10.raman生物醫(yī)學(xué)工程系

1、Raman scattering席 鵬北京大學(xué)工學(xué)院生物醫(yī)學(xué)工程系Quantum Mechanics: Normal ModesOnly certain vibrational frequencies and atomic displacements allowedLinear molecules: 3N-5Non-linear molecules: 3N-6ExamplesStretching between 2 atomsSymmetric and asymmetric stretching with 3 atomsBending amongst 3 atomsOut of plane d

2、eformationsVibrational energies are sensitive toAtomic mass Molecular structure and geometryBond strengthBond orderEnvironmentHydrogen bonding2Units & Dimensional AnalysisSpectroscopic frequencies reported in wavenumbers cm-1, proportional to transition energy :Raman frequencies are independent of e

3、xcitation wavelength and reported as shiftsWavenumbers relative to excitation frequency:3Units & Dimensional AnalysisExampleNIR excitation at 830 nm: 12,048 cm-1 Typical Raman shift: 1000 cm-1R = 905 nmSharp biological Raman linewidths 10 cm-1 FWHMR= 0.69 nm4Raman Spectrum of Cholesterol5Hanlon et a

4、l. “Prospects for in vivo Raman spectroscopy,” Phys Med Biol 45: R1 (2000)Intensity of Raman signalI - Raman signal intensityC - is a constantN - the number density, I0- the laser intensity, - the Raman cross- section - the scattering solid angle,l- the length of the observed segment of the laser be

5、am, F(T) - a temperature dependent factor determined by the spectral width and resolution of the detection system and the investigated molecule6RamanshiftPracticeIf excited with 532nm laser, calculate the Raman signal peak for oxygen. If the Raman signal for nitrogen is 1000counts，then how much woul

6、d be the Raman signal for oxygen?Simulation 1Use Matlab to calculate the Raman peaks of the previous exercise.Input: nmRaman: cm-1function y=ramanpeak_cal(wavei,ramanshift)%Calculate the raman shift wavelength%wavei: nm%ramanshift: cm-1y=1E7./(1E7./wavei-ramanshift); ramanpeak_cal(532,1556)ans = 580

7、.01Generate a GUI of Raman calculatorGUIDE or file-new-blank GUIDraw 3 text inputs and corresponding static texts.Set the default textset(handles.edit2, String,testtest);Important: initializefunction initialize_gui(fig_handle, handles, isreset)% If the metricdata field is present and the reset flag

8、is false, it means% we are we are just re-initializing a GUI by calling it from the cmd line% while it is up. So, bail out as we dont want to reset the data.if isfield(handles, metricdata) & isreset return;end handles.metricdata.wavei = 550;handles.metricdata.ramanshift = 1100; set(handles.edit1, St

9、ring, handles.metricdata.wavei);set(handles.edit2, String, handles.metricdata.ramanshift);set(handles.edit3, String, 0); % Update handles structureguidata(handles.figure1, handles);% - Executes just before Raman1 is made visible.function Raman1_OpeningFcn(hObject, eventdata, handles, varargin)% This

10、 function has no output args, see OutputFcn.% hObject handle to figure% eventdata reserved - to be defined in a future version of MATLAB% handles structure with handles and user data (see GUIDATA)% varargin command line arguments to Raman1 (see VARARGIN) % Choose default command line output for Rama

11、n1handles.output = hObject; % Update handles structureguidata(hObject, handles);initialize_gui(hObject, handles, false);Update a certain fieldfunction edit1_Callback(hObject, eventdata, handles)% hObject handle to edit1 (see GCBO)% eventdata reserved - to be defined in a future version of MATLAB% ha

12、ndles structure with handles and user data (see GUIDATA) % Hints: get(hObject,String) returns contents of edit1 as text% str2double(get(hObject,String) returns contents of edit1 as a doublewavei = str2double(get(hObject, String);if isnan(wavei) set(hObject, String, 0); errordlg(Input must be a numbe

