東南大學(xué)電子學(xué)院《現(xiàn)代光學(xué)基礎(chǔ)》補(bǔ)充內(nèi)容3-顯微鏡

第七章共焦顯微光學(xué)

Chapter7

ConfocalMicroscopyOptics東南大學(xué)先進(jìn)光子學(xué)中心AdvancedPhotonicsCenterSoutheastUniversity崔一平

CUIYipingCyp@http://

OUTLINE7.1OpticalMicroscopy7.2ConfocalMicroscopy7.3ApplicationsofConfocalMicroscopyinBiomedicine7.1OpticalMicroscopyThreegoals:produceamagnifiedimageofthespecimen,separatethedetailsintheimage,renderthedetailsvisibletothehumaneyeorcamera.Simplesinglelensdevicesthatareoftenhand-held,suchasamagnifyingglass.Multiple-lensdesignswithobjectivesandcondensers(compound)Microscopy:HistorySimpleCompoundMicroscopy:HistoryMicroscopy:HistoryMicroscopy:ImportanceBiomedicalsciences:overallmorphologicalfeaturesofspecimens;quantitativetooladvancesinfluorochromestainsandmonoclonalantibodytechniques:explosivegrowthintheuseoffluorescencemicroscopyinbothbiomedicalanalysisandcellbiology.opticalmicroscopemostimportantbiomedicalopticExplosivegrowthinphysicalandmaterialssciences;semiconductorindustry,

observesurfacefeaturesofhigh-techmaterialsandintegratedcircuitsForensicscientists:hairs,fibers,clothing,bloodstains,bullets,andotheritemsassociatedwithcrimesSimpleMagnificationSo>>>2fSo>2fSo=2ff<So<2fImageformationonRetinado~25cmCombinationofLensandEyeSimplemicroscope:bi-convexlens,imageperceivedbyeyeasifitwereatadistanceof10inchesor25centimeters(nearpoint)Imageappearsonsamesideoflensasobject,cannotbeprojectedontoascreen:virtualimage(upright,notinverted).Lightreflectedfromtheroseentersthelensinstraightlines,refractedandfocusedbythelenstoproduceavirtualimageontheretina.Imageoftherosemagnified:perceiveactualsizeofobjecttobeatinfinity;eyestracelightraysbackinstraightlinestovirtualimage

CombinationofLensandEyeSofSilLMP=doDatl=f;DecreaselorL,increaseMP:MP=doD+1atl=0;L=doIncreaselorL,decreaseMP(D=1/f);do=nearpt~25cmCompoundMicroscopeLensclosesttotheobject:objective.Lightfromcondenser,formslightconeconcentratedontotheobject(specimen).Lightpassesthroughthespecimenandintotheobjectiveprojectsareal,inverted,andmagnifiedimageofthespecimentoafixedplanewithinthemicroscope:intermediateimageplaneCompoundMicroscopeLfofeEyeorcameraMP=M(obj)M(e)=(-L/fo)(254/fe)Airydisksandresolution.(a-c)Airydisksizeandrelatedintensityprofile(pointspreadfunction)asrelatedtoobjectivenumericalaperture,whichdecreasesfrom(a)to(c)asnumericalapertureincreases.(e)TwoAirydiskssoclosetogetherthattheircentralspotsoverlap.(d)Airydisksatthelimitofresolution.AirydiskResolutionr=1.2/2NA7.2ConfocalMicroscopyPrincipleofConfocalMicroscopy

PrincipleofCMThekeytechniquetoconfocalmicroscopy

SpatialFilteringTechniquesAdvantages(overconventionalwidefieldopticalmicroscopy)ControldepthoffieldEliminationorreductionofbackgroundinformationawayfromthefocalplaneCapabilitytocollectserialopticalsectionsfromthickspecimensHumanMedullaRabbitsunflowerMuscleFiberspollengrainLasersThefarfieldbeginsatadistance,z,definedby(A(0)isthebeamdiameterattheexitapertureandlisthelaserwavelength)z=A02/l

