东南大学电子学院《现代光学基础》补充内容3-显微镜

第七章共焦显微光学

Chapter7

ConfocalMicroscopyOptics东南大学先进光子学中心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荧光探针（Fluorescentprobe）

WhyneedFluorescentprobe?Stainsinfixedtissuesandlivingcells

Labelingantibodieswithfluorescentdyes－Immunofluorescence

Dyes,QuantumDots,….FluorescenceExcitationandEmissionFundamentals

Luminescence

Photolumine-scence

FluorescencePhosphorescence

单光子激发荧光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双光子吸收（TPA）与双光子激发荧光ApplicationsofTPAOpticalpowerlimitingFluorescenceImagingPhotodynamiccancertherapy

Microfabrication3Dopticaldatastorage双光子荧光成像（续）

Hela细胞的双光子荧光像。染料为trans-4-[p-(9-ethylcarbazde)vinyl]-N-methypyrid-iniumIodide

量子点

又称半导体纳米微晶粒，直径在1－100nm之间，能够吸收激发光产生荧光的半导体纳米颗粒

量子点荧光材料准零维尺度人造原子量子点的结构（续）

CoreShellsCoatingPlay量子点的性质与其它发光材料最大的区别：一种材料发多色光1）在发光范围内为目标光可连续可调2）通过调节量子点大小对发光谱调制Play我们制备的不同尺寸的CdSe量子点在同一激发波长（365nm）下发出的荧光。

实验结果样品（上图左起2、4、6）的紫外吸收光谱以及相应的荧光发射光谱（PL）样品2、4、6的荧光峰值和FWHM分别为：544.8nm、581.5nm、609.8nm和20.2、17.4、21.9实验结果ApplicationsLSCM的高灵敏度、高分辨率、高放大倍数，提供了光学显微镜无法显示的结构，使细胞生物学研究上了一个台阶。目前我们可以在亚细胞水平进行动态实验，检测细胞生物质和离子通道的变化，观察细胞在生理、病理和药理情况下对外界因素作用所产生的快速反应，进行定性、定量、定时和定位的分析测量。最常用的功能是细胞三维重建，细胞荧光检测和细胞显微操作等

细胞的三维重建（3-DReconstruction）LSCM能以0.1μm的步距沿轴向对细胞进行分层扫描，得到一组光学切片。通过计算机进行不同的三维重建算法，可作单色或双色图象处理，组合成细胞真实的三维结构。旋转不同角度可观察各侧面的表面形态，也可不同的断面观察细胞内部结构，测量细胞的长宽高、体积和断层面积等形态学参数。通过角度旋转和细胞位置变化可产生三维动画效果。细胞定量荧光测定LSCM以激光为光源，对细胞分层扫描，单独测定，经积分后能得到细胞荧光的准确定量，重复性极佳。它适于活细胞的定量分析。适用于快速高灵敏度测量，减少光粹灭的影响，在定量免疫荧光测定方面应用广泛，如作各种肿瘤组织切片抗原表达的定量分析，监测肿瘤相关抗原表达的定位定量信息，监测药物对肌体免疫功能的作用等。细胞定量荧光测定可选用单荧光。双荧光和三荧光方式，能自动测定细胞面积，平均荧光强度，积分荧光强度及形状因子等多种参数。

细胞内钙离子PH值和其它离子的动态分析通过Indo－1、Fluo－2、Fluo－3、Calciumgreen、SNARF等多种荧光探针，可对细胞内钙离子、钠离子及PH值等作荧光标记并对它们进行比率值和浓度梯度变化测定。由于细胞内钙离子为传递信息的第二信使，对细胞生长分化起着重要作用，通过对细胞内钙离子和其它离子的荧光强度和分布精确测定，测定样品达到毫秒级的快速变化。借助光学切片功能可以测量样品深层的荧光分布以及细胞光学切片的生物化学特性的变化。细胞胞间通讯（CellCommunication）和膜的流动性动物和植物细胞中缝隙连接介导的胞间通讯在细胞增殖和分化中起着重要作用。通过测量细胞缝隙连接分子的转移，可以研究肿瘤启动因子和生长因子对缝隙连接介导的胞间通讯的抑制作用及细胞内钙离子、pH值等对缝隙连接作用的影响，并监测环境毒素和药物在细胞增殖和分化中所起到的作用。细胞膜荧光探针受到极化光线激发后，发射光极性依赖于荧光分子的旋转，这种有序的运动自由度取决于荧光分子周围的膜流动性，所以极性测量能间接反映细胞膜的流动性。

荧光光漂白恢复（FluorescenceRedistributionAfterPhotobleaching，FRAP）FRAP是用来测定活细胞的动力学参数，借助于高强度脉冲激光来照射细胞某一区域，造成该区域荧光分子的光粹灭，该区域周围的非粹灭荧光分子会以一定的速率向受照射区域扩散，这个扩散速率可通过低强度激光扫描探测,因而可得到活细胞的动力学参数。LSCM可以控制光粹灭作用，实时监测分子扩散率和恢复速率，反映细胞结构和活动机制。广泛用于研究细胞骨架构成，核膜结构跨膜大分子迁移率，细胞