raman-spectroscopy拉曼光谱

BiomedicalRamanSpectroscopyJasonT.MotzHarvardMedicalSchoolandTheWellmanCenterforPhotomedicineMassachusettsGeneralHospital11April20062ANewTypeofSecondaryRadiationC.V.RamanandK.S.Krishnan,Nature,121(3048):501-502,March31,1928IfweassumethattheX-rayscatteringofthe'unmodified'typeobservedbyProf.Comptoncorrespondstothenormaloraveragestateoftheatomsandmolecules,whilethe'modified'scatteringofalteredwave-lengthcorrespondstotheirfluctuationsfromthatstate,itwouldfollowthatweshouldexpectalsointhecaseofordinarylighttwotypesofscattering,onedeterminedbythenormalopticalpropertiesoftheatomsormolecules,andanotherrepresentingtheeffectoftheirfluctuationsfromtheirnormalstate.Itaccordinglybecomesnecessarytotestwhetherthisisactuallythecase.Theexperimentswehavemadehaveconfirmedthisanticipation,andshownthatineverycaseinwhichlightisscatteredbythemoleculesindust-freeliquidsorgases,thediffuseradiationoftheordinarykind,havingthesamewave-lengthastheincidentbeam,isaccompaniedbyamodifiedscatteredradiationofdegradedfrequency.Thenewtypeoflightscatteringdiscoveredbyusnaturallyrequiresverypowerfulilluminationforitsobservation.Inourexperiments,abeamofsunlightwasconvergedsuccessivelybyatelescopeobjectiveof18cm.apertureand230cm.focallength,andbyasecondlenswasplacedthescatteringmaterial,whichiseitheraliquid(carefullypurifiedbyrepeateddistillationinvacuo)oritsdust-freevapour.Todetectthepresenceofamodifiedscatteredradiation,themethodofcomplementarylight-filterswasused.Ablue-violetfilter,whencoupledwithayellow-greenfilterandplacedintheincidentlight,completelyextinguishedthetrackofthelightthroughtheliquidorvapour.Thereappearanceofthetrackwhentheyellowfilteristransferredtoaplacebetweenitandtheobserver'seyeisproofoftheexistenceofamodifiedscatteredradiation.Spectroscopicconfirmationisalsoavailable.Somesixtydifferentcommonliquidshavebeenexaminedinthisway,andeveryoneofthemshowedtheeffectingreaterorlessdegree.Thattheeffectisatruescattering,andsecondlybyitspolarisation,whichisinmanycasesquirestrongandcomparablewiththepolarisationoftheordinaryscattering.Theinvestigationisnaturallymuchmoredifficultinthecaseofgasesandvapours,owingtotheexcessivefeeblenessoftheeffect.Nevertheless,whenthevapourisofsufficientdensity,forexamplewithetheroramylene,themodifiedscatteringisreadilydemonstrable.3ProfessorSirC.V.Raman1888-1970TheNobelPrizeinPhysics1930forhisworkonthescatteringoflightandforthediscoveryoftheeffectnamedafterhimFirstphotographedRamanspectraBangalore,India4TheRamanEffect:InelasticScatteringhnih(ni-nR)hni3210S13210S0EnergyVirtualLevelRayleighRaman(inelastic)(elastic)ScatteringScatteringInelasticScattering•Energytransferredfromincidentlighttomolecularvibrations•Emittedlighthasdecreasedenergy(iR)differenceinenergy5SomeVibrationsinBenzeneBreathingChubbyCheckerKekule40060080010001200140016001800012345x104RamanShift(cm-1)Intensity(CCDCounts)6EvolutionofRamanSpectroscopy•1928~1960–Minorexperimentaladvances•1960–Inventionoflaserexpandsscopeexperiments•1980s:rapidtechnologicaladvances–FourierTransformspectroscopy–ChargeCoupledDevice(CCD)detectors–Holographicanddielectricfilters–Near-Infrared(NIR)lasers•Late1980s1990s–Biomedicalinvestigations–Advanceddispersivespectrometers•2000–Invivoapplication–Opticalfiberprobes–Non-linearspectroscopy7Outline•TheRamanEffect–Theory–Techniques&Applications•BiomedicalRamanSpectroscopy–History–Excitationwavelengthselection–AdvantagesofRamanspectroscopy•Instrumentation–Laboratory–Clinical:opticalfiberprobes•CaseStudy:Atherosclerosis–Diseasebackground–ImpactofRamanspectroscopy•Frontiers8ClassicalRamanPhysics•Interactionbetweenelectricfieldofincidentphotonandmolecule–Electricfieldoscillatingwithincidentfrequencyvi:–Inducesmolecularelectricdipole(p):•Proportionaltomolecularpolarizability,–easewithwhichtheelectroncloudaroundamoleculecanbedistorted–PolarizationresultsinnucleardisplacementpE0cos(2)iiEEtn0cos2Rqqtn9ClassicalRamanPhysics•Forsmalldistortions,polarizabilityislinearlyproportionaltothedisplacement•Resultantdipole:000...qq00cos2ipEEtnRayleighScatteringAnti-StokesRamanStokesRaman0001cos2cos22iRiREqttqnnnn103210n1Auto-IRRayleighStokesAnti-StokesNIRFluorescenceAbsorptionScatteringRamanScatteringFluorescenceEnergyVirtualLevels210n0210n’1Photo-MolecularInteractions-2000-1000010002000020406080100RamanShift(cm-1)IntensityAnti-StokesStokesRayleighScatteringE=hnR11ClassicalRamanPhysics•Ramanscatteringoccursonlywhenthemoleculeis‘polarizable’•Ramanintensityn4–Classicaldipoleradiation–StokesshiftedRamanismoreintensethananti-StokesbyBoltzmannfactor:•Consistentwithotherscatteringphenomena,oftenreportedintermsofcross-section([cm2]),orprobabilityofscattering:–:densityofmolecules–dz:pathlength0dq4RhiRAkTSiRIeInnnnn0IIdz12CharacteristicsofRamanScattering•Veryweakeffect–Only1in107photonsisRamanscattered–NIRelasticscatteringintissue:–NIRabsorptionintissue:–Redabsorptionintissueorwater:–Ramanscatteringintissueorwater:•Truescatteringprocess–Virtualstateisashort-liveddistorti