東南大學電子學院《現代光學基礎》補充內容3-顯微鏡

第七章共焦顯微光學

Chapter7

ConfocalMicroscopyOptics東南大學先進光子學中心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細胞的雙光子熒光像。染料為trans-4-[p-(9-ethylcarbazde)vinyl]-N-methypyrid-iniumIodide

量子點

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

量子點熒光材料準零維尺度人造原子量子點的結構（續(xù)）

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

實驗結果樣品（上圖左起2、4、6）的紫外吸收光譜以及相應的熒光發(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實驗結果ApplicationsLSCM的高靈敏度、高分辨率、高放大倍數，提供了光學顯微鏡無法顯示的結構，使細胞生物學研究上了一個臺階。目前我們可以在亞細胞水平進行動態(tài)實驗，檢測細胞生物質和離子通道的變化，觀察細胞在生理、病理和藥理情況下對外界因素作用所產生的快速反應，進行定性、定量、定時和定位的分析測量。最常用的功能是細胞三維重建，細胞熒光檢測和細胞顯微操作等

細胞的三維重建（3-DReconstruction）LSCM能以0.1μm的步距沿軸向對細胞進行分層掃描，得到一組光學切片。通過計算機進行不同的三維重建算法，可作單色或雙色圖象處理，組合成細胞真實的三維結構。旋轉不同角度可觀察各側面的表面形態(tài)，也可不同的斷面觀察細胞內部結構，測量細胞的長寬高、體積和斷層面積等形態(tài)學參數。通過角度旋轉和細胞位置變化可產生三維動畫效果。細胞定量熒光測定LSCM以激光為光源，對細胞分層掃描，單獨測定，經積分后能得到細胞熒光的準確定量，重復性極佳。它適于活細胞的定量分析。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用廣泛，如作各種腫瘤組織切片抗原表達的定量分析，監(jiān)測腫瘤相關抗原表達的定位定量信息，監(jiān)測藥物對肌體免疫功能的作用等。細胞定量熒光測定可選用單熒光。雙熒光和三熒光方式，能自動測定細胞面積，平均熒光強度，積分熒光強度及形狀因子等多種參數。

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

熒光光漂白恢復（FluorescenceRedistributionAfterPhotobleaching，FRAP）FRAP是用來測定活細胞的動力學參數，借助于高強度脈沖激光來照射細胞某一區(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以一定的速率向受照射區(qū)域擴散，這個擴散速率可通過低強度激光掃描探測,因而可得到活細胞的動力學參數。LSCM可以控制光粹滅作用，實時監(jiān)測分子擴散率和恢復速率，反映細胞結構和活動機制。廣泛用于研究細胞骨架構成，核膜結構跨膜大分子遷移率，細胞