Wavelength(nm)BeamDiameter(mm)FarFieldDistance(cm)Argon-Ion4880.6745141.0195Helium-Neon5430.4305940.7836120.7806320.778Nd:YAG3553.025355321.0188Ti:Sapphire7902.05063952.010127900.881PinholeScanningSystemDetectorsResolutionandContrastResponseofAOpticalSystemPointSpreadFunction(PSF)Thepropertiesoftheintensitypointspreadfunctionintheimageplaneaswellasintheaxialdirectionaremajorfactorsindeterminingtheresolutionofamicroscope.IntensitypointspreadfunctionextendsinallthreedimensionsLateralcomponentsoftheintensitydistribution:Airydisk.ResolutionandContrastResolutionandContrastOrderZeroCross-ingsPeaksI13.810027.01.7310.20.4413.30.2ResolutionandContrastResolutionandContrastInWidefieldMicroscope

Rayleighcriterion

forresolutionstatesthattwopointsareresolvedwhenthefirstminimum(zerocrossing)ofoneAirydiskisalignedwiththecentralmaximumofthesecondAirydisk.Thecontrastvalueis26.4percent

rlateral=0.6l/NA

ResolutionandContrastInconfocalconfigurations

PointwiseIlluminationScanning+PointwiseDetection

PSFconf.=PSFillum.*PSFdetc.~70%*PSFwidefieldTherefore,

rlateral=0.4l/NA

ResolutionandContrastAxialResolutionWidefieldMicroscope:Noopticalsectioningcapability(a)

ConfocalMicroscope:

Opticalsectioningcapability(b)BenefitsofConfocalMicroscopyReducedblurringoftheimagefromlightscatteringIncreasedeffectiveresolutionImprovedsignaltonoiseratioZ-axisscanning,DepthperceptioninZ-sectionedimagesMagnificationcanbeadjustedelectronicallyDrawbacksofConfocalMicroscopySloweracquisition-needtocollectonepixelatatimeIncreasedphotodamage(photobleaching)duetolongerexposuretoexcitinglightHistoricalPerspective

ThebasicconceptofconfocalmicroscopyMarvinMinskyinthemid-1950s(patentedin1957)

M.DavidEggerandMojmir

Petran

multiple-beamconfocalmicroscopeinthelate1960s

M.DavidEggerThefirstmechanicallyscannedconfocallasermicroscopeThefirstrecognizableimagesofcellsin1973.Thelate1970sandthe1980s

Growinginterestinconfocalmicroscopy.HistoricalPerspectiveApplicationInstrumentsG.FredBrakenhoffdevelopedascanningconfocalmicroscopein1979whilealmostsimultaneously,ColinSheppardcontributedtothetechniquewithatheoryofimageformation.TonyWilson,BradAmos,andJohnWhitenurturedtheconceptandlater(duringthelate1980s)demonstratedtheutilityofconfocalimagingintheexaminationoffluorescentbiologicalspecimens.Thefirstcommercialinstrumentsappearedin1987.Duringthe1990s,thenumberofapplicationsthatcouldbetargetedwithlaserscanningconfocalmicroscopy.Modernconfocalmicroscopes

Integratedelectronicsystemswheretheopticalmicroscopeplaysacentralroleinaconfigurationoneormoreelectronicdetectors,acomputer(forimagedisplay,processing,output,andstorage)severallasersystemscombinedwithwavelengthselectiondevicesabeamscanningassembly.ModernconfocalmicroscopesAentireconfocalmicroscopeisoftencollectivelyreferredtoasadigitalorvideoimagingsystemcapableofpro-ducingelectronicimages.Employedforroutineinvestigationsonmolecules,cells,andlivingtissuesnow7.3ApplicationsinBiomedicine熒光探針（Fluorescentprobe）

WhyneedFluorescentprobe?Stainsinfixedtissuesandlivingcells

Labelingantibodieswithfluorescentdyes－Immunofluorescence

Dyes,QuantumDots,….FluorescenceExcitationandEmissionFundamentals

Luminescence

Photolumine-scence

FluorescencePhosphorescence

單光子激發(fā)熒光TimescaleRangeforFluorescenceProcessesTransitionProcessRateConstantTimescale

