电镜技术及应用B.doc

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1、电镜技术及应用B•NegativeStainEMnnr•CryoEM•CorrelativeLight-ElectronMicroscopy•Symmetry•SingleParticlesDataProcessing•AutomationMacromolecularImaging•ADDINGCONTRAST(withheavymetals)>negativestain>immuno-labeling•USINGINTRINSICCONTRAST>particlesinthinfilmofvitrifiediceNegativeStainAsampledep

2、ositedonacarboncoatedgridandsurroundedbystain(1ightgrayshading)interactingwithanelectronbeam(arrows).Phosphotungstateuranylactetate/formatemolybdate(ammonium)Benefits:Veryhighcontrast..Radiationdamagecauseslesschangeinthestainarea.Thesampleiseasytoprepare.Drawbacks:Theparticleisdist

3、ortedduringthestainingprocess.Artifactscanariseifthestainisuneven.Theresolutionislimitedtoapproximately20A.NegativestainingTypicaltransmissionelectronmicrographofnegativelystained,purifiedviruspreparation-calicivirus免疫电镜技术是免疫化学技术与电镜技术结合的产物，是在超微结构水平研究和观察抗原、抗体结合定位的一种方法学。主要分为两大类：一类是免疫凝

4、集电镜技术，即采用抗原抗体凝集反应后，再经负染色直接在电镜下观察；另一类则是伽疫电镜定位技术。该项技术是利用带有特殊标记的抗体与相应抗原相结合，在电子显微镜下观察，由于标准物形成一定的电子密度而指示出相应抗原所在的部位。免疫电镜的应用，使得抗原和抗体定位的研究进入到亚细胞的水平。Immuno-EMjjTlTImmuno-labeling•usingimmunogoldlabelstolocalizeepitopes>widelyusedincellbiologymacromoleculeAusphereMAbepitopeEpitopelocalization

5、inanengineeredvaccine•anewvaccineforHepatitisBcontains3antigens,S,SI&S2,withepitopesoneach•doeseveryparticlecontainall3oftheseepitopes?•MAbsagainstS,SI&S2havebeenmade&conjugatedwithgold:Immunolabellingofoneepitope(SI)using10nm-AulabelledMAbTriplelabellingof3epitopesononesinglepartic

6、leCryo-EM•Negativestain-Easytoprepare-Goodcontrast-Preservation-SampledistortionResolutionlimitedtoabout20angstroms•Cryo-Difficultsampleprep-Lowcontrast-BestpreservationandthereforeresolutionCryoprepusingholeyfilmFlashfreezeinliquidethaneVitrobotDatacollectionImagerecordingFilm•High

7、densitycontent(~20kx16kpixels)•Slow(developmenttime,drying)•Requiresdigitization(scanningtakeshours)CCD(charge-coupleddevice)•Lowdensitycontent(4kx4kpixels)•Fast(mstosec)CMOS(Complementarymetal-oxide-semiconductor)•Fast•HighnoiseimmunityCryoEM•冷冻（低温）:样品经快速冷冻后，结构以接近天然状态保存-成像：电子穿过样品后的

8、成像深浅与样品自身的电子密度分布相关-