Scanning electron microscope studies of human metaphase chromosomes

Abstract

Scanning electron microscopy (SEM) is used to evaluate potential chromosome preparations and staining methods for application in high-resolution three-dimensional X-ray imaging. Our starting point is optical fluorescence microscopy, the standard method for chromosomes, which only gives structural detail at the 200 nm scale. In principle, with suitable sample preparation protocols, including contrast enhancing staining, the surface structure of the chromosomes can be viewed at the 1 nm level by SEM. Here, we evaluate a heavy metal nucleic-acid-specific stain, which gives strong contrast in the backscattered electron signal. This study uses SEM to examine chromosomes prepared in different ways to establish a sample preparation protocol for X-rays. Secondary electron and backscattered electron signals are compared to evaluate the effectiveness of platinum-based stains used to enhance the contrast.

Keywords: chromosomes; layer structure; scanning electron microscopy.

Figure 1.

(a) SE image of a chromosome prepared without Cytoclear and the layer surrounding it. (b) SE image of chromosome in (a) at 80 000× magnification. (c) SE image of a chromosome prepared with Cytoclear at 80 000× magnification. (d) SE image of the mesh layer around the chromosome in (c) at 80 000× magnification.

Figure 2.

Histograms of the globular subunit diameter (a) of a chromosome prepared without Cytoclear, (b) in a chromosome prepared with Cytoclear and (c) in the mesh layer surrounding the chromosome prepared with Cytoclear. (Online version in colour.)

Figure 3.

(a) BSE and (b) SE images of unstained human chromosomes. (c) BSE and (d) SE images of human chromosomes stained with platinum(II)bis(acetamide) complex.

Figure 4.

Phase shift from the chromosome in figure 3c unstained (dashed) and stained with platinum atoms based on the binding ratio of the DNA-specific stain chloro(2,2′:6′,2′′-terpyridine)platinum(II) chloride (dotted). For comparison, a stain with a binding ratio of one platinum ion per base pair is plotted (solid). Calculated with the CXRO online tool (X-ray optical constants data from http://henke.lbl.gov/optical_constants/) [22].