Biomedical Engineering Reference
In-Depth Information
Many techniques require preparing dry, flat or thin specimens for characterization.
Preparing these without introducing artifacts is a challenge because biomass is naturally
wet and fibrous. Confirmation from multiple independent techniques is often necessary
before one can be confident that the observation is not an artifact of specimen prepara-
tion. We suggest an excellent review of microscopic techniques used to probe cell wall
structure (57).
3.4.1.1
Drying Issues
Although drying invariably changes biomass structure, understanding the nature of those
changes can minimize the impact and guide interpretation of results. Analysis of biomass
in the wet state is usually preferred, but some analytical techniques require high vacuum.
Even the simple task of getting the dry weight of a specimen can ruin the specimen
for other analyses, as the act of drying causes irreversible changes in cell wall nano-
structure.
Several methods have been used to preserve structures during water removal, and many
topics are devoted to the techniques, as biological specimens for electron microscopy
have always faced this problem (58, 59). Two principal mechanisms cause specimen
alteration during drying: changing solvent properties and surface tension. Although
surface tension can be avoided, biomass structures surrounded by air or a different
solvent have a different energy, and so have different stability, than in water. A stable
molecular conformation in water could be unstable after water removal. Therefore, even
the best water removal methods have the potential to modify specimens.
The simplest drying strategy is simply to let water evaporate, for example in an oven
at 105 C. This is an easy method but causes the pores to collapse from surface tension.
To avoid this, specimens are either frozen while wet or the solvent is exchanged. Solvent
exchange is often followed by critical point drying or embedding.
In freeze drying, specimens are frozen while wet and placed under vacuum so that ice
sublimes, completely avoiding surface tension. Standard freezing techniques cause ice
crystal formation that partially dehydrates the specimen, causing some fiber shrinkage,
collapse, and artifacts from crystal formation (60). Because of the interaction with
wood polymers, the freezing point of some bound water is as low as
30 C (31),
confirming empirical observations that specimens must be kept extremely cold throughout
the entire freeze-drying process. Partial freeze drying is also common, especially in
electron microscopies with a cold stage. In this technique, some surface water is allowed
to sublime by heating the specimen to ca.
80 C, and then the specimen is cooled again
to prevent further water loss during imaging.
Water can also be removed by deep freezing followed by flooding the specimen with
dry acetone or ethanol (59, 61). This is often followed by embedding procedures, carried
out at low temperature up to the point of polymerizing the embedding medium.
Under proper conditions, damage by ice crystal formation can be avoided by producing
vitreous ice (ice without crystals). Vitreous ice is formed when cooling rates approach
10 6 C/s, 61 and the water is diluted with 10-15% of a solute such as sucrose (62, 63).
Samples kept at high pressure during freezing can have vitreous layers ca.
10 times
thicker than samples frozen at atmospheric pressure (200
m) (62). Fast
freezing is achieved by placing thin specimens in contact with liquids or plates cooled
+
vs.
20
µ
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