Fluorescence-Spectroscopy-朝阳科技大学荧光光谱朝阳科技大学课件

FluorescenceSpectroscopy-朝陽科技大學荧光光谱朝陽科技大學16、自己选择的路、跪着也要把它走完。17、一般情况下)不想三年以后的事，只想现在的事。现在有成就，以后才能更辉煌。18、敢于向黑暗宣战的人，心里必须充满光明。19、学习的关键--重复。20、懦弱的人只会裹足不前，莽撞的人只能引为烧身，只有真正勇敢的人才能所向披靡。FluorescenceSpectroscopy-朝陽科技大學荧光光谱朝陽科技大學FluorescenceSpectroscopy-朝陽科技大學荧光光谱朝陽科技大學16、自己选择的路、跪着也要把它走完。17、一般情况下)不想三年以后的事，只想现在的事。现在有成就，以后才能更辉煌。18、敢于向黑暗宣战的人，心里必须充满光明。19、学习的关键--重复。20、懦弱的人只会裹足不前，莽撞的人只能引为烧身，只有真正勇敢的人才能所向披靡。FluorescenceSpectroscopyPartI.BackgroundPerrin-JablonskidiagramFluorescenceSpectroscopyPartI.BackgroundPerrin-JablonskidiagramSissingletandTistriplet.TheS0stateisthegroundstateandthesubscriptnumbersidentifyindividualstates.n

→p*

<p→p*

<n

→s*

<s→p*

<s→s*EnergylevelofMODS0Singlet&TripletCharacteristicsofExcitedStatesEnergyLifetimeQuantumYieldPolarizationStokesshiftTheStokesshiftisthegapbetweenthemaximumofthefirstabsorptionbandandthemaximumofthefluorescencespectrumlossofvibrationalenergyintheexcitedstateasheatbycollisionwithsolventheatExample:7-amino-4-methylcoumarin(AMC)ExampleExamplefluorophoresfluoresceinethidiumbromideboundtoDNA.LifetimeLifetimeExcitedstatesdecayexponentiallywithtime–I=I0e-t/t

I0

istheinitialintensityattimezero,

I

istheintensityatsomelatertimet

t

isthelifetimeoftheexcitedstate.

kF=1/t,wherekFistherateconstantforfluorescence.QuantumYieldQuantumYield=FF•FF=numberoffluorescencequantaemitteddividedbynumberofquantaabsorbedtoasingletexcitedstate•FF=ratioofphotonsemittedtophotonsabsorbedQuantumyieldistheratioofphotonsemittedtophotonsabsorbedbythesystem:QuantumYieldQuantumYield&StructurerigidityPolarizationMoleculeofinterestisrandomlyorientedinarigidmatrix(organicsolventatlowtemperatureorroomtemperaturepolymer).Andplanepolarizedlightisusedastheexcitationsource.DegreeofpolarizationisdefinedasPI||andI^

aretheintensitiesoftheobservedparallelandperpendicularcomponents,a

istheanglebetweentheemissionandabsorptiontransitionmoments.Ifais0°thanP=+1/2,andifais90°thanP=-1/3.•Steady-statemeasurements:F,I•Time-Resolvedmeasurements:tExperimentalMeasurementsInstrumentsInnerFilterEffectAtlowconcentrationtheemissionoflightisuniformfromthefronttothebackofsamplecuvette.Athighconcentrationmorelightisemittedfromthefrontthantheback.SinceemittedlightonlyfromthemiddleofthecuvetteisdetectedtheconcentrationmustbelowtoassureaccurateFFmeasurements.InnerFilterEffectIf(em)

=IAbs(ex).

f

.f(em).

