SEM样品处理方法.docVIP

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SEM样品处理方法

Electron microscopy. Exponential-phase cultures of different M. smegmatis strains were harvested, washed three times with 0.15 M cacodylate buffer, treated with 1% osmium tetroxide for 1 h at room temperature, centrifuged, and the collected cells were fixed with 0.15 M cacodylate buffer containing 2% glutaraldehyde for 2 h at room temperature. Cells were again collected after centrifugation and fixed overnight with 0.15 M cacodylate buffer containing 1% osmium tetroxide at 4 ℃. The cells were dehydrated using graded ethanol (10 %, 30 %, 50 %, 70% and 100 %), mounted on microscope stubs and sputter coated on the sputter coater. The samples were then examined under a Leica S440 scanning electron microscope. 2.5. Scanning electron microscope(SEM) For SEM analysis, cells were fixed with2.5%(v/v) glutaraldehyde for 2–3 h. The fixed cells were washed with 0.1Mphosphate- buffered saline(PBS)(pH7.3) for three times(10mineach). Subsequently, ethanol concentration gradient(v/v)of 50%,70%, 80%, 90%and100%was used to dehydrate the fixed cells in a sequential way. Finally, the resulted cell samples were further dehydrated by two more100% ethanol treatments. After that, tertiary butyl alcohol mixed with ethanol in a ratio of 1:1and pure tertiary butyl alcohol was applied to achieve metathesis of ethanol in the cells. At last, cells were mixed in tertiary butyl alcohol and lyophilized for imaging. 2.4.2. Electronmicroscopyanalysis Cells were harvested by centrifugation at 5000g for 3min, washed three times with PBS(phosphatebufferpH7.2).Subsequently, the cells were fixed with 2%glutaraldehyde(pH7.2)at room temperature for 2h and washed again as above. The cells were prepared for scanning or transmission electron microscopy as described previously (Denner etal.,1994). 真空扫描式电子显微镜 JSM-6360LV 扫描电子显微镜0020 S-3400N 二、扫描电子显微镜 扫描电子显微镜(scanning electron microscope,SEM)于20世纪60年代问世,用来观察标本的表面结构。其工作原理是用一束极细的电子束扫描样品,在样品表面激发出次级电子,次级电子的多少与电子束入射角有关,也就是说与样品的表面结构有关,次级电子由探测体收集,并在那里被闪烁器

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