显微镜知识讲座

-1-显微镜知识讲座欢迎光临本次讲座主题:显微镜基础知识和应用20040101-2-显微镜知识讲座•讲座内容•显微镜基础知识介绍•显微镜种类介绍•新型显微镜的发展方向•新的应用技术-3-显微镜知识讲座一些误区放大倍数越高，显微镜越好光学显微镜可以放大1500-2000倍物镜的质量决定一切倒置显微镜可以代替正立显微镜-4-显微镜基本概念显微镜定义:光学显微镜:利用可见光作为光源存在各种相差（Abbreviation）如：色差、球差、畸变等2步放大（物镜和目镜）-5-显微镜基本概念分辨率(可以分辨的两个目标间的最小距离):两个目标间的最小距离DD=0.61Wavelength/N.A(物镜)无效放大:可以放大,但不提高分辨率有效放大:通过提高物镜的N.A来实现的放大-6-显微镜的分类正立显微镜•最常用的显微镜,清晰的成像质量•物镜在样品上面•用于观察动植物切品•物镜如样品间的距离较小-7-显微镜分类-正立显微镜-8-显微镜分类倒置显微镜•适合活体细胞样品观察•优化折中的性能(较长的光路,较底的数值孔径的聚光镜,和长工作距离低数值孔径的的物镜•长工作距离适合于培养皿观察和显微操作•物镜在样品下面,•相对较弱的荧光(因为光路较长)-9-显微镜分类-倒置显微镜-10-显微镜分类体式显微镜•较低的放大倍数•图象有3维空间感•冷光源照明避免对样品的伤害•丰富多样的底座用于各种实际需要•生物需要:•解剖•绿色荧光蛋白GFP(fluorescenthook-up)-11-显微镜分类–体式显微镜-12-显微镜基础–观察方式观察方式:A.明视野观察(brightfieldobservation)•彩色样品•H&E染色,Gram染色,等.•最常见的观察方式B.暗视野观察•黑色背景,样品发光•特别适合对微小样品的外部形态的观察•细菌观察bacteriology(Treponema),Silvergainetc-13-显微镜基础–观察方式C.相差(Phasecontrast)•光线通过样品时产生相位的变化•通过相位的变化产生光的干涉,从而反应样品的密度的结构•最适合于观察透明的活体细胞•细胞周围有光晕l(细胞越后光晕越明显)•用绿色滤光片提高反差-14-显微镜基础–观察方式D.微分干涉相称DIC(DifferentialandInterferenceContrast)•利用Normaski棱镜将光分为两束偏振光•两束偏振光通过样品后发生干涉,产生图象•可以获得完美的3D图象,•没有光晕,适合各种薄厚标本(6microns).•比相差观察的分辨率更高.•适合细胞注射.•Problem:对塑料培养皿有偏色-15-BasicMicroscopy--ContrastingmethodE.HoffmanmodulationorVarel(Reliefcontrast)•AkindoOpaqueillumination•Illuminatedfromonesidegiveshadowand3Dlikeimage,NotuseonPollight•ExcellentreplacementofDICforplasticmediumbutlesscontrast,homogeneousbackgroundandresolution.•BestforIVF(ICSI)ascanbeusedonplasticpetridish•Varelsimpleandcheaperbutsimilarresult,abitmoredifficulttogethomogeneousilluminationbutsampledependent.F.PolarizingContrast(notmentionedhere,moreformaterial)-16-BasicMicroscopy–Application-17-BasicMicroscopy–Application-18-显微镜基础–观察方式G.落射荧光•用于植物的自发荧光观察•免疫荧光,荧光原位杂交(FISH)或者GFP•首先用一种短波长的光(蓝光)照射用荧光染色的标本,使标本发出较长波长的光(绿光),这种荧光染料是FITC.•黑色的背景,彩色的图象-19-BasicMicroscopy–Application-20-显微镜基础—观察方式-21-显微镜基础—观察方式G.落射荧光•汞灯光源•激发块包括激发镜（―excitationfilter‖）;分光镜（“dichloricmirror‖）;透射镜（emissionfilter）,和支架•黑色的背景是关键•广泛用于免疫荧光,FISH,M-FISH,GFPrelated,FRET,FRAP,FLIP,钙离子浓度测量等领域（Calciumandratioimaging)•基于荧光技术的激光共聚焦技术-22-荧光原理-23-显微镜基础–共聚焦显微镜激光共聚焦显微镜（ConfocalLaserscanningmicroscopy）•用激光作光源•通过扫描获得图象•通过PMTsensor获得图象•通过针孔（confocalpinhole），一个时间只获得一个焦平面的图象.