电镜技术及应用课2013B.pdf

电镜技术及应用B 陈荣 rongchen@ 结构病毒学研究组 上海巴斯德所 2013-11-7 • Negative Stain EM • Immuno-EM • CryoEM • Correlative Light-Electron Microscopy • Symmetry • Single Particles Data Processing • Automation Macromolecular Imaging • ADDING CONTRAST (with heavy metals) negative stain immuno-labeling • USING INTRINSIC CONTRAST particles in thin film of vitrified ice Negative Stain A sample deposited on a carbon coated grid and surrounded by stain (light gray shading) interacting with an electron beam (arrows). Phosphotungstate uranyl actetate/formate molybdate (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 20Å. Negative staining Typical transmission electron micrograph of negatively stained, purified virus preparation – calicivirus Immuno-EM 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-labeling • using immunogold labels to localize epitopes widely used in cell biology Au sphere MAb