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

0.1N:f=0.93MeasurementoffluorescencequantumyieldsTheTCSPCmeasurementreliesontheconceptthattheprobabilitydistributionforemissionofasinglephotonafteranexcitationyieldstheactualintensityagainsttimedistributionofallthephotonsemittedasaresultoftheexcitation.Bysamplingthesinglephotonemissionafteralargenumberofexcitationflashes,theexperimentconstructsthisprobabilitydistribution.Timecorrelatedsinglephotoncounting:#events....t(nsec)differentexcitationflashesStartPMTStopPMTsampleexc.monochromatoremissionmonochromatorpulsedsourcetMeasurementoffluorescencelifetimesLifetimens

AbsorptionFluorescenceWavelengthnmAbsorptivityWavelengthnmQuantumTryptophan2.62805,6003480.20Tyrosine3.62741,4003030.14Phenylalanine6.42572002820.04IntrinsicFluorescenceofProteinsandPeptidesTryptophan,thedominantintrinsicfluorophore,isgenerallypresentatabout1mol%inproteins.AproteinmaypossessjustoneorafewTrpresidues,whichfacilitatesinterpretationofthespectraldata.Tryptophanisverysensitivetoitslocalenvironment.Itispossibletoseechangesinemissionspectrainresponsetoconformationalchanges,subunitassociation,substratebinding,denaturation,andanythingthataffectsthelocalenvironmentsurrondingtheindolering.Also,Trpappearstobeuniquelysensitivetocollisionalquenching,eitherbyexternallyaddedquenchers,orbynearbygroupsintheprotein.Tryptophan

fluorescencecanbeselectivelyexcitedat295-305nm.(toavoidexcitationofTyr)TryptophanIIIIIIIVVExample:

TyrosineanditsderivativesIIIIIIIIIIIVIVIIVVEmissionspectraofPseudomonasfluorescensazurinPfl.For275-nmexcitation,apeakisobservedduetothetyrosineresidue(s)

Thepositionandstructureofthefluorescencesuggeststhattheindoleresidueislocatedinacompletelynonpolarregionoftheprotein.TheseresultsagreewithX-raystudies,whichshowthattheindolegroupislocatedinthehydrophobiccoreoftheprotein.Inthepresenceofadenaturingagent,theTrpPemissionlosesitsstructureandshiftsto351nm,characteristicofafullyexposedTrpresidue.ChangesinemissionspectracanbeusedtofollowproteinunfoldingResolutionofthecontributionsofindividualtryptophanresiduesinmulti-tryptophanproteins.I(,t)=i()exp(-t/i)i1=2ns,

2=5ns1=2ns2=5nst(ns)Fluorescenceintensity(A.U.)Fluorescenceintensity(A.U.)wavelength(nm)emExampleTime-resolvedproteinfluorescenceIsolatedfromthePacificjellyfishAequoreavictoriaandnowplayscentralrolesinbiochemistryandcellbiologyduetoitswidespreaduseasaninvivoreporterofgeneexpression,celllineage,protein­proteininteractionsandproteintraffickingOneofthemostimportantattributesofGFPwhichmakesitsousefulinthelifesciencesisthattheluminescentchromophoreisformedinvivo,andcanthusgeneratealabeledcellularmacromoleculewithoutthedifficultiesoflabelingwithexogenousagents.Greenfluorescentprotein(abbreviatedGFPThestructureofGFP:eleven-strandbeta-barrelwrappedaroundacentralalpha-helixcore.ThiscentralcorecontainsthechromophorewhichisspontaneouslyformedfromachemicalreactioninvolvingresiduesSer65,Tyr66,andGly67(SYG)ThereiscyclizationofthepolypeptidebackbonebetweenSer65andGly67toforma5-memberedring,followedbyoxidationofTyr66.ThehighquantumyieldofGFPfluorescenceprobablyarisesfromthenearlycompleteprotectionofthefluorophorefromquenchingwateroroxygenmoleculesbyburialwithinthebeta-barrel.RibbondiagramoftheGreenFluorescentProtein(GFP)drawnfromthewild-typecrystalstructure.Theburiedchromophore,whichisresponsibleforGFP'sluminescence,isshowninfullatomicdetail.WildtypeGFPfromjellyfishhastwoexcitationpeaks,amajoroneat395nmandaminoroneat475nmwithextinctioncoefficientof30,000and7,000M-1cm-1,respectively.Itsemissionpeakisa