Whole-mount Immunoelectron Tomography of Chromosomes and Cells.
From: Haartman Institute, Department of Pathology and Virology, Universityof Helsinki, Finland.
Methods in molecular biology (Clifton, N.J.)
- Publish Date: 2007
- ISSN: 1064-3745
- Volume: 369
- Issue:
- Pages: 387-405
- Medium: Print
- Language: English
- Citation (JAMA): Engelhardt Peter, Meriläinen Jari, Zhao Fang, et al. Whole-mount Immunoelectron Tomography of Chromosomes and Cells.. Methods Mol. Biol. 2007;369:387-405
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.
Mesh Headings (Keywords): Animals, Cell Line, Cells, Chick Embryo, Chromosomes, Chromosomes, Human, Gold Colloid, Hela Cells, Humans, Imaging, Three-Dimensional, Microscopy, Immunoelectron, Tomography
Check for Full Text / PubMed Unique Identifier (PMID): 17656761
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