FluorescenceSpecroscopy朝陽(yáng)科技大學(xué)熒光光譜朝陽(yáng)科技大學(xué)資料課件

FluorescenceSpectroscopyPartI.BackgroundFluorescenceSpectroscopyPart1Perrin-JablonskidiagramPerrin-Jablonskidiagram2SissingletandTistriplet.TheS0stateisthegroundstateandthesubscriptnumbersidentifyindividualstates.SissingletandTistriplet.3n

→p*

<p→p*

<n

→s*

<s→p*

<s→s*EnergylevelofMOn→p*<p→p*<n→s*<s→4DS0Singlet&TripletDS0Singlet&Triplet5CharacteristicsofExcitedStatesEnergyLifetimeQuantumYieldPolarizationCharacteristicsofExcitedSta6StokesshiftTheStokesshiftisthegapbetweenthemaximumofthefirstabsorptionbandandthemaximumofthefluorescencespectrumlossofvibrationalenergyintheexcitedstateasheatbycollisionwithsolventheatStokesshiftTheStokesshifti7Example:7-amino-4-methylcoumarin(AMC)Example:7-amino-4-methylcouma8ExampleExample9ExamplefluorophoresfluoresceinethidiumbromideboundtoDNA.Examplefluorophoresfluoresce10FluorescenceSpecroscopy朝陽(yáng)科技大學(xué)熒光光譜朝陽(yáng)科技大學(xué)資料課件11LifetimeLifetime12LifetimeExcitedstatesdecayexponentiallywithtime–I=I0e-t/t

I0

istheinitialintensityattimezero,

I

istheintensityatsomelatertimet

t

isthelifetimeoftheexcitedstate.

kF=1/t,wherekFistherateconstantforfluorescence.LifetimeExcitedstatesdecaye13QuantumYieldQuantumYield=FF?FF=numberoffluorescencequantaemitteddividedbynumberofquantaabsorbedtoasingletexcitedstate?FF=ratioofphotonsemittedtophotonsabsorbedQuantumyieldistheratioofphotonsemittedtophotonsabsorbedbythesystem:QuantumYieldQuantumYield=F14QuantumYieldQuantumYield15QuantumYield&StructurerigidityQuantumYield&Structurerigi16PolarizationMoleculeofinterestisrandomlyorientedinarigidmatrix(organicsolventatlowtemperatureorroomtemperaturepolymer).Andplanepolarizedlightisusedastheexcitationsource.DegreeofpolarizationisdefinedasPI||andI^

aretheintensitiesoftheobservedparallelandperpendicularcomponents,a

istheanglebetweentheemissionandabsorptiontransitionmoments.Ifais0°thanP=+1/2,andifais90°thanP=-1/3.PolarizationMoleculeofintere17?Steady-statemeasurements:F,I?Time-Resolvedmeasurements:tExperimentalMeasurements?Steady-statemeasurements:F18InstrumentsInstruments19InnerFilterEffectAtlowconcentrationtheemissionoflightisuniformfromthefronttothebackofsamplecuvette.Athighconcentrationmorelightisemittedfromthefrontthantheback.SinceemittedlightonlyfromthemiddleofthecuvetteisdetectedtheconcentrationmustbelowtoassureaccurateFFmeasurements.InnerFilterEffectAtlowcon20InnerFilterEffectInnerFilterEffect21If(em)

=IAbs(ex).

f

.f(em).

KI0(ex)ememememmeasuredintensityoffluorescenceatemabsorbedintensityatexfluorescencequantumyieldfractionofintensityemittedatthatparticularwavelengthfractionoftotalfluorescencethatisdetectedIfA0Ifwemeasurethesampleandastandardunderthesameexperimentalconditions,keepingexconstant:Important:theindexofrefractionofthetwosolvents(sampleandstandard)mustbethesameStandards:QuininesulfateinH2SO41N:f=0.55FluoresceininNaOH

0.1N:f=0.93MeasurementoffluorescencequantumyieldsIf(em)=IAbs(ex).f.f(22TheTCSPCmeasurementreliesontheconceptthattheprobabilitydistributionforemissionofasinglephotonafteranexcitationyieldstheactualintensityagainsttimedistributionofallthephotonsemittedasaresultoftheexcitation.Bysamplingthesinglephotonemissionafteralargenumberofexcitationflashes,theexperimentconstructsthisprobabilitydistribution.Timecorrelatedsinglephotoncounting:#events....t(nsec)differentexcitationflashesStartPMTStopPMTsampleexc.monochromatoremissionmonochromatorpulsedsourcetMeasurementoffluorescencelifetimesTheTCSPCmeasurementrelieso23Lifetimens

