Biomedical Engineering Reference
In-Depth Information
Isolated cells or monolayer cells, as well as isolated organelles (mitochondria,
DNA, RNA, etc.) and viruses are very easy to fix (cases 1 and 3).
Bacteria and yeasts, as well as certain protozoa, are unicellular organisms,
endowed with walls that make penetration by liquids more difficult (case 2). It is
often necessary to dissolve these membranes using enzymatic digestion.
Cells are usually combined with one another to form a tissue, and often several
tissues are combined to form an organ (case 4). The connective tissue that is often
associated is a water trap that will require increased dehydration time.
Plant cells are surrounded with a relatively complex and impermeable wall
(case 5). These walls are often poorly permeable and necessitate allowing longer
action times than for animal cells. Animal and plant cells can also contain air
(case 6). Air must be replaced under vacuum by the fixation liquid.
Support tissues such as cartilage, bone, tooth, cuticle will present difficulties for
the embedding. It is often necessary to soften these tissues by enzymatic or chemical
digestion (cases 7 and 7a).
Very small organisms (worms, insects, parasites, and plant nodules) have imper-
meable, rigid membranes in which one must make openings using microdissection
in order to ensure penetration of the preparation liquids (case 8).
The preparation protocol must be adapted each time to the different types of cells
or organisms to be dealt with.
6.2 Dehydration Principles
As seen earlier, dehydration consists of gradually replacing water with a solvent.
This solvent must be miscible with both water and the embedding resin. Sometimes,
two successive solvents must be used, the first one miscible with water and the
second miscible with the first and the resin.
These solvents are alcohols, acetone, or aromatic compounds (1,2 epoxy,
propane, styrene, etc.).
Dehydration is performed gradually in successive baths of increasingly concen-
trated solvents; it is completed in baths of pure solvent.
The solvent (alcohol or acetone) is harmful to organic molecules. It coagulates
proteins and dissolves lipids, unless chemical fixation is carried out beforehand.
6.3 Infiltration Principles
Infiltration consists of replacing a liquid or gaseous phase within a material with
a liquid polymer that will then be hardened to produce a sample of homogeneous
hardness.
In the case of porous materials, resin will replace the air. If the porosity is coarse,
the air is replaced directly with resin under a primary vacuum. If the porosity is fine,
the air is replaced under vacuum with a solvent or water and then substitution baths
are used as in the case of hydrated samples.
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