Keene Douglas R, Tufa Sara F, Lunstrum Gregory P, Holden Paul, Horton William A
Research Department, Shriners Hospital for Children, Portland, OR 97239, USA.
Microsc Microanal. 2008 Aug;14(4):342-8. doi: 10.1017/S1431927608080306.
Genetic manipulation allows simultaneous expression of green fluorescent protein (GFP) and its derivatives with a wide variety of cellular proteins in a variety of living systems. Epifluorescent and confocal laser scanning microscopy (confocal) localization of GFP constructs within living tissue and cell cultures has become routine, but correlation of light microscopy and high resolution transmission electron microscopy (TEM) on components within identical cells has been problematic. In this study, we describe an approach that specifically localizes the position of GFP/yellow fluorescent protein (YFP) constructs within the same cultured cell imaged in the confocal and transmission electron microscopes. We present a simplified method for delivering cell cultures expressing fluorescent fusion proteins into LR White embedding media, which allows excellent GFP/YFP detection and also high-resolution imaging in the TEM. Confocal images from 0.5-microm-thick sections are overlaid atop TEM images of the same cells collected from the next serial ultrathin section. The overlay is achieved in Adobe Photoshop by making the confocal image somewhat transparent, then carefully aligning features within the confocal image over the same features visible in the TEM image. The method requires no specialized specimen preparation equipment; specimens are taken from live cultures to embedding within 8 h, and confocal transmission overlay microscopy can be completed within a few hours.
基因操作能够使绿色荧光蛋白（GFP）及其衍生物与多种细胞蛋白在多种活体系统中同时表达。在活组织和细胞培养物中，通过落射荧光显微镜和共聚焦激光扫描显微镜（共聚焦显微镜）对GFP构建体进行定位已成为常规操作，但在相同细胞内对光学显微镜和高分辨率透射电子显微镜（TEM）的组件进行关联却存在问题。在本研究中，我们描述了一种方法，可在共聚焦显微镜和透射电子显微镜成像的同一培养细胞内特异性定位GFP/黄色荧光蛋白（YFP）构建体的位置。我们提出了一种将表达荧光融合蛋白的细胞培养物导入LR White包埋介质的简化方法，该方法能实现出色的GFP/YFP检测，还能在TEM中进行高分辨率成像。从0.5微米厚切片获得的共聚焦图像叠加在从下一个连续超薄切片收集的相同细胞的TEM图像之上。通过在Adobe Photoshop中使共聚焦图像稍微透明，然后将共聚焦图像中的特征与TEM图像中可见的相同特征仔细对齐来实现叠加。该方法不需要专门的标本制备设备；标本从活培养物中获取，8小时内即可完成包埋，共聚焦透射叠加显微镜检查可在数小时内完成。