AbsorptionFluorescenceWavelengthnmAbsorptivityWavelengthnmQuantumTryptophan2.62805,6003480.20Tyrosine3.62741,4003030.14Phenylalanine6.42572002820.04IntrinsicFluorescenceofProteinsandPeptidesLifetimeAbsorptionFluorescence24FluorescenceSpecroscopy朝陽(yáng)科技大學(xué)熒光光譜朝陽(yáng)科技大學(xué)資料課件25FluorescenceSpecroscopy朝陽(yáng)科技大學(xué)熒光光譜朝陽(yáng)科技大學(xué)資料課件26Tryptophan,thedominantintrinsicfluorophore,isgenerallypresentatabout1mol%inproteins.AproteinmaypossessjustoneorafewTrpresidues,whichfacilitatesinterpretationofthespectraldata.Tryptophanisverysensitivetoitslocalenvironment.Itispossibletoseechangesinemissionspectrainresponsetoconformationalchanges,subunitassociation,substratebinding,denaturation,andanythingthataffectsthelocalenvironmentsurrondingtheindolering.Also,Trpappearstobeuniquelysensitivetocollisionalquenching,eitherbyexternallyaddedquenchers,orbynearbygroupsintheprotein.Tryptophanfluorescencecanbeselectivelyexcitedat295-305nm.(toavoidexcitationofTyr)TryptophanTryptophan,thedominantintri27IIIIIIIVVExample:

TyrosineanditsderivativesIIIIIIIVVExample:Tyrosineand28IIIIIIIIIIIVIVIIVVIIIIIIIIIIIVIVIIVV29FluorescenceSpecroscopy朝陽(yáng)科技大學(xué)熒光光譜朝陽(yáng)科技大學(xué)資料課件30EmissionspectraofPseudomonasfluorescensazurinPfl.For275-nmexcitation,apeakisobservedduetothetyrosineresidue(s)Thepositionandstructureofthefluorescencesuggeststhattheindoleresidueislocatedinacompletelynonpolarregionoftheprotein.TheseresultsagreewithX-raystudies,whichshowthattheindolegroupislocatedinthehydrophobiccoreoftheprotein.Inthepresenceofadenaturingagent,theTrpPemissionlosesitsstructureandshiftsto351nm,characteristicofafullyexposedTrpresidue.ChangesinemissionspectracanbeusedtofollowproteinunfoldingEmissionspectraofPseudomona31FluorescenceSpecroscopy朝陽(yáng)科技大學(xué)熒光光譜朝陽(yáng)科技大學(xué)資料課件32Resolutionofthecontributionsofindividualtryptophanresiduesinmulti-tryptophanproteins.I(,t)=i()exp(-t/i)i1=2ns,

2=5ns1=2ns2=5nst(ns)Fluorescenceintensity(A.U.)Fluorescenceintensity(A.U.)wavelength(nm)emExampleTime-resolvedproteinfluorescenceResolutionofthecontribution33IsolatedfromthePacificjellyfishAequoreavictoriaandnowplayscentralrolesinbiochemistryandcellbiologyduetoitswidespreaduseasaninvivoreporterofgeneexpression,celllineage,protein-proteininteractionsandproteintraffickingOneofthemostimportantattributesofGFPwhichmakesitsousefulinthelifesciencesisthattheluminescentchromophoreisformedinvivo,andcanthusgeneratealabeledcellularmacromoleculewithoutthedifficultiesoflabelingwithexogenousagents.Greenfluorescentprotein(abbreviatedGFPIsolatedfromthePacificjell34ThestructureofGFP:eleven-strandbeta-barrelwrappedaroundacentralalpha-helixcore.ThiscentralcorecontainsthechromophorewhichisspontaneouslyformedfromachemicalreactioninvolvingresiduesSer65,Tyr66,andGly67(SYG)ThereiscyclizationofthepolypeptidebackbonebetweenSer65andGly67toforma5-memberedring,followedbyoxidationofTyr66.ThehighquantumyieldofGFPfluorescenceprobablyarisesfromthenearlycompleteprotectionofthefluorophorefromquenchingwateroroxygenmoleculesbyburialwithinthebeta-barrel.RibbondiagramoftheGreenFluorescentProtein(GFP)drawnfromthewild-typecrystalstructure.Theburiedchromophore,whichisresponsibleforGFP'sluminescence,isshowninfullatomicdetail.ThestructureofGFP:eleven-35WildtypeGFPfromjellyfishhastwoexcitationpeaks,amajoroneat395nmandaminoroneat475nmwithextinctioncoefficientof30,000and7,000M-1cm-1,respectively.Itsemissionpeakisat509nminthelowergreenportionofthevisiblespectrum.ForwildtypeGFP,excitingtheproteinat395nmleadstorapidquenchingofthefluorescencewithanincreaseinthe475nmexcitationband.ThisphotoisomerizationeffectisprominentwithirradiationofGFPbyUVlight.InawiderangeofpH,increasingpHleadstoareductioninfluorescenceby395nmexcitationandanincreasedsensitivityto475nmexcitation.WildtypeGFPfromjellyfishh36MelittinGIGAVLKVLTTGLPALISWIKRKRQQX

