Comparisons of Techniques - Sample Preparation Topic for Transmission Electron Microscopy - page 223

Physical method: cryofixation (“Techniques” Chapter 2, Section 12) and substi-
tution, infiltration, and embedding in cryogenic mode (“Techniques” Chapter
2, Section 10)
Ultramicrotomy (“Techniques” Chapter 4, Section 4) Positive-staining contrast
(“Techniques” Chapter 7, Section 3)
Bulk material: liver cell (Figs. 8.41, 8.42, and 8.43)

Comparison discussion : The liver cell can be well identified using all three tech-
niques. The membranes are different depending on the case. They are darkened by
the presence of osmium in the chemical fixation and are very well marked in the
nuclear membrane as well as the rough endoplasmic reticulum and mitochondria
(Fig. 8.41) . With cryofixation, the membranes are not as visible as such, but they
are marked because they are highlighted by the surface glycoproteins, especially at
the junction between two cells. Along the cell membranes (M1 and M2), a desmo-
some before the biliary canaliculus (c) can be distinguished. The nuclear membrane
is not visible in the nucleus, and the placement of the nuclear pores is marked by
the absence of heterochromatin (hC) in the nucleus at this level. The mitochondrial
crests are dark by inverting the contrast, the membranes are bright, and the matrix
is dark (Fig. 8.42) .

Fig. 8.41 Chemical fixation,
substitution-impregnation,
and embedding in epoxy resin
at room temperature,
ultramicrotomy and positive
contrast (uranyl in alcohol
solution + lead); hC

=

heterochromatin, L = lipidic

phase, m = mitochondria,

N rough

endoplasmic reticulum, black
arrows

= nucleus, r =

nuclear pores. ( J.
Boumendil UCB-Lyon 1 )

=

Freeze fracture penetrates the membrane in the double phospholipidic layer.
Thus, in the nucleus, the external face (Fe) and the internal face (Fi) of the nuclear
envelope can be seen, as well as the nuclear pores resembling buttons on the nucleus.
They constitute a kind of canaliculi across the double nuclear membrane. This type
of image helps to provide a good approach to the distribution of nuclear pores on a
cell nucleus to take counts and to establish relative proportions (Fig. 8.43) .
The chemical method is particularly used for investigating structure and for
immunolabeling. The freeze-fracture technique is used for 3D viewing and the
approach to intramembrane structures.