Engelhardt Peter, Meriläinen Jari, Zhao Fang, Uchiyama Susumu, Fukui Kiichi, Lehto Veli-Pekka
Haartman Institute, Department of Pathology and Virology, Universityof Helsinki, Finland.
Methods Mol Biol. 2007;369:387-405. doi: 10.1007/978-1-59745-294-6_19.
Standard immunogold-labeling methods in transmission electron microscopy (TEM) are unable to locate immunogold particles in the depth direction. This inability does not only concern bulky whole mounts, but also sections. A partial solution to the problem is stereo inspection. However, three-dimensional reconstruction of immunogold-labeled structures, that is, immuno-electron tomography (IET), is a correct solution for this inconsistency. Striking improvement in resolution is achieved: the 1.4-nm immunogold particles are shown in IET that are not detected in the original tilt series. IET is not restricted to laboratories with advanced medium- or high-voltage TEM and super-computing facilities; the methods we have developed for whole-mounted chromosomes and also for whole-mounted cytoskeleton of fibroblasts work remarkably well with ordinary 80-kV TEMs equipped with a goniometer to collect tilt series for IET on film. In addition, free programs are available to produce three-dimensional reconstructions even without high-performance computers. These improvements make it possible to many laboratories without modern facilities to perform IET reconstruction with standard TEM apparatus.
透射电子显微镜（TEM）中的标准免疫金标记方法无法在深度方向定位免疫金颗粒。这种无能不仅涉及庞大的整装标本，也涉及切片。该问题的部分解决方案是立体检查。然而，免疫金标记结构的三维重建，即免疫电子断层扫描（IET），是解决这种不一致性的正确方法。分辨率有了显著提高：在IET中显示出了原始倾斜系列中未检测到的1.4纳米免疫金颗粒。IET并不局限于拥有先进的中压或高压TEM以及超级计算设施的实验室；我们为整装染色体以及成纤维细胞的整装细胞骨架开发的方法，在配备用于在胶片上收集IET倾斜系列的测角仪的普通80 kV TEM上效果非常好。此外，即使没有高性能计算机，也有免费程序可用于生成三维重建。这些改进使得许多没有现代设施的实验室能够使用标准TEM设备进行IET重建。
