasurements:thetime-domain(TD)andfrequency-domain(FD)methods.Therearealsoseveralvariationstoeachapproach.Sincethepreviousedi-tionofthisbooktherehavebeenadvancesinbothmethods.Thetime-domaintechnologyhasbecomesmaller,lessexpensive,andmorereliable.Wewillalsodescribesomeoftheearlierapproaches,whichclarifywhyspecificproce-dureshavebeenselected.Wealsodiscusstheimportanttopicofdataanalysis,whichisessentialforusingtheexten-sivedatafrommoderninstruments,andavoidingmisuseoftheresultsbyover-interpretationofthedata.4.1.OVERVIEWOFTIME-DOMAINANDFREQUENCY-DOMAINMEASUREMENTSTwomethodsofmeasuringtime-resolvedfluorescenceareinwidespreaduse:thetime-domainandfrequency-domainmethods.Intime-domainorpulsefluorometry,thesampleisexcitedwithapulseoflight(Figure4.1).Thewidthofthepulseismadeasshortaspossible,andispreferablymuchshorterthanthedecaytimeτofthesample.Thetime-dependentintensityismeasuredfollowingtheexcitationpulse,andthedecaytimeτiscalculatedfromtheslopeofaplotoflogI(t)versust,orfromthetimeatwhichtheintensitydecreasesto1/eoftheintensityatt=0.Theinten-sitydecaysareoftenmeasuredthroughapolarizerorientedat54.7Efromtheverticalz-axis.Thisconditionisusedtoavoidtheeffectsofrotationaldiffusionand/oranisotropyontheintensitydecay(Chapter11).Thealternativemethodofmeasuringthedecaytimeisthefrequency-domainorphase-modulati