Abstract
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.
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Engelhardt, P., Meriläinen, J., Zhao, F., Uchiyama, S., Fukui, K., Lehto, VP. (2007). Whole-Mount Immunoelectron Tomography of Chromosomes and Cells. In: Kuo, J. (eds) Electron Microscopy. Methods in Molecular Biology™, vol 369. Humana Press. https://doi.org/10.1007/978-1-59745-294-6_19
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DOI: https://doi.org/10.1007/978-1-59745-294-6_19
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