Bridging the resolution gap: Imaging the same transcription factories in cryosections by light and electron microscopy
Bridging the resolution gap: Imaging the same transcription factories in cryosections by light and electron microscopy
复制标题
DOI：
10.1177/002215549904700405
复制
发表时间：
1999-04-01
影响因子：
3.2
通讯作者：
Cook, PR
中科院分区：
文献类型：
--
作者：
Pombo, A;Hollinshead, M;Cook, PR
传统光学显微镜的分辨率在x轴和y轴上限制在200 nm左右，在z轴上限制在500 nm左右。提高z轴分辨率的一个简单方法是分析100- 200nm的薄片。这种方法的效用是通过参考人类细胞核冷冻切片成像的转录位点来说明的。细胞被渗透，允许在Br-UTP中扩展新生转录本，固定，冷冻切片，并用荧光染料和金颗粒对br - rna进行免疫标记。正如预期的那样，物理切片提高了分辨率并带来了其他优势。首先，切片允许改进抗体获取和更好的免疫标记。其次，现在可以在较低的背景下解析更多的站点（具有更具代表性的强度范围），从而促进定量分析。第三，当收集同一物体的两幅不同颜色的图像时，可以通过在图像收集之间重新聚焦来避免色差问题。第四，完全相同的位置可以通过光学和电子显微镜成像，从而可以直接比较两种技术。免疫金标记和电子显微镜提供了最准确的位点数计数。结果证实，核质中的新生转录本被限制在几千个位点，或“工厂”，直径约为40纳米。
The resolution of conventional light microscopy is limited to similar to 200 nm in the x- and y-axes and >500 nm in the z-axis. A simple way of improving z-axis resolution is to analyze thin sections of 100-200 nm. The utility of such an approach is illustrated by reference to transcription sites imaged in cryosections of human nuclei. Cells are permeabilized, allowed to extend nascent transcripts in Br-UTP, fixed, cryosectioned, and Br-RNA-immunolabeled with fluorochromes and gold particles. As expected, physical sectioning improves resolution and brings other advantages. First, sections allow improved antibody access and better immunolabeling. Second, more sites (with a more representative range of intensities) can now be resolved against lower backgrounds, facilitating quantitative analysis. Third, problems associated with chromatic aberration when two differently colored images of the same objects are collected can be sidestepped by refocusing between image collection. Fourth, exactly the same sites can be imaged by light and electron microscopy, allowing direct comparison between the two techniques. Immunogold labeling and electron microscopy provided the most accurate counts of site number. The results confirm that nascent transcripts in the nucleoplasm are confined to several thousand sites, or "factories," with diameters of similar to 40 nm.