13、r,Error);endhandles.metricdata.wavei = wavei;guidata(hObject,handles)Update a certain fieldfunction edit1_Callback(hObject, eventdata, handles)% hObject handle to edit1 (see GCBO)% eventdata reserved - to be defined in a future version of MATLAB% handles structure with handles and user data (see GUI

14、DATA) % Hints: get(hObject,String) returns contents of edit1 as text% str2double(get(hObject,String) returns contents of edit1 as a doublewavei = str2double(get(hObject, String);if isnan(wavei) set(hObject, String, 0); errordlg(Input must be a number,Error);endhandles.metricdata.wavei = wavei;guidat

15、a(hObject,handles)Update a certain fieldfunction edit1_Callback(hObject, eventdata, handles)% hObject handle to edit1 (see GCBO)% eventdata reserved - to be defined in a future version of MATLAB% handles structure with handles and user data (see GUIDATA) % Hints: get(hObject,String) returns contents

16、 of edit1 as text% str2double(get(hObject,String) returns contents of edit1 as a doublewavei = str2double(get(hObject, String);if isnan(wavei) set(hObject, String, 0); errordlg(Input must be a number,Error);endhandles.metricdata.wavei = wavei;guidata(hObject,handles)Please update the edit2_Callback

17、accordingly.Press the push button% - Executes on button press in pushbutton1.function pushbutton1_Callback(hObject, eventdata, handles)% hObject handle to pushbutton1 (see GCBO)% eventdata reserved - to be defined in a future version of MATLAB% handles structure with handles and user data (see GUIDA

18、TA)ramanpeak=1E7/(1E7./handles.metricdata.wavei-handles.metricdata.ramanshift);set(handles.edit3, String, ramanpeak);Specific Raman Spectroscopic TechniquesNon-resonant Raman spectroscopyVisibleNear-infrared(UV) Resonance Raman spectroscopy Raman microscopy/imagingFiber optic samplingTime resolved (

19、pulsed) Raman spectroscopyHigh-wavenumber Raman spectroscopySERS: Surfaced Enhanced Raman SpectroscopyNon-linear Raman spectroscopyCARS: Coherent Anti-Stokes Raman Spectroscopy21General Applications of Raman SpectroscopyStructural chemistrySolid stateAnalytical chemistryApplied materials analysisPro

20、cess controlMicrospectroscopy/imagingEnvironmental monitoringBiomedical22Comparison to Infrared Absorption(N)IR absorption directly interrogates molecular vibrationsRequires change in dipole momentNo symmetric stretches observedNo diatomic activityOnly observed in NIR and IR spectral regionsHigh wat

21、er absorptionBroad spectral featuresRaman requires a change in polarizability with vibrational motionOccurs at all wavelengthsWeak signalSharp spectral features for molecular fingerprintingComplementary techniquesSymmetric molecules with a center of inversion have vibrations which are either Raman o

22、r IR active, but not both (e.g. benzene) Molecules with no symmetry are active in both methods 23Comparison to Infrared Absorption24 OutlineThe Raman EffectTheoryTechniques & ApplicationsBiomedical Raman SpectroscopyHistoryExcitation wavelength selectionAdvantages of Raman spectroscopyInstrumentatio

23、nLaboratoryClinical: optical fiber probes Case Study: AtherosclerosisDisease backgroundImpact of Raman spectroscopyFrontiers25History of Biological Raman Spectroscopy1970: Lord and Yu record 1st protein spectrum from lysozyme using HeNe excitationEvolution to NIR excitationDecreased fluorescenceIncr

24、eased penetration (mm)1980s: FT Raman with Nd:YAG and cooled InGaAs detectorslong collection times (30 min)Clarke (1987-1988): visible excitation of arterial calcium hydroxyapatite and carotenoids1990s, advances in:LasersDetectorsDispersive spectrometersFiltersChemometrics26UV, Visible, and NIR Exci

