Whole-mount immunoelectron tomography of chromosomes and cells

Methods Mol Biol. 2007:369:387-405. doi: 10.1007/978-1-59745-294-6_19.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cells / ultrastructure
  • Chick Embryo
  • Chromosomes / ultrastructure*
  • Chromosomes, Human / ultrastructure
  • Gold Colloid
  • HeLa Cells
  • Humans
  • Imaging, Three-Dimensional / methods*
  • Microscopy, Immunoelectron / methods*
  • Tomography / methods

Substances

  • Gold Colloid