ASSESSING HARDWOOD PULP FIBRE ULTRASTRUCTURE
chemical and cryo-methods for assessing hardwood pulp fibre ...
chemical and cryo-methods for assessing hardwood pulp fibre ...
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CHEMICAL AND CRYO-METHODS FOR<br />
<strong>ASSESSING</strong> <strong>HARDWOOD</strong> <strong>PULP</strong> <strong>FIBRE</strong><br />
<strong>ULTRASTRUCTURE</strong><br />
PRABASHNI LEKHA 1,2 , TAMARA BUSH 1,3 PATRICIA BERJAK 2 AND NORMAN PAMMENTER 2<br />
1<br />
CSIR/UKZN, Forestry and Forest Products Research Centre, 2 School of Biological and Conservation Sciences, 3 School of Chemistry, UKZN, Durban, South Africa.<br />
INTRODUCTION<br />
• Various techniques and methods are available for sample preparation but their use is limited by the sensitivity to the<br />
specimen.<br />
• Biological material, including wood and pulp fibres, constitute the most difficult specimens to prepare for high resolution<br />
microscopy because of the complex and intricate structural detail.<br />
• Accurate imaging and analysis of the fibre wall ultrastructure depends extensively on the sample preparation method used.<br />
• For the production of novel materials from wood and cellulose, it is important to understand how different processes affect<br />
fibre wall ultrastructure.<br />
• This study used chemical and cryo-methods to obviate artefact induction during sample preparation aimed at elucidating<br />
‘true’ ultrastructural detail.<br />
CHEMICAL SAMPLE PREPARATION<br />
MATERIALS AND METHODS<br />
A<br />
Fibres were<br />
dehydrated in<br />
acetone (30, 50,<br />
75, 100%) for<br />
1 h<br />
CRYO SAMPLE PREPARATION (A) CRYO-FRACTURING, (B) CRYO-SECTIONING<br />
Fibres were<br />
loosened and<br />
strung out onto<br />
pieces of foil<br />
Prolonged<br />
infiltration with<br />
low viscosity<br />
epoxy resin<br />
for 3 days<br />
Fibre clumps<br />
were then<br />
submerged in<br />
supercooled<br />
liquid nitrogen @<br />
-210°C<br />
Fibres were<br />
placed in 100%<br />
fresh resin and<br />
polymerised for<br />
8 h at 70°C<br />
A sharp metal<br />
blade cooled<br />
with liquid<br />
nitrogen was<br />
then used to<br />
fracture the fibre<br />
clumps<br />
Fractured fibres<br />
were then freeze<br />
dried using a<br />
freeze drier<br />
RESIN<br />
BLOCK<br />
Polished block face<br />
post microtoming<br />
Glass cover slip<br />
coated with Haupt’s<br />
adhesive<br />
0.5 - 2 µm thick resin<br />
sections were tested<br />
Freeze dried<br />
fractured fibres<br />
were then<br />
mounted onto<br />
carbon stubs<br />
using carbon<br />
fluid<br />
Resin block face<br />
and sections<br />
were etched with<br />
potassium<br />
methoxide for<br />
1 min to dissolve<br />
resin<br />
Sonicated with<br />
methanol for<br />
2 min<br />
to remove<br />
remaining resin<br />
from tissue<br />
All sample preparations were sputter<br />
coated with carbon and then viewed at<br />
2 - 10 kV by use of a Carl Zeiss Ultra<br />
FEG-SEM<br />
Resin blocks and<br />
etched sections<br />
on cover slips<br />
were left to dry<br />
under a fume<br />
hood<br />
Resin blocks,<br />
cover slips and<br />
formvar grids<br />
were mounted<br />
onto metal stubs<br />
with carbon tape<br />
Chemical<br />
Concentration<br />
B<br />
Fibres were<br />
loosened and<br />
treated with<br />
various<br />
cryoprotectant<br />
solutions for<br />
different times<br />
30 min – 24 h<br />
1-hexadecen<br />
sucrose<br />
glycerol<br />
polyvinylpyrrollidone (PVP)<br />
PVP<br />
PVP<br />
3.5 M<br />
2.3 M<br />
