Introduction to Materials - Sample Preparation Topic for Transmission Electron Microscopy - page 25

of evolution, living organisms (both plants and animals) are very complex and var-
ied. Despite this, the basis of organization on the structural scale remains simple:
the cell.
The use of an electron beam propagating inside the vacuum of a TEM repre-
sents a dual difficulty with regard to the observation of biological structures: the
sensitivity of organic molecules to electron irradiation and the need to eliminate
water before observation. These problems require complex preparations, leading
to the complete shutdown of the vital process. The challenge with these observa-
tions follows the same principle as in any other investigation conducted at any given
moment: the researcher obtains precise but incomplete information that does not
take constant remodeling into account. Information must often be supplemented by
kinetic studies and coupled with biochemical studies. The second difficulty comes
from the fact that certain chemical systems do not shut down instantaneously upon
the death of an organism. These systems continue to function and they deteriorate
the structure before it can be observed in its natural state. In this case, fixation, the
step that stops biochemical activities, must be especially fast and effective at the
system core in order for it to be studied properly.
Structure has no meaning only insofar as one or more functions associated with
it can be recognized. The approach to synthetic phenomena or the degradation
of cellular compounds couples observations with labeling-preparation techniques
(labeling using gold or platinum particles functionalized for specific enzymatic
sites).
In TEM, biological research is conducted at three levels: (1) on the microstruc-
tures of protein or nucleic macromolecules; (2) on cell infrastructures, their internal
organization, their interconnections, and their relationship with their immediate
surroundings; and (3) on the microstructures that correspond to tissue organization.
In the first case, isolation and concentration techniques for a macromolecule
made up of cells (proteins or nucleic acids) are used to infer its 3D structure using
mathematical reconstruction. Their dimensions are too small to enable them to be
characterized directly. This constitutes structural biology.
In the second case, structural recognition is used to deduce their biological func-
tions, metabolic or catabolic activities, and energetic reactions. By investigating
these morphologies in the normal state compared to the morphology of pathologi-
cal tissues, it is possible to deduce the damage caused by these pathologies, define
their causes, and select the appropriate treatments. It is also possible to determine,
at the cellular scale, the action of medicines used, their sites of action, and possible
damaging effects. The study of virus or bacteria microstructures enables an initial
approach to determine these microorganisms' families. Recognizing them within a
tissue can help to confirm a diagnosis and identify their means of attacking cells,
method of replication, and dispersion within the tissue. This constitutes cytology.
A cell must always be considered in its immediate surroundings, i.e., observing
the other cells with which it makes contact, thereby creating tissue organizations
that have a particular function. It is highly dependent on other cells, which first
influence its differentiation, then its activities and therefore its role. All these lead to
diverse and varied microstructures. Cells are bathed in a common milieu intérieur ,