25、tationRaman signals have a constant shift can vary excitation wavelengthUV: resonance enhanced, RF, photo damage, low penetrationVisible: Raman -4, fluorescence overlaps with Raman signalNIR: low fluorescence, deep penetration, Raman -427UV, Visible, and NIR Applications UVRRBiological macromolecule

26、s: nucleic acids, proteins, lipidsOrganelles, cells, micro-organisms, bacteria, phytoplankton neurotoxins, virusesClinically limited: photomutagenicityVisibleCells (minimal fluorescence)DNA in chromosomes, pigment in granulocytes and lymphocytes, RBCs, hepatocytesFirst artery studies: hydroxyapatite

27、 and carotenoids (Clarke 1987, 1988)NIRHirschfeld & Chase, 1986: FT-RamanTissue: artery, cervix, skin, breast, blood, GI, esophagus, brain tumor, Alzheimers, prostate, bone28Raman Spectra: Fingerprinting a MoleculeRaman spectra are molecule specificSpectra contain information about vibrational modes

28、 of the moleculeSpectra have sharp features, allowing identification of the molecule by its spectrum29Examples of analytes found in blood which are quantifiable with Raman spectroscopy Spectroscopic Advantages of NIR Raman30Narrow vibrational bands are chemical specific and rich in informationFreedo

29、m to choose excitation wavelengthminimize unwanted tissue fluorescenceoptimize sampling depthutilize CCD technology350 mW1 min10020050010002 0001021104106Molar extinction coefficient (10-3M-1cm-1); H20 (cm-1)H2OHbOHbOVisible10-2NIR ExcitationMelaninWavelength (nm)3210 n13210 n0EnergyVirtual LevelFlu

30、orescence Raman (inelastic)ScatteringDiagnostic Advantages of Raman SpectroscopyWavelength selectionNo biopsy requiredDirectly measures moleculesSmall concentrationsChemical compositionMorphological analysisQuantitative analysisIn vivo diagnosis31OutlineThe Raman EffectTheoryTechniques & Application

31、sBiomedical Raman SpectroscopyHistoryExcitation wavelength selectionAdvantages of Raman spectroscopyInstrumentationLaboratoryClinical: optical fiber probes Case Study: AtherosclerosisDisease backgroundImpact of Raman spectroscopyFrontiers32Laser Sources for Raman SpectroscopySourceWavelength (nm)Ar+

32、488.0, 514.5Kr+530.9, 647.1He: Ne632.8Ti: Al2O3 (cw)720-1000Diode (InGaAs)785, 830Nd:YAG106433In Vitro Experimental Raman System34CCDCollimating lensesBand-pass filterNotch filterTi: sapphirelaserArgon ion pump laserNIR excitation (830 nm)f/4SpectrographCCDDichroic beam-splitterConfocalpinholeMotori

33、zed translation stageCCD CameraIn Vitro Turbid Liquid Analysisppm accuracy for precise quantitative measurements35Hanlon et al. “Prospects for in vivo Raman spectroscopy,” Phys Med Biol 45: R1 (2000)Clinical Raman Systems36CCD830 nmbandpassfilternotch filterholographic gratingRaman probeshutterDiode

34、 LaserCurrent Raman InstrumentationLaser diodesCompactStable narrow lineNIRHigh throughput spectrographs (f/1.8)Holographic elementsBandpass filters (eliminates spontaneous emission of lasing medium)Notch filters (106 rejection of Rayleigh scattered laser line)Large area, highly efficient transmissi

35、on gratingsCCD detectorsHigh QE (back-thinned, deep-depletion)Low noise (LN2 cooled)Multichannel detection High throughput, filtered fiber optics probesNIR FT and scanning PMT systems no longer useful37Early in vivo Data Simple 6-around-1 optical fiber probe100 mW excitation, 3 second collection38Op

36、tical Fiber ProbesProblemsFiber backgroundDistorts signalAdds shot-noiseLow signal collectionRaman effect is weakTissue is highly diffusive39Fiber background NA2Solution #1: Reduce Fiber BackgroundFiber background produced equally in excitation and collection fibersExcitation laser of power Po gener