(Seconds)S(0)=>S(1)orS(n)Absorption(Excitation)Instantaneous10-15S(n)=>S(1)InternalConversionk(ic)10-14

to10-10S(1)=>S(1)VibrationalRelaxationk(vr)10-12

to10-10S(1)=>S(0)Fluorescencek(f)orG10-9

to10-7S(1)=>T(1)IntersystemCrossingk(pT)10-10

to10-8S(1)=>S(0)Non-RadiativeRelaxation

Quenchingk(nr),k(q)10-7

to10-5T(1)=>S(0)Phosphorescencek(p)10-3

to100T(1)=>S(0)Non-RadiativeRelaxation

Quenchingk(nr),k(qT)10-3

to100StokesShiftandMirrorImageRuleWhatisTPA?S1S0S10S1vS1S0S10S1vVirtualStateProcessofTwo-PhotonAbsorption雙光子吸收（TPA）與雙光子激發(fā)熒光ApplicationsofTPAOpticalpowerlimitingFluorescenceImagingPhotodynamiccancertherapy

Microfabrication3Dopticaldatastorage雙光子熒光成像（續(xù)）

Hela細(xì)胞的雙光子熒光像。染料為trans-4-[p-(9-ethylcarbazde)vinyl]-N-methypyrid-iniumIodide

量子點(diǎn)

又稱半導(dǎo)體納米微晶粒，直徑在1－100nm之間，能夠吸收激發(fā)光產(chǎn)生熒光的半導(dǎo)體納米顆粒

量子點(diǎn)熒光材料準(zhǔn)零維尺度人造原子量子點(diǎn)的結(jié)構(gòu)（續(xù)）

CoreShellsCoatingPlay量子點(diǎn)的性質(zhì)與其它發(fā)光材料最大的區(qū)別：一種材料發(fā)多色光1）在發(fā)光范圍內(nèi)為目標(biāo)光可連續(xù)可調(diào)2）通過調(diào)節(jié)量子點(diǎn)大小對發(fā)光譜調(diào)制Play我們制備的不同尺寸的CdSe量子點(diǎn)在同一激發(fā)波長（365nm）下發(fā)出的熒光。

實(shí)驗(yàn)結(jié)果樣品（上圖左起2、4、6）的紫外吸收光譜以及相應(yīng)的熒光發(fā)射光譜（PL）樣品2、4、6的熒光峰值和FWHM分別為：544.8nm、581.5nm、609.8nm和20.2、17.4、21.9實(shí)驗(yàn)結(jié)果ApplicationsLSCM的高靈敏度、高分辨率、高放大倍數(shù)，提供了光學(xué)顯微鏡無法顯示的結(jié)構(gòu)，使細(xì)胞生物學(xué)研究上了一個(gè)臺(tái)階。目前我們可以在亞細(xì)胞水平進(jìn)行動(dòng)態(tài)實(shí)驗(yàn)，檢測細(xì)胞生物質(zhì)和離子通道的變化，觀察細(xì)胞在生理、病理和藥理情況下對外界因素作用所產(chǎn)生的快速反應(yīng)，進(jìn)行定性、定量、定時(shí)和定位的分析測量。最常用的功能是細(xì)胞三維重建，細(xì)胞熒光檢測和細(xì)胞顯微操作等