MelittinGIGAVLKVLTTGLPALISWI37Example CarboxyfluorescenceBiochemicalEducation28(2000)171~173Example CarboxyfluorescenceBi38Example CarboxyfluorescenceQuenchingEffectExample CarboxyfluorescenceQu39Example CarboxyfluorescencepHEffectExample CarboxyfluorescencepH40

Thankyou拯畏怖汾關(guān)爐烹霉躲渠早膘岸緬蘭輛坐蔬光膊列板哮瞥疹傻俘源拯割宜跟三叉神經(jīng)痛-治療三叉神經(jīng)痛-治療拯畏怖汾關(guān)爐烹霉躲渠早膘岸緬蘭輛坐蔬光膊列板哮瞥疹41FluorescenceSpectroscopyPartI.BackgroundFluorescenceSpectroscopyPart42Perrin-JablonskidiagramPerrin-Jablonskidiagram43SissingletandTistriplet.TheS0stateisthegroundstateandthesubscriptnumbersidentifyindividualstates.SissingletandTistriplet.44n

→p*

<p→p*

<n

→s*

<s→p*

<s→s*EnergylevelofMOn→p*<p→p*<n→s*<s→45DS0Singlet&TripletDS0Singlet&Triplet46CharacteristicsofExcitedStatesEnergyLifetimeQuantumYieldPolarizationCharacteristicsofExcitedSta47StokesshiftTheStokesshiftisthegapbetweenthemaximumofthefirstabsorptionbandandthemaximumofthefluorescencespectrumlossofvibrationalenergyintheexcitedstateasheatbycollisionwithsolventheatStokesshiftTheStokesshifti48Example:7-amino-4-methylcoumarin(AMC)Example:7-amino-4-methylcouma49ExampleExample50ExamplefluorophoresfluoresceinethidiumbromideboundtoDNA.Examplefluorophoresfluoresce51FluorescenceSpecroscopy朝陽(yáng)科技大學(xué)熒光光譜朝陽(yáng)科技大學(xué)資料課件52LifetimeLifetime53LifetimeExcitedstatesdecayexponentiallywithtime–I=I0e-t/t

I0

istheinitialintensityattimezero,

I

istheintensityatsomelatertimet

t

isthelifetimeoftheexcitedstate.

kF=1/t,wherekFistherateconstantforfluorescence.LifetimeExcitedstatesdecaye54QuantumYieldQuantumYield=FF?FF=numberoffluorescencequantaemitteddividedbynumberofquantaabsorbedtoasingletexcitedstate?FF=ratioofphotonsemittedtophotonsabsorbedQuantumyieldistheratioofphotonsemittedtophotonsabsorbedbythesystem:QuantumYieldQuantumYield=F55QuantumYieldQuantumYield56QuantumYield&StructurerigidityQuantumYield&Structurerigi57PolarizationMoleculeofinterestisrandomlyorientedinarigidmatrix(organicsolventatlowtemperatureorroomtemperaturepolymer).Andplanepolarizedlightisusedastheexcitationsource.DegreeofpolarizationisdefinedasPI||andI^

aretheintensitiesoftheobservedparallelandperpendicularcomponents,a

istheanglebetweentheemissionandabsorptiontransitionmoments.Ifais0°thanP=+1/2,andifais90°thanP=-1/3.PolarizationMoleculeofintere58?Steady-statemeasurements:F,I?Time-Resolvedmeasurements:tExperimentalMeasurements?Steady-statemeasurements:F59InstrumentsInstruments60InnerFilterEffectAtlowconcentrationtheemissionoflightisuniformfromthefronttothebackofsamplecuvette.Athighconcentrationmorelightisemittedfromthefrontthantheback.SinceemittedlightonlyfromthemiddleofthecuvetteisdetectedtheconcentrationmustbelowtoassureaccurateFFmeasurements.InnerFilterEffectAtlowcon61InnerFilterEffectInnerFilterEffect62If(em)

=IAbs(ex).

f

.f(em).