•优点:•没有杂散的荧光的干扰•3D图象•其他应用-24-显微镜基础–共聚焦显微镜ConfocalLaserscanningmicroscopy•Therefore,goodratioimagingtooltogivemeasurementandimage•On-linefastdatameasurementbyline-measured•Disadv:•Expensive•UVexcitationisexpensiveanddiffficult•Newtechnology:•NLOforvariousapplication•Meta:unmixofoverlappedFLdye,GFPandFITCcanbeusedtogether,replaceUVneeded-25-BasicMicroscopy–LSMMetaGFPandFITC(Distanceofemissionpeaks7nm)CulturedfibroblastsexpressingaGFP-Histone2Bfusionprotein(green),actinfilamentsstainedwithFITC-phalloidin(red)ClearseparationofsignalsdespiteheavyspectralandspatialoverlapofemissionspectraSample:MaryDickinson,PhD,Caltech,Pasadena,USAGFPFITC-26-BasicMicroscopy–LSMMetaCFP,GFPandYFP•CulturedcellsexpressingECFP-RanGAP1,EGFP-emerinundEYFP-SUMO1•MixtureofNIH3T3cellsexpressingeitherCFP,GFPorYFPCourtesy:Prof.Y.Hiraoka,KARC,Kobe,Japan;MaryDickinson,PhD,Caltech,Pasadena,USAGFPYFPCFPCFP,GFP,YFP-27-BasicMicroscopy–ApplicationGFP•Genomeincorporatetothetargetandexpressfluorescence•Excellentlivingdyeandwidelyusedinvariousapplication•Problem:highlyoverlappingspectrumFRET•Fluorescentresonanceenergytransfer•Theenergyemissionofonedye(notemit)butresonanceabsorbedbythedyenexttoit,andemitfluorescence•Onlyiftwodyeareveryclosed•Co-localizationstudyandmobilityordynamictest-28-BasicMicroscopy–Application–FRETFRETsignalnoFRETsignalCFPYFPexemCFPYFPexememFRETsignal•CFPisexcitedbylightbutdoesemitlittlelight•CFPisincloseproximity(1-10nm)toacceptorYFP•YFPisnotexcitedbylightbutdoesemitlightNoFRETsignal•CFPisexcitedbylightandemitslight•CFPismorethan10nmdistantfromYFP•YFPisnotexcitedanddoesnotemitlightEnergydiagramofCFP/YFPFRETCFP(donor)isexcitedbutmostofitsenergyisnotresultingincyanemission,insteaditistransferredtotheYFP(acceptor),thus,theresultingemissionisyellow.-29-BasicMicrsocopy–Application--FRET•Fluorescentproteins(FPs)areveryattractiveforFRETexperimentsonlivingcells.•FPscanbegeneticallyfusedtoproteinsofinterestandexpressedasreportersforgeneexpressionandproteinlocalizationinlivingcells.•CFP(donor)/YFP(acceptor)isthemostpopularFRETpair413458514ExcitationWavelength527475EmissionWavelengthCFPYFPCFPYFPExcitationEmissionCrosstalkbetweenCFPandYFPonexcitationandemissonlevelGFPstructure-30-BasicMicroscopy–Application–FRET(Y.Hiraoka,KARC,Kobe,Japan/A.Miyawaki,RIKEN,Wako,Japan)SpectralSignaturesStep3:CFP/YFPratioTimeIntensityCFP/YFPratioStimulusInformationdensity...LambdaStacksFRETandionimagingCa2+imagingusingtheFRETprobeYellowCameleon2-31-BasicMicroscopy–ApplicationFRAP•Fluorescencerecoveryafterphoto-bleaching•Tobleachthefluorescenceinaparticularareabystrongemissionlight,toseehowlongitrecovery.•Recoveryduetochemicaltransportorre-productionofprotein,FLIP•FluorescentLife-timeMethod•SameasFRETbutworkbackward.-32-Basicmicroscopy–Application–FRAPWhatisFRAP?FRAPistherecoveryoffluorescenceinadefinedregionafterableachingeventduetofluorophoresthatmovefromthe