Martin Sonya, Failla Antonio Virgilio, Spöri Udo, Cremer Christoph, Pombo Ana
MRC, Clinical Sciences Centre, Faculty of Medicine, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, United Kingdom.
Mol Biol Cell. 2004 May;15(5):2449-55. doi: 10.1091/mbc.e04-01-0045. Epub 2004 Mar 12.
Spatially modulated illumination fluorescence microscopy can in theory measure the sizes of objects with a diameter ranging between 10 and 200 nm and has allowed accurate size measurement of subresolution fluorescent beads ( approximately 40-100 nm). Biological structures in this size range have so far been measured by electron microscopy. Here, we have labeled sites containing the active, hyperphosphorylated form of RNA polymerase II in the nucleus of HeLa cells by using the antibody H5. The spatially modulated illumination-microscope was compared with confocal laser scanning and electron microscopes and found to be suitable for measuring the size of cellular nanostructures in a biological setting. The hyperphosphorylated form of polymerase II was found in structures with a diameter of approximately 70 nm, well below the 200-nm resolution limit of standard fluorescence microscopes.
空间调制照明荧光显微镜理论上能够测量直径在10至200纳米之间的物体大小，并且已实现对亚分辨率荧光微珠（约40 - 100纳米）的精确尺寸测量。迄今为止，这个尺寸范围内的生物结构一直是通过电子显微镜进行测量的。在这里，我们使用抗体H5标记了HeLa细胞核中含有活性、高度磷酸化形式的RNA聚合酶II的位点。将空间调制照明显微镜与共聚焦激光扫描显微镜和电子显微镜进行了比较，发现它适用于在生物环境中测量细胞纳米结构的大小。发现聚合酶II的高度磷酸化形式存在于直径约为70纳米的结构中，远低于标准荧光显微镜200纳米的分辨率极限。
