光学显微及电子成像

FundamentalsofLightMicroscopyJunleQuE-mail:[e-mail]深圳大学光电工程学院Chapter12Microscope-显微镜Theoldestpublishedimageknowntohavebeenmadewithamicroscope(1630)ItisoftensaidthatDutchspectacle-makersHansJanssenandhissonZachariasJanssentoinventthefirstcompoundmicroscopein1600sMicroscope:instrumenttoseeobjectstoosmallforthenakedeye.Itisdifficulttosaywhoinventedthecompoundmicroscope.HistoryofinventionM:produceamagnifiedimageofthespecimenR:separatethedetailsintheimageC:renderthedetailsvisibletothehumaneyeorcamera.MRC:Magnification,Resolution,Contrast4HistoryofLightMicroscopyAsimplecompoundmicroscopeinventedbyBritishmicroscopistRobertHookesometimeinthe1660s.567HistoryofLightMicroscopy8InventedbyRobertHookein1600sMicroscope20thcenturymicroscopes21thcenturymicroscopes9Microscope&Microscopy&Microscopic1.Microscope(显微镜)instrumenttoseeobjectstoosmallforthenakedeye.2.Microscopy(显微成像方法)thescienceofinvestigatingsmallobjectsusingsuchaninstrument.3.Microscopic(用显微镜可见的)invisibletotheeyeunlessaidedbyamicroscope.10MicroscopeElementsOcularlens(eyepiece)(1)-目镜Objective(3)–物镜Diaphragmandcondenserlens(8)光栏和聚光镜Lightsource,alightoramirror(7)BasicopticaltransmissionmicroscopeelementsObjectiveturret(2)（目镜架）Stage(toholdthesample)(6)、(9)样品台Focuswheel（聚焦轮）tomovethestage(4-coarseadjustment,5-fineadjustment)Basiccomponentsofthelightpath：光路部分构成“Structural”components:结构部分11Whatisaneyepiece(orocular)目镜?Aneyepiece,orocularlens:isatypeoflensthatisattachedtoavarietyofopticaldevicessuchastelescopes（望远镜)andmicroscopes(显微镜）.namedbecauseitisusuallythelensthatisclosesttotheeyeAcollectionofdifferenttypesofeyepiecesA25mmKellnereyepiece12Whatiseyepiece(orocular)?Aneyepieceisusuallydescribedbybarreldiameterandfocallength.Commonbarreldiametersare0.96,1.25(standard)and2.Commonlyavailableeyepiecefocallengthsrangefrom2mmto40mm.1-Realimage2-Fielddiaphragm3-Eyerelief4-ExitpupilTheeyepieceisplacedatthefocalpointoftheobjective13Whatisobjective?Objective:theopticalelementthatgathers(收集)lightfromtheobjectbeingobservedandfocusesthelightraystoproducearealimage(实像）.namedbecauseitisusuallythelensthatisclosesttotheobjectLeicaoilimmersionmicroscopeobjective1415Howimagebecomesmagnifiedinmicroscope?16Howimagebecomesmagnifiedinmicroscope?17Howimagebecomesmagnifiedinmicroscope?18Howimagebecomesmagnifiedinmicroscope?19Howimagebecomesmagnifiedinmicroscope?20BasicConfiguration:InvertedMicroscope（倒置显微镜）invertedmicroscopeforfluorescencemicroscopy（倒置荧光显微镜）.Invertedmicroscope:lightsourceandcondenseronthetop,abovethestagepointingdown,whiletheobjectivesandturretarebelowthestagepointingup.invertedmicroscopefortissueculture（组织培养用倒置显微镜）.21BasicConfiguration:UprightMicroscope（正置显微镜）Uprightmicroscope:lightsourceandcondenseronthebottom,belowthestagepointingup,whiletheobjectivesandturretareabovethestagepointingdown.22PrincipleofKoeherIllumination:AgivenpointinthespecimenisilluminatedbyeverypointinthelightsourceAgivenpointinthelightsourceilluminateseverypointinthespecimen.Koeher(科勒)IlluminationSystem照明系统Koehlerillumination,inventedin1893byAugustKoehlerHowtoachieveKoeherIllumination?23Koeher(科勒)IlluminationSystem科勒照明的实现：把光源的发光体正好摆在聚光镜下方的前焦平面Koeher照明光路发光体上点光源变成一束平行光（光源上一点照明样品的每一点）整个发光体变成无数束平行光（样品上一点由光源每一点照明）样品上无灯丝像，无条纹24Koeher(科勒)IlluminationSystemFieldplanesandApertureplaneseyepieceseyepiecetelescope孔径光阑视场光阑25Diaphragm光阑分为孔径光阑和视场光阑、渐晕光阑和杂消光光阑等孔径光阑：用于确定辐射测量的面积的光阑（改变成象光束的大小和控制进入光学系统的光通亮）视场光阑：限制成像范围的光阑26Fieldstopdiaphragm(视场光阑)Limittheareaandthepositionofilluminationonthespecimen.LimittheamountoflightcastEliminateflaresandghosting,leadingtosharperimages.CanNotadjusttheNAofthecondenserlensandobjectiveCanNotaffecttheResolutionFunctionofthefieldstopdiaphragminMicroscope27Aperturediaphragm(孔径光阑)Limitstheprojectionofthelightsource(filament),whichenablesadjustmentofbrightnessofthefieldofview.Adjusttheangleofincidence.Adjustthecontrastresolutionandfocaldepth.CanNotaffecttheviewingfieldFunctionoftheaperturediaphragminMicroscope28AperturediaphragmFunctionoftheaperturediaphragminMicroscopeAdjusttheNAofthecondenserlensNA=1.2NA=0.6NA=0.329AperturediaphragmFunctionoftheaperturediaphragminMicroscopeAdjustthecontrastresolution聚光镜孔径光阑=NA聚光镜孔径光阑=0.8NA聚光镜孔径光阑=0.6NA聚光镜孔径光阑的大小缩小到等于所用物镜出射光瞳的2/3或4/5效果好,这是在最大分辩率与最佳反差间所作的折衷.30AdjustingMicroscopeforKoeherIllumination1.Preliminariesspecimenslidecondenserobjectivediaphragmslamppowerspecimenplaneeyepointimageformedbyobjectivecondenseraperturediaphragmfieldstopdiaphragm31AdjustingMicroscopeforKoeherIllumination2.LampfillsthefrontapertureofthecondenserPurpose:toachieveKoeherIlluminationTool:eyepiecetelescopeorBertrandlenscondenserobjectivediaphragmslamppower32AdjustingMicroscopeforKoeherIllumination3.FocusthespecimenPurpose:toachieveKoeherIlluminationTool:eyepiecetelescopeorBertrandlensobjectivefocusdialsstagecontrolsNOTED:avoidobjectiveplungeintospecimenslide33AdjustingMicroscopeforKoeherIllumination4.FocusandcenterthecondenserPurpose:reducescatteredlightandimproveimagecontrastco