天津大学《科学仪器》课件3 光学显微成像仪器.pptxVIP

第三章

光学显微技术及仪器3.1基本概念3.2光学显微镜的结构3.3各种光学显微镜3.4荧光显微技术3.5单分子成像3.6超高分辨率成像OpticalMicroscopyandInstruments

3.1基本概念1.光的特性光的波动性：波长、振幅、相位和偏振

Violet(400nm)?Red(700nm)

22:39:37光的偏振Y-线偏X-线偏圆偏线偏

RefractionReflectionincidentlightreflectedlightscatteredlighttransmittedlighttissue2.光与物质相互作用（Light-MatterInteractions）

弹性散射非弹性散射Absorptiona(n)=absorptioncross-sectionaS=scatteringcross-sectionI(z)=intensityindepthzI0=incidentintensityI(n)=transmittedintensity

荧光

衍射光学双折射（Birefringent）

BrightfieldAmplitudedifference3.光学对比度(1)强度(亮场)UniformbrightfieldimageBrightfieldimageofMossreeds

Darkfield(2)强度(暗场)

PhasecontrastDifferentialphasecontrastPhasedifferenceRefractiveindices(3)相位(相衬、相差)

(4)吸收对比度(光热、光声成像)nn’PumpProbeProbe

(5)荧光对比度(荧光成像)ExcitationEmission

(6)非弹性散射对比度(拉曼光谱成像)ExcitationScattering

(7)偏振对比度west(left)east(right)LightvibratingE-WUnpolarizedlightLightvibratinginmanyplanesandwithmanywavelengthsLightandcolorsreacheye!

3.2光学显微镜的结构显微镜的功能：放大、分辨率、对比度1.显微原理单透镜系统

2.照明系统Kohlerillumination

ArrowsmarkconjugateplanesKohlerIlluminationgivesthemostuniformilluminationEachpartofthelightsourcedivergestowholespecimenEachpartofthespecimengetslightthatconvergesfromthewholelightsource视场光阑孔径光阑

视场光阑

22:39:37孔径光阑

3.成像光路有限与无限远校正显微物镜

4.物镜

1)数值孔径(NA)与分辨率

分辨率：

2)球差和修正环

3)散光

消色差（achromats）色差4)色差

复消色差的（apochromatic）萤石（fluorite）

5)彗差

6)像场弯曲和平场物镜

Acentralobstructionblocksthecentralcone.Thesampleisonlyilluminatedbythemarginalrays.Thesemarginalraysmustbeatanglestoolargefortheobjectivelenstocollect.Onlylightscatteredbytheobjectiscollectedbythelens.1.暗场显微镜(Dark-FieldOpticalMicroscopy)3.3各种光学显微镜

2.相衬显微镜(Phasecontrastillumination)

3.差分干涉显微镜(DifferentialInterferenceContrast)

4.光热显微镜(Photothermalimaging)

3.4荧光显微技术1.荧光现象

22:39:37

GreenFluorescentProtein(GFP)

TheFluoroporeActiveSiteSer65-Tyr66-Gly67Deprotonatedphenol