37、ates Raman scattered light from tissuePosxlx: fraction of laser light Raman scattered and transmitted by excitation fiber*Bx=Posxlxes: Raman background detected from excitation fiberes: fraction of light elastically scattered (and collected) from sample Poes: intensity of scattered excitation light

38、gathered by collection fibersBc=Poessclc: intensity of background generated and transmitted by collection fibers*BT=Bx+Bc=Poes(sxlx+sclc)40Tissue Samplexc* NA2From McCreery RL “Raman Spectroscopy for Chemical Analysis,” 2000. Excitation laserTissue Raman Fiber backgroundxcTissue SampleFilter Transmi

39、ssion41Region of InterestSolution #1: FilteringProblemsFiber backgroundDistorts signalAdds shot-noiseLow signal collectionRaman effect is weakTissue is highly diffusiveSolutionsMicro-optical filtersShort-pass excitation filterLong-pass collection filterOptimize optical designCharacterize distributio

40、n of Raman light in tissueDefine optimal geometryDesign collection optics42Excitation Light Diffusing Through Tissue43Monte Carlo1 mmExperimentalSolution #2: Optical DesignProblemsFiber backgroundDistorts signalAdds shot-noiseLow signal collectionRaman effect is weakTissue is highly diffusiveSolutio

41、nsMicro-optical filtersShort-pass excitation filterLong-pass collection filterOptimize optical designCharacterize distribution of Raman light in tissueDefine optimal geometryDesign collection optics44Raman Probe Design GoalsRestricted geometry for clinical useTotal diameter 2mm for access to coronar

42、y arteriesFlexibleAble to withstand sterilization Designed to work with 830 nm excitationHigh throughputData accumulation in 1 or 2 secondsSafe power levelsSNR similar to open-air optics laboratory systemAccurate application of models45Raman Probe Design462 mmretainingsleeveball lens1 mm1.75 mmcolle

43、ction fibers0.70long-passfilter tubemetal sleevealuminum jacket0.55excitation fibershort-passfilter rodSingle Ring Probe has 15 FibersMotz et al. Appl Opt 43: 52 (2004)Calcified Aorta47OutlineThe Raman EffectTheoryTechniques & ApplicationsBiomedical Raman SpectroscopyHistoryExcitation wavelength sel

44、ectionAdvantages of Raman spectroscopyInstrumentationLaboratoryClinical: optical fiber probes Case Study: AtherosclerosisDisease backgroundImpact of Raman spectroscopyFrontiers48The Burden of Cardiovascular Disease71,300,000 people in United States afflicted910,600 deaths per year1 out of every 2.7

45、deathsCoronary artery disease claims 653,000 lives annually1 out of every 5 deathsEconomic cost: greater than $142.5 billion49American Heart Association, Heart and Stroke Statistics-2006 UpdateArterial Anatomy50LumenIntimaAdventitiaMediaTAtheromaNCFibrous CapNormalMildly Atherosclerotic PlaqueRuptur

46、ed PlaqueIntima: innermost layer of arterial wallcomposed of a single layer of endothelia cells in normal arteryregion of artery involved in atherosclerotic diseaseMedia: arterial layer composed primarily of smooth muscle cellsconstricts and dilates to control blood flowin large arteries (e.g. aorta

47、) this layer is largely composed of elastinAdventitia: outermost layer of arterial wallconnective tissue and fatT: thrombusNC: necrotic coreSome Current Challenges in CardiologyEvaluation and development of therapeuticsEtiology of atherosclerosisMechanisms of re-stenosisPost-angioplastyTransplant va

48、sculopathy Detection of vulnerable atherosclerotic plaquesPrediction/prevention of cardiac events51Vulnerable PlaquesAccount for majority of sudden cardiac deathFrequently occur in clinically silent vessels 50% stenosisEffective treatments unknown Characterized by:Biochemical changesFoam cellsLipid

