电镜技术及应用B

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

2、positedonacarboncoatedgridandsurroundedbystain(lightgrayshading)interactingwithanelectronbeam(arrows).Phosphotungstateuranylactetate/formatemolybdate(ammonium)Benefits:Veryhighcontrast..Radiationdamagecauseslesschangeinthestainarea.Thesampleiseasytoprepare.Drawbacks:Theparticleisdistorted

3、duringthestainingprocess.Artifactscanariseifthestainisuneven.Theresolutionislimitedtoapproximately20Å.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：一类是免疫凝集电镜技术，即采用抗原

4、抗体凝集反应后，再经负染色直接在电镜下观察；另一类则是免疫电镜定位技术。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-EMImmuno-labeling•usingimmunogoldlabelstolocalizeepitopes>widelyusedincellbiologymacromoleculeAusphereMAbepitopeEpitopelocalizationinanengineeredvaccine•

5、anewvaccineforHepatitisBcontains3antigens,S,S1&S2,withepitopesoneach•doeseveryparticlecontainall3oftheseepitopes?•MAbsagainstS,S1&S2havebeenmade&conjugatedwithgold:S15nmS110nmS25nmImmunolabellingofoneepitope(S1)using10nm-AulabelledMAbTriplelabellingof3epitopesononesingleparticleCryo-EM•Ne

6、gativestain–Easytoprepare–Goodcontrast–Preservation–Sampledistortion–Resolutionlimitedtoabout20angstroms•Cryo–Difficultsampleprep–Lowcontrast–BestpreservationandthereforeresolutionCryoprepusingholeyfilmFlashfreezeinliquidethaneVitrobotDatacollectionImagerecordingFilm•Highdensitycontent(~2

7、0kx16kpixels)•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