ASSESSING HARDWOOD PULP FIBRE ULTRASTRUCTURE

CHEMICAL AND CRYO-METHODS FOR
ASSESSING HARDWOOD PULP FIBRE
ULTRASTRUCTURE
PRABASHNI LEKHA 1,2 , TAMARA BUSH 1,3 PATRICIA BERJAK 2 AND NORMAN PAMMENTER 2
1
CSIR/UKZN, Forestry and Forest Products Research Centre, 2 School of Biological and Conservation Sciences, 3 School of Chemistry, UKZN, Durban, South Africa.
INTRODUCTION
• 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.
• Accurate imaging and analysis of the fibre wall ultrastructure depends extensively on the sample preparation method used.
• For the production of novel materials from wood and cellulose, it is important to understand how different processes affect
fibre wall ultrastructure.
• This study used chemical and cryo-methods to obviate artefact induction during sample preparation aimed at elucidating
‘true’ ultrastructural detail.
CHEMICAL SAMPLE PREPARATION
MATERIALS AND METHODS
A
Fibres were
dehydrated in
acetone (30, 50,
75, 100%) for
1 h
CRYO SAMPLE PREPARATION (A) CRYO-FRACTURING, (B) CRYO-SECTIONING
Fibres were
loosened and
strung out onto
pieces of foil
Prolonged
infiltration with
low viscosity
epoxy resin
for 3 days
Fibre clumps
were then
submerged in
supercooled
liquid nitrogen @
-210°C
Fibres were
placed in 100%
fresh resin and
polymerised for
8 h at 70°C
A sharp metal
blade cooled
with liquid
nitrogen was
then used to
fracture the fibre
clumps
Fractured fibres
were then freeze
dried using a
freeze drier
RESIN
BLOCK
Polished block face
post microtoming
Glass cover slip
coated with Haupt’s
adhesive
0.5 - 2 µm thick resin
sections were tested
Freeze dried
fractured fibres
were then
mounted onto
carbon stubs
using carbon
fluid
Resin block face
and sections
were etched with
potassium
methoxide for
1 min to dissolve
resin
Sonicated with
methanol for
2 min
to remove
remaining resin
from tissue
All sample preparations were sputter
coated with carbon and then viewed at
2 - 10 kV by use of a Carl Zeiss Ultra
FEG-SEM
Resin blocks and
etched sections
on cover slips
were left to dry
under a fume
hood
Resin blocks,
cover slips and
formvar grids
were mounted
onto metal stubs
with carbon tape
Chemical
Concentration
B
Fibres were
loosened and
treated with
various
cryoprotectant
solutions for
different times
30 min – 24 h
1-hexadecen
sucrose
glycerol
polyvinylpyrrollidone (PVP)
PVP
PVP
3.5 M
2.3 M
10.6 M
0.5 mM
1 mM
1.5 mM
1. CHEMICAL SAMPLE PREPARATION TECHNIQUES
Small fibre
clumps were
then adhered to a
metal pin using
tissue adhesive
and was
subsequently
frozen in liquid
nitrogen
RESULTS
The pin was
transferred into
the gas chamber
of a cryomicrotome
(Reichert-Jung,
Type 652701,
Austria)
500 nm cryosections
were
obtained using a
cutting speed
between 0.4 to
0.8 mm sec -1 and
the sample was
at 80°C
Unbleached Eucalyptus pulp fibres were used throughout.
The sections
were transferred
using a metal
loop and 1.5 mM
PVP to grids
coated with
formvar on which
carbon had been
deposited
a
b
c
lumen
S2 layer
S2 layer
10 µm
0.1 µm
0.2 µm
Figure 1. (a), (b) and (c) etched resin block showing fibre cross-sections.
The etching of resin blocks resulted in limited ultrastructural detail being observed. The cross-sectioned fibre surface was
fused and there was no observable detail of the microfibrils within the different layers (Figs 1b and c).

a
b
RESULTS cont.
S1 layer
c
S2 layer
S2 layer
S3 layer
10 µm
0.4 µm
0.2 µm
Figure 2. (a), (b) and (c) etched 1.5 µm thick resin sections of Eucalyptus pulp fibres.
Etching of resin-embedded sections of different thickness on glass cover slips showed more ultrastructural detail compared
with etched resin blocks, 1.5 µm thick sections being the best (cf. Figs 1 and 2). However, microfibril ultrastructure was
obscured within the fibre wall when using chemical sample preparation techniques (Fig. 2c).
2. CRYO-SAMPLE PREPARATION TECHNIQUES
a
b
S1 layer
c
S1 layer
S2 layer
S2 layer
S2 layer
S2 layer
S1 layer
S1 layer
lumen
2 µm
0.2 µm
0.4 µm
Figure 3. (a), (b) and (c) cryo-fractures of Eucalyptus pulp fibres without pre-treatment.
Cryo-fracturing Eucalyptus pulp fibres did not result in proper cross-sections (Fig. 3a). The fractured surface curled obscuring
internal ultrastructural detail (Figs 3b and c).
a
S1 layer
b
c
S2 layer
S2 layer
S2 layer
S1 layer
0.5 µm
0.4 µm
0.1 µm
Figure 4. Cryo-sections of Eucalyptus pulp fibres, (a) 2.3 M sucrose, (b) and (c) 1.5 mM PVP pre-treatment.
Pre-treatment of fibres with 2.3 M sucrose following the standard cryo-sectioning method resulted in crevices being present
across the fibre cross-section (Fig. 4a). The use of polyvinylpyrrollidone (PVP) as a cryoprotectant facilitated intact
cryo-sections (Fig. 4b), all other cryoprotectants tested not achieving this. From the three concentrations (0.5, 1.0 and 1.5 mM)
and different exposure times tested, 30 min pre-treatment of 1.5 mM PVP produced the best results.
CONCLUSIONS
• The level of ultra-structural detail obtained using cryo-sectioning is superior to that obtained using chemical methods or
cryo-fracturing.
• Cryo-sectioning of hardwood pulp fibres promises to be beneficial for the assessment of mechanical and chemically
treated fibres.
ACKNOWLEDGEMENTS: We thank UKZN, CSIR and SAPPI Chemical Cellulose for funding.
Corresponding email address: plekha@csir.co.za, 201295678@ukzn.ac.za.