Comparisons of Techniques - Sample Preparation Topic for Transmission Electron Microscopy - page 206

Fig. 8.12 Metallic rotary
shadowing under a
unidirectional angle of 45 ◦ ,
observed in bright-field and
inverted image mode (f =

flagella). ( A. Ryter, Institut
Pas t eur Par i s )

2.4 Comparison of “Positive-Staining” and Decoration-Shadowing
Contrast Techniques

2.4.1 Positive-Staining (“Techniques” Chapter 7, Section 2)
and Decoration-Shadowing (“Techniques” Chapter 7, Section 1)
Contrast Techniques

Fine particle material: relaxed circular DNA molecules (pBR 322 plasmid,
4,361 bp)

Comparison discussion : The observation of DNA molecules requires the spread-
ing of the macromolecule, which is negatively charged. Historically, the first type
of DNA spreading was done using the cytochrome c technique, which forms a thin
film on the surface of an aqueous hypophase and complexes with the DNA. There
are several variants of this method (see fine particle dispersion technique). Next,
the spread DNA can be shadowed or positively stained in order to be viewed. The
contrast depends on these methods and the observation in bright-field or dark-field
mode. For dark-field imaging, we can make a tilted dark field, or use the electron
energy loss, either by using elastically transmitted electrons or on the uranium peak
(Figs. 8.13 and 8.14) .
The second type of DNA spreading uses the technique of a preliminary coating
of a pentylamine film (Dubochet method) on the carbon grid, followed by positive
staining using a uranyl acetate rinse. Positive staining can highlight the conforma-
tion of the DNA. This type of preparation can also be metalized i.e., shadowing as
described above (Fig. 8.14) .
Both techniques, “shadowing after spreading of cytochrome c and positive stain-
ing,” yield the same information on the conformation of the DNA, with contrasts
and resolutions depending on the sharpness of the shadowing and on the observation
mode.