Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.
Photoelectron microscopy and immunofluorescence microscopy of cytoskeletal elements in the same cells.
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同一细胞中细胞骨架元件的光电子显微镜和免疫荧光显微镜。
DOI：
10.1073/pnas.80.13.4012
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发表时间：
1983
影响因子：
11.1
通讯作者：
Griffith,OH
中科院分区：
文献类型：
--
作者：
Nadakavukaren,KK;Chen,LB;Habliston,DL;Griffith,OH
Pt K2大鼠袋鼠上皮细胞和rat -1成纤维细胞生长在导电玻璃圆盘上，固定并渗透，通过间接免疫荧光观察细胞骨架元件肌动蛋白、角蛋白和波形蛋白。荧光显微镜观察后，将细胞后固定并脱水，用于光电子显微镜观察。在这些光电子显微照片的对比主要是地形的起源，在低密度的荧光染料的存在不贡献显著的材料对比。通过与在同一细胞上获得的荧光显微图进行比较，在光电子显微图中鉴定出含有肌动蛋白的应力纤维、角蛋白丝和静脉蛋白丝。光电子显微照片所显示的细胞骨架网络所占的表观体积比荧光显微照片所显示的要小得多，因为光电子显微镜的高分辨率显示纤维更接近其真实尺寸。光电子显微镜是一种表面技术，图像突出暴露的细胞骨架结构，并抑制那些沿着细胞核以下的底物延伸的结构。本文报道的结果表明光电子显微照片的图像质量有了显著的改善，并且该技术有可能有助于细胞骨架结构的高分辨率研究。
Pt K2 rat kangaroo epithelial cells and Rat-1 fibroblasts were grown on conductive glass discs, fixed, and permeabilized, and the cytoskeletal elements actin, keratin, and vimentin were visualized by indirect immunofluorescence. After the fluorescence microscopy, the cells were postfixed and dehydrated for photoelectron microscopy. The contrast in these photoelectron micrographs is primarily topographical in origin, and the presence of fluorescent dyes at low density does not contribute significantly to the material contrast. By comparison with fluorescence micrographs obtained on the same individual cells, actin-containing stress fibers, keratin filaments, and vimentin filaments were identified in the photoelectron micrographs. The apparent volume occupied by the cytoskeletal network in the cells as judged from the photoelectron micrographs is much less than it appears to be from the fluorescence micrographs because the higher resolution of photoelectron microscopy shows the fibers closer to their true dimensions. Photoelectron microscopy is a surface technique, and the images highlight the exposed cytoskeletal structures and suppress those extending along the substrate below the nuclei. The results reported here show marked improvement in image quality of photoelectron micrographs and that this technique has the potential of contributing to higher resolution studies of cytoskeletal structures.