24 July, 2012
IAWS 2011, Stockholm, August 31 to September 2
Chemical and Cryo-methods for assessing hardwood pulp fibre ultrastructure
Prabashni Lekha(1,2), Tamara Bush(1,3), Patricia Berjak (2), Norman Pammenter (2)
(1)CSIR/UKZN, Forestry and Forest Product Research Centre, Natural resources and
environment, P.O. Box 17001, Congella, 4013, South Africa, (2)University of KwaZulu-
Natal(UKZN), School of Biological and Conservation Sciences, Durban, 4041, South
Africa. (3)UKZN, School of Chemistry, Durban, 4041, South Africa.
Accurate imaging and analysis of the fibre wall ultrastructure depends extensively on the sample preparation method used (Chinga-Carrasco, 2010). Various techniques and methods are available for sample preparation but their use is limited by the sensitivity to the specimen. Biological material, including wood and pulp fibres constitute the most difficult specimens to prepare for high resolution microscopy because of the complex
and intricate structural detail.
For the production of novel materials from wood and cellulose, it is important to understand how different processes affect fibre wall ultrastructure. The approach used in this study was to use different sample preparation techniques that would obviate artefact induction during sample preparation to elucidate ‘true' ultrastructural detail. Most preparation techniques require a series of steps to enable final processing, e.g. resin embedding of pulp fibres requires a preceding dehydration step, which can be artefact inducing, e.g. shrinkage of the fibre wall.
Unbleached Eucalyptus fibres were used as the experimental specimen. For the chemical preparation of fibres, a serial dilution of acetone solutions (30, 50, 70, and 100%) was used to dehydrate the fibres followed by slow infiltration with Spurr's epoxy resin (Spurr, 1969). Following polymerisation, different thickness (0.5 - 2 μm) of resin sections were cut using an ultra-microtome and adhered to glass slides coated with Haupt's adhesive (Haupt, 1930). Thereafter the sections were etched with potassium methoxide for 3 min and were sonicated with methanol for 2 min. For cryofracturing, the fibres were frozen in liquid nitrogen and fractured with a super-cooled steel blade. Thereafter, the fibre fractures were freeze dried and mounted onto carbon stubs. Cryosectioning involved the pre-treatment of fibres with a range of cryo-protectant solutions; thereafter the fibres were mounted onto a steel pin and frozen in liquid nitrogen. The sample was then transferred into the gas chamber of a cryo-microtome (Reichert-Jung, Austria) and sections (100 - 300 nm) were collected on formvar-coated grids. For all sample preparation methods the fibres were sputter coated with carbon and then viewed at 2 kV by use of a Carl Zeiss Ultra FEG-SEM.
The cryo-sectioning protocol of Tokayasu (1980) involves the pre-treatment of the specimen in 2.3 M sucrose. When applied to Eucalyptus unbleached fibres, crevices were observed across the fibre wall. High quality pulp fibre cross-sections (Fig. 1a) were obtained when 1.5 mM polyvinylpyrrollidone (PVP) was applied for 30 min prior to cryo-sectioning. The ultrastructural detail observed for cryo-sectioned fibres was superior to that observed for acetone dehydrated resin embedded sections that had been etched with potassium methoxide (Fig. 2a). The ultrastructure of the cellulose microfibrils were obscured with the latter method (cf. Fig. 1b and 2b). Cryo-fracturing of fibres proved to be inconsistent for producing high-quality cross-sections.
The level of ultra-structural detail obtained using cryo-sectioning is superior to that obtained using cryo-fracturing (cf. Fig. 1 and 3). Cryo-sectioning of hardwood pulp fibres can prove to be extremely beneficial in the assessment of mechanical and chemically treated fibres.
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Microscopy: Science, Technology, Applications and Education, p. 2182.
- Haupt AW (1930): A gelatin fixative for paraffin sections. Biotechnic and Histochemistry 5, p. 97.
- Spurr AR (1969): A low-viscosity epoxy resin embedding medium for electron microscopy. Journal of Ultrastructure Research 26, p. 31.