THE LDPE RESIN-CASTING METHOD APPLIED TO VESSEL CHARACTERISATION - Brill
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IAWA Journal, Vol. 21 (3), 2000: 347–359
THE LDPE RESIN-CASTING METHOD APPLIED TO
VESSEL CHARACTERISATION
by
Tomoyuki Fujii 1 & Yasunori Hatano2
Wood Anatomy Laboratory1 & Adhesion Laboratory2, Forestry and Forest Products Research
Institute, Tsukuba Norin P.O. Box 16, Ibaraki 305-8687, Japan
SUMMARY
Low-density polyethylene (LDPE) was used as a casting medium to
study vessel attributes. The LDPE had a low crystallinity and melted
around 102 °C. The glass transition point was very low (-20 °C) in com-
parison to polystyrene (105 °C). The viscosity decreased exponentially
as the temperature was raised.
Dry sample blocks of selected hardwoods, fixed in tubes and previ-
ously heated, were impregnated with melted LDPE under vacuum or
under compression. After the removal of cell-wall materials, the resin
casts obtained almost entirely consisted of vessels clearly showing three-
dimensional arrangement. These casts were dissected with a thin steel
knife for SEM observation. Vessel-casts were usually not accompanied
by casts of vasicentric elements. Resin-casts of fibres, tracheids and
parenchyma cells were observed only near the surface of the blocks.
Cell wall sculpturing such as pits and helical thickenings were observed
in detail. The depth of LDPE drawn into vessel lumina depended on the
time of evacuation and diameter of vessels.
Key words: Resin casting method, LDPE, thermoplastic polymer, SEM,
vessel network.
INTRODUCTION
Vessels are the most important flow path for longitudinal water conduction in the tree
trunk and also for the penetration of liquid in wood quality improvement. Vessels
may be short to very long and are composed of few to very many vessel elements
(Zimmermann 1983). Depending on the species, some vessel elements have dense
pitting on their lateral walls connecting to neighbouring vessel elements (jointly form-
ing multiples as seen in cross section). As has been clearly indicated by the recon-
struction of three-dimensional vessel orientation from serial sections (Zimmerman
1983), vessels are connected with each other through intervessel pitting and comprise
a three-dimensional vessel.
The resin-casting method using styrene monomer as injection medium has been
successfully applied to the scanning electron microscopic study of cell wall sculptur-
ing and three-dimensional arrangement of elements such as vessels, fibres, and pa-
renchyma cells (Fujii 1993a). The method has been expanded to fresh samples using
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a solvent exchange method to replace water with styrene monomer (Mauseth & Fujii
1994; Fujii 1994). As has been described in detail, these resin-casts should be son-
icated to remove a major part of fibres and parenchyma cells covering vessel casts
(Fujii 1993a).
For resin-casts specially aimed at the study of vessels, polyester sucked into wood
blocks and subsequenly polymerised clearly illustrates the ramifications of vessels
and fine wall sculptures (Stieber 1981; Fujii 1993b). Artefacts such as moulds of gas
bubbles on vessel lumen surfaces, possibly caused by the surface tension, cannot be
avoided using this method (Fujii 1993a), although they are only infrequent. Other
synthesised polymers have been inspected for their ability as injecting media for resin-
casting in wood anatomy. We have had excellent results using Mercox (Oken Ltd.)
which is a commercial preparation for resin-casting of blood vessels (Fujii 1993b;
Mauseth & Fujii 1994). Partially polymerised styrene has a higher viscosity and the
resin injection was limited to only around the vessels (Fujii 1994), and sometimes
one vessel in a multiple in the cast was only filled with a thin resin layer while others
were completely filled.
These above-mentioned polymers usually give us excellent casts of xylem ele-
ments, but they are not easily applicable to the study of three-dimensional networks
of vessels in larger wood volumes. The casts are so brittle that they often fracture dur-
ing the chemical treatments to remove cell wall materials (Fujii 1993a, b; Mauseth &
Fujii 1994).
Silicon elastomers have given us resin-casts of vessels of almost full length from
herbaceous materials and from monocotyledons (André 1993, 1998), but the casts did
not give enough information on intervessel pit connections, which form the three-
dimensional vessel network within wood. Because the injected medium cannot pen-
etrate through pit membrane and the casts obtained are so flexible, the original con-
nections in vessel multiples are not preserved.
