光学显微与电子成像1

27五月2023光学显微与电子成像1Microscope-显微镜Theoldestpublishedimageknowntohavebeenmadewithamicroscope(1630)ItisoftensaidthatDutchspectacle-makersHansJanssenandhissonZachariasJanssentoinventthefirstcompoundmicroscopein1600s

Microscope:instrumenttoseeobjectstoosmallforthenakedeye.Itisdifficulttosaywhoinventedthecompoundmicroscope.

Historyofinvention

/wiki/MicroscopeThreeGoalsofMicroscopyM:produceamagnifiedimageofthespecimenR:separatethedetailsintheimageC:renderthedetailsvisibletothehumaneyeorcamera.MRC:Magnification,Resolution,ContrastHistoryofLightMicroscopyAsimplecompoundmicroscopeinventedbyBritishmicroscopistRobertHookesometimeinthe1660s.HistoryofLightMicroscopyInventedbyRobertHookein1600s

Microscope20thcenturymicroscopes

21thcenturymicroscopes

Microscope&Microscopy&MicroscopicMicroscope(显微镜)instrumenttoseeobjectstoosmallforthenakedeye.Microscopy(显微成像方法)thescienceofinvestigatingsmallobjectsusingsuchaninstrument.Microscopic(用显微镜可见的)invisibletotheeyeunlessaidedbyamicroscope.MicroscopeElementsOcularlens(eyepiece)(1)-目镜Objective(3)–物镜Diaphragmandcondenserlens(8)光栏和聚光镜Lightsource,alightoramirror(7)BasicopticaltransmissionmicroscopeelementsObjectiveturret(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

Whatisaneyepiece(orocular)目镜?Aneyepiece,orocularlens:isatypeoflensthatisattachedtoavarietyofopticaldevicessuchastelescopes（望远镜)andmicroscopes(显微镜）.namedbecauseitisusuallythelensthatis

closesttotheeyeAcollectionofdifferenttypesofeyepiecesA25mmKellnereyepieceWhatiseyepiece(orocular)?Aneyepieceisusuallydescribedbybarreldiameterandfocallength.Commonbarreldiametersare0.96",1.25"(standard)and2".Commonlyavailableeyepiecefocallengthsrangefrom2mmto40mm.

1-Realimage2-Fielddiaphragm3-Eyerelief4-ExitpupilTheeyepieceisplacedatthefocalpointoftheobjectiveWhatisobjective?Objective:theopticalelementthatgathers(收集)lightfromtheobjectbeingobservedandfocusesthelightraystoproducearealimage(实像）.namedbecauseitisusuallythelensthatis

closesttotheobjectLeicaoilimmersionmicroscopeobjectiveHowimagebecomesmagnifiedinmicroscope?Howimagebecomesmagnifiedinmicroscope?Howimagebecomesmagnifiedinmicroscope?Howimagebecomesmagnifiedinmicroscope?Howimagebecomesmagnifiedinmicroscope?BasicConfiguration:InvertedMicroscope（倒置显微镜）invertedmicroscopeforfluorescencemicroscopy（倒置荧光显微镜）.Invertedmicroscope:

lightsourceandcondenseronthetop,abovethestagepointingdown,whiletheobjectives

andturretarebelowthestagepointingup.invertedmicroscopefortissueculture（组织培养用倒置显微镜）.

BasicConfiguration:UprightMicroscope（正置显微镜）Uprightmicroscope:

lightsourceandcondenseronthebottom,belowthestagepointingup,whiletheobjectives

andturretareabovethestagepointingdown.

PrincipleofKoeherIllumination:

AgivenpointinthespecimenisilluminatedbyeverypointinthelightsourceAgivenpointinthelightsourceilluminateseverypointinthespecimen.Koeher(科勒)IlluminationSystem照明系统Koehlerillumination,inventedin1893byAugustKoehlerHowtoachieveKoeherIllumination?Koeher(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Koeher(科勒)IlluminationSystemFieldplanesandApertureplaneseyepieceseyepiecetelescope孔径光阑视场光阑Diaphragm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Fieldstopdiaphragm(视场光阑)Limittheareaandthepositionofilluminationonthespecimen.LimittheamountoflightcastEliminateflaresandghosting,leadingtosharperimages.

CanNotadjusttheNAofthecondenserlensandobjectiveCanNotaffecttheResolutionFunctionofthefieldstopdiaphragminMicroscope

Aperturediaphragm(孔径光阑)Limitstheprojectionofthelightsource(filament),whichenablesadjustmentofbrightnessofthefieldofview.Adjusttheangleofincidence.

Adjustthecontrastresolutionandfocaldepth.CanNotaffecttheviewingfieldFunctionoftheaperturediaphragm

inMicroscope

AperturediaphragmFunctionoftheaperturediaphragm

inMicroscope

AdjusttheNAofthecondenserlensNA=1.2

NA=0.6

NA=0.3

AperturediaphragmFunctionoftheaperturediaphragm

inMicroscope

Adjustthecontrastresolution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.

AdjustingMicroscopeforKoeherIllumination1.Preliminaries

specimenslidecondenserobjectivediaphragmslamppowerspecimenplaneeyepointimageformedbyobjectivecondenseraperturediaphragmfieldstopdiaphragmAdjustingMicroscopeforKoeherIllumination2.Lampfillsthefrontapertureofthecondenser

Purpose:toachieveKoeherIlluminationTool:eyepiecetelescopeorBertrandlenscondenserobjectivediaphragmslamppowerAdjustingMicroscopeforKoeherIllumination3.Focusthespecimen

Purpose:toachieveKoeherIlluminationTool:eyepiecetelescopeorBertrandlensobjectivefocusdialsstagecontrolsNOTED:avoidobjectiveplungeintospecimenslideAdjustingMicroscopeforKoeherIllumination4.Focusandcenterthecondenser

Purpose:reducescatteredlightandimproveimagecontrastcondenserfielddiaphragmlamppowerAdjustingMicroscopeforKoeherIllumination5.Adjustthecondenserdiaphragm

Purpose:toobtainthebestresolutionandcontrastTool:eyepiecetelescopeorBertrandlens

condenserdiaphragmNOTED:~3/4ofthemaximumdiameteroftheapertureAdjustingMicroscopeforKoeherIllumination6.Adjustthebrightness

Purpose:toobtainoptimalimagebrightnessvoltageneutraldensityfiltersNOTED:aperturediaphragmshouldneverbecloseddownsasawaytoreducelightintensityDetectorsforOpticalMicroscopyFilmphotomicrographyVideoimaging(CloseCircuitTV)Digitalimagingwithachargecoupleddevice(CCD)ScanningmicroscopyPhotomultipliertube(PMT)-光电倍增管Avalanchephotodiode(APD)-雪崩光电二极管WhatisContrast（对比度）?Differencesinlightintensity,phase,polarization,colorcreateimagecontrast,whichallowsindividualfeaturesanddetailsofthespecimentobecomevisible.Contrastisdefinedasthedifferenceinlightintensitybetweentheobjectandtheadjacentbackgroundrelativetothe