KI0(ex)ememememmeasuredintensityoffluorescenceatemabsorbedintensityatexfluorescencequantumyieldfractionofintensityemittedatthatparticularwavelengthfractionoftotalfluorescencethatisdetectedIfA0Ifwemeasurethesampleandastandardunderthesameexperimentalconditions,keepingexconstant:Important:theindexofrefractionofthetwosolvents(sampleandstandard)mustbethesameStandards:QuininesulfateinH2SO41N:f=0.55FluoresceininNaOH

0.1N:f=0.93MeasurementoffluorescencequantumyieldsIf(em)=IAbs(ex).f.f(63TheTCSPCmeasurementreliesontheconceptthattheprobabilitydistributionforemissionofasinglephotonafteranexcitationyieldstheactualintensityagainsttimedistributionofallthephotonsemittedasaresultoftheexcitation.Bysamplingthesinglephotonemissionafteralargenumberofexcitationflashes,theexperimentconstructsthisprobabilitydistribution.Timecorrelatedsinglephotoncounting:#events....t(nsec)differentexcitationflashesStartPMTStopPMTsampleexc.monochromatoremissionmonochromatorpulsedsourcetMeasurementoffluorescencelifetimesTheTCSPCmeasurementrelieso64Lifetimens

AbsorptionFluorescenceWavelengthnmAbsorptivityWavelengthnmQuantumTryptophan2.62805,6003480.20Tyrosine3.62741,4003030.14Phenylalanine6.42572002820.04IntrinsicFluorescenceofProteinsandPeptidesLifetimeAbsorptionFluorescence65FluorescenceSpecroscopy朝陽(yáng)科技大學(xué)熒光光譜朝陽(yáng)科技大學(xué)資料課件66FluorescenceSpecroscopy朝陽(yáng)科技大學(xué)熒光光譜朝陽(yáng)科技大學(xué)資料課件67Tryptophan,thedominantintrinsicfluorophore,isgenerallypresentatabout1mol%inproteins.AproteinmaypossessjustoneorafewTrpresidues,whichfacilitatesinterpretationofthespectraldata.Tryptophanisverysensitivetoitslocalenvironment.Itispossibletoseechangesinemissionspectrainresponsetoconformationalchanges,subunitassociation,substratebinding,denaturation,andanythingthataffectsthelocalenvironmentsurrondingtheindolering.Also,Trpappearstobeuniquelysensitivetocollisionalquenching,eitherbyexternallyaddedquenchers,orbynearbygroupsintheprotein.Tryptophanfluorescencecanbeselectivelyexcitedat295-305nm.(toavoidexcitationofTyr)TryptophanTryptophan,thedominantintri68IIIIIIIVVExample:

TyrosineanditsderivativesIIIIIIIVVExample:Tyrosineand69IIIIIIIIIIIVIVIIVVIIIIIIIIIIIVIVIIVV70FluorescenceSpecroscopy朝陽(yáng)科技大學(xué)熒光光譜朝陽(yáng)科技大學(xué)資料課件71EmissionspectraofPseudomonasfluorescensazurinPfl.For275-nmexcitation,apeakisobservedduetothetyrosineresidue(s)Thepositionandstructureofthefluorescencesuggeststhattheindoleresidueislocatedinacompletelynonpolarregionoftheprotein.TheseresultsagreewithX-raystudies,whichshowthattheindolegroupislocatedinthehydrophobiccoreoftheprotein.Inthepresenceofadenaturingagent,theTrpPemissionlosesitsstructureandshiftsto351nm,characteristicofafullyexposedTrpresidue.ChangesinemissionspectracanbeusedtofollowproteinunfoldingEmissionspectraofPseudomona72FluorescenceSpecroscopy朝陽(yáng)科技大學(xué)熒光光譜朝陽(yáng)科技大學(xué)資料課件73Resolutionofthecontributionsofindividualtryptophanresiduesinmulti-tryptophanproteins.I(,t)=i()exp(-t/i)i1=2ns,

2=5ns1=2ns2=5nst(ns)Fluorescenceintensity(A.U.)Fluorescenceintensity(A.U.)wavelength(nm)emExampleTime-resolvedproteinfluorescenceResolutionofthecontribution74IsolatedfromthePacificjellyfishAequoreavictoriaandnowplayscentralrolesinbiochemistryandcellbiologyduetoitswidespreaduseasaninvivoreporterofgeneexpression,celllineage,protein-proteininteractionsandproteintraffickingOneofthemostimportantattributesofGFPwhichmakesitsousefulinthelifesciencesisthattheluminescentchromophoreisformedinvivo,andcanthusgeneratealabeledcellularmacromoleculewithoutthedifficultiesoflabelingwithexogenousagents.Greenfluorescentprotein(abbreviatedGFPIsolatedfromthePacificjell75ThestructureofGFP:eleven-strandbeta-barrelwrappedaroundacentralalpha-helixcore.Thiscentral