电镜技术及应用B.docx

电镜技术及应用B?Negative Stain EM?Immuno-EM?CryoEM?Correlative Light-Electron Microscopy?Symmetry?Single Particles Data Processing?AutomationMacromolecular Imaging?ADDING CONTRAST (with heavy metals) negative stain immuno-labeling? USING INTRINSIC CONTRAST particles in thin film of vitrified iceNegative StainA sample deposited on a carbon coated gridand surrounded by stain (light gray shading)interacting with an electron beam (arrows).Phosphotungstateuranyl actetate/formatemolybdate (ammonium)Benefits:Very high contrast..Radiation damage causes less change in the stain area.The sample is easy to prepare.Drawbacks:The particle is distorted during the staining process.Artifacts can arise if the stain is uneven.The resolution is limited to approximately 20?.Negative stainingTypical transmission electronmicrograph of negatively stained,purified virus preparation –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?using immunogold labels to localize epitopes widely used in cell biologymacromoleculeAu sphereMAbepitopeEpitope localization in an engineered vaccine?a new vaccine for Hepatitis B contains 3 antigens, S, S1 S2,with epitopes on each? does every particle contain all 3 of these epitopes ?? MAbs against S, S1 S2 have been made conjugated withgold:S 15 nmS1 10 nmS2 5 nmImmunolabelling of one epitope (S1) using 10 nm-Au labelled MAbTriple labelling of 3 epitopes on one single particleCryo-EM?Negative stain–Easy to prepare–Good contrast–Preservation–Sample distortion–Resolution limited to about 20 angstroms?Cryo–Difficult sample prep–Low contrast–Best preservation and therefore resolutionCryo prep using holey filmFlash freeze in liquid ethaneVitrobotData collectionImage recordingFilm? High density content (~20k x 16k pixels)? Slow (development time, drying)? Requires digitization (sc