The following characteristics are required for a resin to be applicable to the three-
dimensional analysis of vessels in wood; among them 1–3 are essential for the casting
medium and 4–5 are preferable for easy handling:
1) Sufficiently viscous to mould cell-wall sculpturing and cavities in wood at a tem-
perature where cell-wall materials are not degraded seriously.
2) Sufficiently resistant against chemical treatments to remove cell-wall materials
such as concentrated sulphuric acid to remove crystalline polysaccharides and mix-
tures of hydrogen peroxide and acetic acid to remove lignin.
3) To be stable in shape and size during casting and the chemical treatments.
4) To be flexible and not too fragile (brittle) during handling at room temperature.
5) Rheological properties should be controllable for the selected casting of vessels.
Synthesised resins used for casting in the previous studies have mostly adequate quality
satisfying criteria 1–3. Only Mercox is not so suitable for resin-casting of wood, be-
cause the casts become soft in acid solutions and are easily deformed. Polystyrene
and polymethacrylate resins are brittle (criterion 4) and monomers of these resins
have such a low viscosity that these polymers are not suitable (criterion 5).
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In this study thermoplastic resin was selected as one of the resin-casting media for
testing. The thermoplastic characteristics of the resin were analysed and the resin-casts
were observed by scanning electron microscopy (SEM). Pilot studies using LDPE-
resin (Low-density Polyethylene) were earlier published by Fujii and Hatano (1995),
Nakajima et al. (1995), and Lee and Fujii (1996).
Theoretical background of the depth of resin casts
According to Muller and Stauff (1963), the depth of resin penetration (h) is related
to the diameter of cavities (d), the viscosity of the resin (η ), pressure applied (P), and
the period (t) of applying pressure differentials in the empirical formula as follows:
h2 = {d • γ L • cos θ + P • (d/2) 2} • t /4 η
Here, γ L is the surface tension of the liquid and θ is the contact angle.
In this study, cavities are cell lumina of xylem elements. Because the diameter (d)
of vessels are usually much wider than those of fibres and parenchyma cells, the depth
of resin (h) moulding vessel lumina is expected to be much greater than in the other
cell types. Here, the other factors should be almost constant, as resin-injection into a
wood block should be carried out at once and temperature and pressure should not
significantly vary within a sample.
MATERIALS AND METHODS
Selection of thermoplastic casting medium
Thin films of several kinds of thermoplastic synthetic polymers were tested for
their chemical durability against concentrated sulphuric acid and hydrogen peroxide/
acetic acid mixture prior to their application for resin-casting of wood.
Commercial hot-melt resins, which were composed mainly of polypropylene with
plasticiser and amorphous polyalpha olefin (polypropylene /ethylene copolymer and
propylene /isobutene copolymer: Ubetac APAO, UT-2115, UT-2215, UT-2304, UT-2315,
UT-2535, UT-2585, UT-2715, UT-2780), were not resistant against the chemical treat-
ments.
Low density polyethylene (LDPE), which is a commercial thermoplastic, synthetic
polymer (Showa Denko Co. Ltd. – M281, Melt Index = 80), was so resistant against
the chemical treatments to remove cell wall materials that further investigations were
carried out using this polymer. Thermoplastic characters of the LDPE were measured
in dynamic elasticity, differential scanning calorimetric spectra and viscosity in rela-
tion to temperature.
Resin casting procedure (Fig. 1)
Longitudinal columnar samples (7 mm in diameter) were punched out using a leather
punch from transverse section of dry wood specimens (c. 1 cm thick) of Pterocarya
rhoifolia Siebold et Zucc. (TWTw 9301), Ilex macropoda Miq. (TWTw 14960), and
Acer pictum Thunb. (TWTw 9335). Transverse surfaces on both ends of the samples
were finished using a new steel knife equipped to a sliding microtome. Each sample
was fixed at an end of polytertrafluoroethylene (Teflon) or glass tube with rapid-cure-
type epoxy adhesive. In the case samples were of irregular shape or of smaller size,
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1. Finish a sample block by
sectioning with a microtome
2. Fix the sample in a
tube with epoxy glue
Epoxy glue
Teflon or 4. Suck or push the
glass tube melted resin into
the sample with
a rotary pump
3. Heat in a mantle heater
Fig. 1. Resin injecting methods.
they were fixed filling the gap between sample and tube with rapid-type epoxy glue,
which was later completely decomposed during the treatment with hydrogen peroxide
and acetic acid mixture.
