Electron Tomography of Chromosome Structure
Authors: Engelhardt Peter 
Published: September 2006

Abstract
Three‐dimensional (3‐D) structures at the molecular level of chromosomes and other cell structures are far beyond the reach of ordinary or confocal (3‐D) light microscopy (LM). The electron tomography method (ETM), i.e. transmission electron microscopy (TEM) tomography, is able to provide methods of 3‐D reconstruction of macromolecular assemblies, providing insights into the qualitative and quantitative spatial comprehension of macromolecular structures. ETM is a powerful method for developing reliable immunoelectron tomography (IET) and in situ hybridization analysis at the molecular level.
With ETM the data are collected with TEM by taking tilt series, i.e. projection images from different viewing angles over the object. By combining such projections taken over a sufficient angular range (from 0 to ±60° manually, e.g. in 3° increments), a wholly transparent 3‐D representation of the object is recovered with necessary details. This requires the use of suitable computational methods.
Automatic single‐axis tilt‐series data registration is possible today that will not only allow the collection of data with increments as small as 1°, for high‐resolution reconstructions of bulky whole mounts, but will also protect the specimen from electron beam damage (dose reduced by a factor of 10–100). Only automation of ETM procedures makes it possible to collect tilt series of cryo‐electron microscopy (EM) preparations of unfixed and unstained preparations in vitrified ice, i.e. fully hydrated samples close to the native state.
Novel methods and tools for electron tomography are available that have been developed in different laboratories. Our electron tomography programs are automated to a high degree and therefore very fast and easy to use, in comparison with other program kits that are available. Specifically, our arsenal includes advanced tomography procedures based on the maximum entropy method (MEM) (e.g. 16‐bit gray‐scale MEM).
A collection of preparative methods for preserving whole‐mounted chromosomes and cells for ETM studies has been developed. Examples of 3‐D reconstructions of human chromosomes are shown at high resolution (3–15 nm).
It is considered that ETM allows 3‐D reconstructions, depending on the thickness of the preparation, with a resolution in the range 2.5–8.5 nm. In this respect, some success was recently made as we were able to detect nanoprobes (1.4‐nm immunogold markers) with the IET methods developed.
ETM of chromosomes involving localized genes with in situ hybridization and gold‐labeling techniques is possible and would allow chromosome mapping in a 3‐D configuration with molecular resolution much more accurately than any standard EM method which are affected by superimposing gold labels with their accompanying structures.
Concerning the structure and folding of the DNA in chromosomes, it is important to note that the linear sequence of bases in DNA is accompanied by higher level organizations, composed of loops, twists and folds in the long DNA chain, and, significantly, by DNA–protein interactions, e.g. nucleosomes, supranucleosomes, together with telomerase, topoisomerases and the new scaffold proteins [structural maintenance of proteins (SMC) family] recently described. These so‐called ‘higher order structures’ are certainly very much involved in the regulation of gene expression in chromosomes. Further, it is easy to realize that genes distantly located in the linear sequence map could in fact be topologically associated when DNA is folded in chromosomes, and thus mutually regulated. Sequencing of the genomes does not include and cannot reveal higher order structures. It is only with ETM that genome maps can be brought in relation to 3‐D molecular configurations, and it can be refined by combination with in situ hybridization to localize gene sequences and with IET to identify chromosomal proteins.