Physical Principles of Electron Microscopy: An Introduction to TEM ...

102 Chapter 4
whose composition or thickness varies between different regions. Thicker
regions of the sample scatter a higher fraction of the incident electrons, many
of which are absorbed by the objective diaphragm, so that the corresponding
regions in the image appear dark, giving rise to thickness contrast in the
image. Regions of higher atomic number also appear dark relative to their
surroundings, due mainly to an increase in the amount of elastic scattering,
as shown by Eq. (4.15), giving atomic-number contrast (Z-contrast). Taken
together, these two effects are often described as mass-thickness contrast.
They provide the information content of TEM images of amorphous
materials, in which the atoms are arranged more-or-less randomly (not in a
regular array, as in a crystal), as illustrated by the following examples.
Stained biological tissue
TEM specimens of biological (animal or plant) tissue are made by cutting
very thin slices (sections) from a small block of embedded tissue (held
together by epoxy glue) using an instrument called an ultramicrotome that
employs a glass or diamond knife as the cutting blade. To prevent the
sections from curling up, they are floated onto a water surface, which
supports them evenly by surface tension. A fine-mesh copper grid (3-mm
diameter, held at its edge by tweezers) is then introduced below the water
surface and slowly raised, leaving the tissue section supported by the grid.
After drying in air, the tissue remains attached to the grid by local
mechanical
and chemical forces.
Tissue sections prepared in this way are fairly uniform in thickness,
therefore almost no contrast arises from the thickness term in Eq. (4.15).
Their atomic number also remains approximately constant (Z � 6 for dry
tissue), so the overall contrast is very low and the specimen appears
featureless in the TEM. To produce scattering contrast, the sample is
chemically treated by a process called staining. Before or after slicing, the
tissue is immersed in a solution that contains a heavy (high-Z) metal. The
solution is absorbed non-uniformly by the tissue; a positive stain, such as
lead citrate or uranyl acetate, tends to migrate to structural features
( organelles) within each cell.
As illustrated in Fig. 4-6, these regions appear dark in the TEM image
because Pb or U atoms strongly scatter the incident electrons, and most of
the scattered electrons are absorbed by the objective diaphragm. A negative
stain (such as phosphotungstic acid) tends to avoid cellular structures, which
in the TEM image appear bright relative to their surroundings, as they
contain fewer tungsten atoms.