Streamlined Embedding of Cell Monolayers on Gridded Glass-Bottom Imaging Dishes for Correlative Light and Electron Microscopy
Streamlined Embedding of Cell Monolayers on Gridded Glass-Bottom Imaging Dishes for Correlative Light and Electron Microscopy
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DOI：
10.1017/s1431927610094092
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发表时间：
2010-12-01
影响因子：
2.8
通讯作者：
Phillips, Greg R.
中科院分区：
文献类型：
--
作者：
Hanson, Hugo H.;Reilly, James E.;Phillips, Greg R.
相关光学和电子显微镜（CLEM）促进了细胞内运输的研究。CLEM的常规应用将有利于许多实验室，但先前描述的技术是特别苛刻的，即使对于那些访问激光扫描共聚焦显微镜（LSCM）和透射电子显微镜（TEM）。我们描述了流线型的方法，TEM的绿色荧光蛋白（GFP）标记的细胞器后，成像LSCM使用网格玻璃底成像皿。用雷帕霉素处理GFP-MAP 1A/1B LC 3（GFP-LC 3）转染的细胞，固定并通过LSCM成像。获得共聚焦图像堆栈，使得能够完全可视化每个GFP-LC 3标记的细胞器。在LSCM之后，使用简化的两步法嵌入细胞以用于TEM，所述两步法稳定玻璃底部，使得块可以通过温和加热与玻璃分离。所有成像和TEM处理都在同一个皿中进行。将LSCM成像的细胞重新定位在块上并连续切片。细胞标志有助于LSCM、DIC和TEM图像的相关性。所有GFP标记的结构都成功地重新鉴定，并通过连续切片TEM成像。这种方法可以使CLEM更容易与基本的电子显微镜专业知识的非专业实验室，并可用于常规确认荧光斑点的细胞器定位。
Correlative light and electron microscopy (CLEM) has facilitated study of intracellular trafficking. Routine application of CLEM would be advantageous for many laboratories but previously described techniques are particularly demanding, even for those with access to laser scanning confocal microscopy (LSCM) and transmission electron microscopy (TEM). We describe streamlined methods for TEM of green fluorescent protein (GFP)-labeled organelles after imaging by LSCM using gridded glass bottom imaging dishes. GFP-MAP 1A/1B LC3 (GFP-LC3) transfected cells were treated with rapamycin, fixed and imaged by LSCM. Confocal image stacks were acquired enabling full visualization of each GFP-LC3 labeled organelle. After LSCM, cells were embedded for TEM using a simplified two step method that stabilizes the glass bottom such that the block can be separated from the glass by mild heating. All imaging and TEM processing are performed in the same dish. The LSCM imaged cells were relocated on the block and serial sectioned. Correlation of LSCM, DIC, and TEM images was facilitated by cellular landmarks. All GFP labeled structures were successfully reidentified and imaged by serial section TEM. This method could make CLEM more accessible to nonspecialized laboratories with basic electron microscopy expertise and could be used routinely to confirm organelle localization of fluorescent puncta.