49、pool Inflammatory cellsThin fibrous cap (65 m)Currently undetectable52Standard Diagnostic TechniquesAngiographySeverity of stenosis, thrombosis, dense calcificationsProvides no biochemical informationAngioscopySurface features of plaque, including colorNo information of sub-surface features Histopat

50、hologyBiochemical and morphological informationRequires excision of tissue53Emerging Diagnostic TechniquesMagnetic resonance imagingExternal ultrasoundPositron emission tomographyElectron beam computed tomographyThermographyElastographyIntravascular ultrasoundOptical coherence tomography 54Non-Invas

51、iveSpectroscopic Diagnostic TechniquesNIR Absorption spectroscopyInhibited by water absorptionBroad spectral featuresFluorescence spectroscopyLimited chemical informationBroad spectral featuresRaman SpectroscopyQuantitative biochemical informationMorphological analysis55 In Vitro Experimental Method

52、sMacroscopic Raman Spectroscopy1 mm3 volumes of excised tissue are examined100-350 mW excitation with 830 nm laser light10 - 100 s collection timesComparison with histopathology for disease classificationPrincipal Component Analysis56Raman Spectral Pathology of Atherosclerosis57Image from medstat.me

53、/WebPath/webpath.htmlnormal arterylipid-rich plaquecalcified plaque collagen elastin actin cholesterol -carotene proteins Ca hydroxyapatite proteinsRaman Spectral ModelingGoal: Diagnose disease by analyzing the complex macroscopic spectra (R) obtained from biopsy samplesStrategy: Develop a

54、 library of microscopic or chemical basis spectra (B) that compose the macroscopic data Implementation: Use ordinary least squares fitting to determine a weighted linear combination of basis spectra to evaluate the biopsies58R(l)artery = wcollagenB(l)collagen+ wcholesterolB(l)cholesterol+ wcalcifica

55、tionB(l)calcification+Morphological Model HypothesisRaman spectroscopy detectsmolecules (chemicals)Every morphological feature has a distinct molecular structureEach morphological feature has a unique Raman spectrum59Chemical analysis of morphological structuresMorphological modeling of coronary art

56、eriesPotential Features for Spectral Identification60CollagenElastinActin Adventitial fat-caroteneFoam cellsCholesterolNecrotic coreCalcificationHemoglobinFibrin In Vitro Experimental MethodsMacroscopic Raman Spectroscopy1 mm3 volumes of excised tissue are examined100-350 mW excitation with 830 nm l

57、aser light10 - 100 s collection timesComparison with histopathology for disease classificationPrincipal Component Analysis Confocal Microscopic Raman Spectroscopy(2x2x2) m3 sampling volume of microscopic structures100 mW excitation of 6 m thick sections with 830 nm laser light10-360 s collection tim

58、esDevelopment of morphological modelSpectroscopic mapping of tissue sections61Atherosclerosis In Vitro: Confocal Microscopy62AdventitiaMediaIntima(2x2x2) m3 Sampling VolumeElastic LaminaFoam CellSmooth Muscle Cell0.1 mmCoronary Artery Morphological Structures63Buschman HPJ, et al. Cardiovascular Pat

59、hology 10(2), 69-82 (2001)Morphological Model of Coronary Arteries64Calcified PlaqueResidualMacroscopic DataMicroscopicModel FitBuschman HPJ, Motz JT, et al. Cardiovascular Pathology 10(2), 59-68 (2001)Normal Coronary ArteryNon-Calcified PlaqueMorphological Assay of Coronary Arteries65Mildly Calcifi

60、ed PlaqueStructure ContributionCollagen 39%Cholesterol 10%Calcification 11%Elastic Lamina 0%Fat 28%Foam Cell / Core 0%-Carotene 0%Smooth Muscle 12%Structure ContributionCollagen 20%Cholesterol 6%Calcification 0%Elastic Lamina 6%Fat 38%Foam Cell / Core 0%-Carotene 3%Smooth Muscle 27%Normal Coronary A