細(xì)胞的三維重建（3-DReconstruction）LSCM能以0.1μm的步距沿軸向?qū)?xì)胞進(jìn)行分層掃描，得到一組光學(xué)切片。通過計(jì)算機(jī)進(jìn)行不同的三維重建算法，可作單色或雙色圖象處理，組合成細(xì)胞真實(shí)的三維結(jié)構(gòu)。旋轉(zhuǎn)不同角度可觀察各側(cè)面的表面形態(tài)，也可不同的斷面觀察細(xì)胞內(nèi)部結(jié)構(gòu)，測量細(xì)胞的長寬高、體積和斷層面積等形態(tài)學(xué)參數(shù)。通過角度旋轉(zhuǎn)和細(xì)胞位置變化可產(chǎn)生三維動(dòng)畫效果。細(xì)胞定量熒光測定LSCM以激光為光源，對細(xì)胞分層掃描，單獨(dú)測定，經(jīng)積分后能得到細(xì)胞熒光的準(zhǔn)確定量，重復(fù)性極佳。它適于活細(xì)胞的定量分析。適用于快速高靈敏度測量，減少光粹滅的影響，在定量免疫熒光測定方面應(yīng)用廣泛，如作各種腫瘤組織切片抗原表達(dá)的定量分析，監(jiān)測腫瘤相關(guān)抗原表達(dá)的定位定量信息，監(jiān)測藥物對肌體免疫功能的作用等。細(xì)胞定量熒光測定可選用單熒光。雙熒光和三熒光方式，能自動(dòng)測定細(xì)胞面積，平均熒光強(qiáng)度，積分熒光強(qiáng)度及形狀因子等多種參數(shù)。

細(xì)胞內(nèi)鈣離子PH值和其它離子的動(dòng)態(tài)分析通過Indo－1、Fluo－2、Fluo－3、Calciumgreen、SNARF等多種熒光探針，可對細(xì)胞內(nèi)鈣離子、鈉離子及PH值等作熒光標(biāo)記并對它們進(jìn)行比率值和濃度梯度變化測定。由于細(xì)胞內(nèi)鈣離子為傳遞信息的第二信使，對細(xì)胞生長分化起著重要作用，通過對細(xì)胞內(nèi)鈣離子和其它離子的熒光強(qiáng)度和分布精確測定，測定樣品達(dá)到毫秒級的快速變化。借助光學(xué)切片功能可以測量樣品深層的熒光分布以及細(xì)胞光學(xué)切片的生物化學(xué)特性的變化。細(xì)胞胞間通訊（CellCommunication）和膜的流動(dòng)性動(dòng)物和植物細(xì)胞中縫隙連接介導(dǎo)的胞間通訊在細(xì)胞增殖和分化中起著重要作用。通過測量細(xì)胞縫隙連接分子的轉(zhuǎn)移，可以研究腫瘤啟動(dòng)因子和生長因子對縫隙連接介導(dǎo)的胞間通訊的抑制作用及細(xì)胞內(nèi)鈣離子、pH值等對縫隙連接作用的影響，并監(jiān)測環(huán)境毒素和藥物在細(xì)胞增殖和分化中所起到的作用。細(xì)胞膜熒光探針受到極化光線激發(fā)后，發(fā)射光極性依賴于熒光分子的旋轉(zhuǎn)，這種有序的運(yùn)動(dòng)自由度取決于熒光分子周圍的膜流動(dòng)性，所以極性測量能間接反映細(xì)胞膜的流動(dòng)性。

熒光光漂白恢復(fù)（FluorescenceRedistributionAfterPhotobleaching，F(xiàn)RAP）FRAP是用來測定活細(xì)胞的動(dòng)力學(xué)參數(shù)，借助于高強(qiáng)度脈沖激光來照射細(xì)胞某一區(qū)域，造成該區(qū)域熒光分子的光粹滅，該區(qū)域周圍的非粹滅熒光分子會(huì)以一定的速率向受照射區(qū)域擴(kuò)散，這個(gè)擴(kuò)散速率可通過低強(qiáng)度激光掃描探測,因而可得到活細(xì)胞的動(dòng)力學(xué)參數(shù)。LSCM可以控制光粹滅作用，實(shí)時(shí)監(jiān)測分子擴(kuò)散率和恢復(fù)速率，反映細(xì)胞結(jié)構(gòu)和活動(dòng)機(jī)制。廣泛用于研究細(xì)胞骨架構(gòu)成，核膜結(jié)構(gòu)跨膜大分子遷移率，細(xì)胞