Physical and Chemical Mechanisms of Preparation Techniques - Sample Preparation Topic for Transmission Electron Microscopy - page 104

Table 5.1 Ice vapor pressure
as a function of temperature

Temperature (K) Ice vapor pressure (mmHg)

183 0.000070

203 0.00194

213 0.00808

233 0.0966

253 0.776

263 1.950

273 4.579

6 Actions Resulting in a Change in Material Properties

Certain techniques require the use of chemical compounds intended to stabilize the
biological material. They create chemical bonds between the biological material and
the additional chemical compound that definitively modify the chemical composi-
tion of the material. This is the case for chemical fixation and “positive-staining”
contrast.
Other techniques result in changes in the material's mechanical behavior during
preparation. This is the case for infiltration and embedding. They require the use of
polymer resins that will be intimately bound to the sample, around it in the case of
embedding or infiltration. For hydrated samples, this step is preceded by dehydration
if embedding takes place in non-polar resins.

6.1 Chemical Fixation Principles

Living matter can be considered as a very hydrated protein gel in which ions, sugars,
lipids, amino acids, nucleic acids, etc. constantly move.
Chemical fixation consists of bridging the proteins, so as to create an artificial
cross-linking of the gel by forming long insoluble chains in order to eliminate the
water, while preserving the original structural network. What follows is a denaturing
of the proteins and a loss of all of the small diffusible elements. By denatur-
ing the proteins, chemical fixation blocks the enzymatic systems, preventing any
subsequent deterioration of the specimen through autolysis.
The fixatives used are aldehydes (paraformaldehyde and glutaraldehyde) or pow-
erful oxidants (osmium tetroxide, ruthenium tetroxide, etc.). The fixative binds to
the protein by an addition reaction at the level of the double bonds or alcohol,
aldehydes, or hydroxyl groupings.
Glutaraldehyde is a dialdehyde with the formula OHC

(CH 2 ) 3 COH. It can react
twice with the H + 's and create bridges between the molecules.
Paraformaldehyde is a monoaldehyde of an indefinite formula (CH 2 O) n ( n

−

=

8-100), because its degree of polymerization is variable, explaining the n index.
Aldehydes are good fixatives for proteins and nucleic acids. Glutaraldehyde can
result in artificial bridges between proteins and free amine groups through the addi-
tion phenomenon. It also causes a change in protein spatial conformation, which