10.6 M<br />
0.5 mM<br />
1 mM<br />
1.5 mM<br />
1. CHEMICAL SAMPLE PREPARATION TECHNIQUES<br />
Small fibre<br />
clumps were<br />
then adhered to a<br />
metal pin using<br />
tissue adhesive<br />
and was<br />
subsequently<br />
frozen in liquid<br />
nitrogen<br />
RESULTS<br />
The pin was<br />
transferred into<br />
the gas chamber<br />
of a cryomicrotome<br />
(Reichert-Jung,<br />
Type 652701,<br />
Austria)<br />
500 nm cryosections<br />
were<br />
obtained using a<br />
cutting speed<br />
between 0.4 to<br />
0.8 mm sec -1 and<br />
the sample was<br />
at 80°C<br />
Unbleached Eucalyptus pulp fibres were used throughout.<br />
The sections<br />
were transferred<br />
using a metal<br />
loop and 1.5 mM<br />
PVP to grids<br />
coated with<br />
formvar on which<br />
carbon had been<br />
deposited<br />
a<br />
b<br />
c<br />
lumen<br />
S2 layer<br />
S2 layer<br />
10 µm<br />
0.1 µm<br />
0.2 µm<br />
Figure 1. (a), (b) and (c) etched resin block showing fibre cross-sections.<br />
The etching of resin blocks resulted in limited ultrastructural detail being observed. The cross-sectioned fibre surface was<br />
fused and there was no observable detail of the microfibrils within the different layers (Figs 1b and c).
a<br />
b<br />
RESULTS cont.<br />
S1 layer<br />
c<br />
S2 layer<br />
S2 layer<br />
S3 layer<br />
10 µm<br />
0.4 µm<br />
0.2 µm<br />
Figure 2. (a), (b) and (c) etched 1.5 µm thick resin sections of Eucalyptus pulp fibres.<br />
Etching of resin-embedded sections of different thickness on glass cover slips showed more ultrastructural detail compared<br />
with etched resin blocks, 1.5 µm thick sections being the best (cf. Figs 1 and 2). However, microfibril ultrastructure was<br />
obscured within the fibre wall when using chemical sample preparation techniques (Fig. 2c).<br />
2. CRYO-SAMPLE PREPARATION TECHNIQUES<br />
a<br />
b<br />
S1 layer<br />
c<br />
S1 layer<br />
S2 layer<br />
S2 layer<br />
S2 layer<br />
S2 layer<br />
S1 layer<br />
S1 layer<br />
lumen<br />
2 µm<br />
0.2 µm<br />
0.4 µm<br />
Figure 3. (a), (b) and (c) cryo-fractures of Eucalyptus pulp fibres without pre-treatment.<br />
Cryo-fracturing Eucalyptus pulp fibres did not result in proper cross-sections (Fig. 3a). The fractured surface curled obscuring<br />
internal ultrastructural detail (Figs 3b and c).<br />
a<br />
S1 layer<br />
b<br />
c<br />
S2 layer<br />
S2 layer<br />
S2 layer<br />
S1 layer<br />
0.5 µm<br />
0.4 µm<br />
0.1 µm<br />
Figure 4. Cryo-sections of Eucalyptus pulp fibres, (a) 2.3 M sucrose, (b) and (c) 1.5 mM PVP pre-treatment.<br />
Pre-treatment of fibres with 2.3 M sucrose following the standard cryo-sectioning method resulted in crevices being present<br />
across the fibre cross-section (Fig. 4a). The use of polyvinylpyrrollidone (PVP) as a cryoprotectant facilitated intact<br />
cryo-sections (Fig. 4b), all other cryoprotectants tested not achieving this. From the three concentrations (0.5, 1.0 and 1.5 mM)<br />
and different exposure times tested, 30 min pre-treatment of 1.5 mM PVP produced the best results.<br />
CONCLUSIONS<br />
• The level of ultra-structural detail obtained using cryo-sectioning is superior to that obtained using chemical methods or<br />
cryo-fracturing.<br />
• Cryo-sectioning of hardwood pulp fibres promises to be beneficial for the assessment of mechanical and chemically<br />
treated fibres.<br />
ACKNOWLEDGEMENTS: We thank UKZN, CSIR and SAPPI Chemical Cellulose for funding.<br />
Corresponding email address: plekha@csir.co.za, 201295678@ukzn.ac.za.