Physical and Chemical Mechanisms of Preparation Techniques - Sample Preparation Topic for Transmission Electron Microscopy - page 121

structure appears bright, revealing its form, dimensions, and external ornamentation.
This staining is not followed by any washing, which would totally eliminate the
stain. This is a purely physical reaction of the adsorption of a chemical compound
(the stain) on the support film. The stain in solution is ionized and binds to the
support film or the sample surface because of its electrical charges. The intensity
of this contrast will depend on the abundance of electrical charges on the sample
surface and the surface of the support film. It also depends on the spatial size of the
staining ion. For a material in aqueous solution, the stain penetrates the polar regions
of the sample and replaces the water, helping to view very fine details on its surface.
The stain is selected based on the sample and the structures to be highlighted. It is
placed in solution with a specific pH that will induce its dissociation and therefore
its electrical charges.
Negative staining has the advantage of protecting the sample from the drawbacks
of desiccation. The stain evaporates faster than the sample and creates an envelope
of stain around it, which maintains a certain degree of humidity.
There is a risk of crushing the sample on the support, increasing its dimensions.
By chemically fixing the material beforehand and/or working with low electron
doses, this phenomenon can be minimized.
The stains used are salts of heavy metals such as uranium, tungsten, and molyb-
denum. They are selected for their high water solubility in order to prevent the
formation of crystals around the sample when drying. Their good stability under
the electron beam depends on their boiling point, which preferably will be high.
For tungsten ions obtained using phosphotungstic acid, the ion size is from 0,8 to
1,5 nm. For uranyl ions, the size is ranging from 0,4 to 0,5 nm.
Uranyl can be used in the form of an acetate, nitrate, or formate.
It is also used for “positive-staining” contrast. Note that in negative staining, a
fraction of positive reactions may occur. To minimize this possibility, work is done
at an acidic pH (less than 5); moreover, for a pH greater than 6, uranyl is unstable.
Furthermore, uranyl has a known role as a fixative, on lipids in particular, but its
mechanism is poorly understood.
Tungsten is most often used in the form of a potassium salt or sodium salt of
phosphotungstic or silicotungstic acid.
Ammonium molybdate seems to perform better than the previous stains for
revealing the structural details of membranes. It would also be less toxic and would
reversibly inhibit metabolic activities (Fig. 5.25) .

a b c d

Fig. 5.25 Different stain-distribution possibilities based on the respective polarities of the support
film and the sample surface: ( a ) polar sample and film, ( b ) polar sample and non-polar film, ( c )
non-polar sample and polar film, and ( d ) non-polar sample and film