The samples were heated in a mantle heater with LDPE pellets. When the tempera-
ture of the resin increased to 210–220 °C, free ends of the tubes were connected to a
rotary pump (MINI-VAC P-15). Then they were evacuated for 2 min. to suck melted
LDPE resin under wood samples or were given pressure for 1 min. to push melted
LDPE resin on the upper surface into cavities in the wood. Injection times of 1 and 2
min. were applied to the wood of Pterocarya to investigate the relation between the
depth of casts and the injection time.
The sample blocks injected with LDPE-resin were treated alternatively and repeat-
edly with a mixture of hydrogen peroxide /acetic acid at 60 °C or more and concen-
trated sulphuric acid at room temperature to remove cell wall materials completely
(Fujii 1993a). They were rinsed well with water and freeze-dried to prevent the dis-
tortion of fine casts during drying. The resin casts obtained were dissected with a thin
steel knife to select adequate portions for SEM observation. These were coated with
Pt-Pd in an ion-sputter coater (Jeol JFC 1100) at 1.5 keV for 3 min. During the ion-
sputter coating, some resin-casts became seriously warped, probably caused by the
increased temperature due to electron current through them due to the inferior elec-
tron insulation of a sample holder. SEM (Jeol JSM 840) was operated at the following
conditions: accelerating voltage 5 keV; probe current c. 1*10-10 A; objective diaphragm
50 microns in diameter; working distance 48 mm.
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RESULTS AND DISCUSSION
Thermoplastic characters of the low density polyethylene (LDPE)
The LDPE is a thermoplastic synthetic polymer of low crystallinity. The tempera-
ture dependence of visco-elastic properties of the polymer used in this study is shown
in Figure 2. The glass transition temperature was suggested by the peak of the loss
modulus (E") which is the indicator of dissipation heat energy transformed from vi-
bration energy, and above this point the micro-Brownian motions of the polymer chain
segments occurs. It is obvious that the glass transition temperature of the LDPE was
very low at -20 °C in comparison to polystyrene (105 °C). The weak shoulder that ap-
peared around 80 °C is supposedly due to the molecular motions of crystalline parts.
This suggests that the amount of crystals in this resin is small.
Results from differential scanning calorimetery shown in Figure 3 indicate that the
crystalline fractions in the polymer melted around 102 °C. The melting viscosity (η o )
10 11
(dyne / cm 2)
1010
10 9
E' & E''
108
$ = E'
% = E"
107
-100 0 100
Temperature (°C)
Fig. 2. Visco-elastic properties of LDPE. E ': storage modulus (nearly equivalent to modulus
of the polymer); E": loss modulus. The peak of E " suggests the glass transition of the polymer.
Relative heat absorption
60 100 140 180
Temperature (°C)
Fig. 3. Differential scanning calorimetry of LDPE. The heat absorption peak around 102 °C
shows the breakdown of the crystalline in the LDPE.
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10 4
s)
103
•
η o (Pa
102
100 150 200
Temperature (°C)
Fig. 4. Melting viscosity of LDPE.
of the polymer was measured in relation to the temperature and clearly decreased
exponentially along with the raise of the temperature (Fig. 4).
Verification of the empirical formula
Resin casts of pores incised by laser-beam radiation into coniferous woods
LDPE-casts used here were those prepared for Nakajima et al. (1995). Tangential
pores were processed with a high power laser beam radiated on tangential surfaces of
coniferous woods forming a long and narrow conical cavity in the woods. In a wood
of Agathis sp. (Fig. 5), tracheids around the laser-incised pore were not fully filled
with the resin and the length decreased gradually from top to bottom in Figure 5. This
result supports the empirical formula and is explained as follows. “As melted resin
reaches each open tracheid sequentially, the time for penetration (t) of individual
tracheid lumina during application of pressure/vacuum decreases resulting in shorter
resin-casts (h).”
In Douglas-fir (Pseudotsuga menziesii) wood (Fig. 6), resin-casts of latewood tra-
cheids were conspicuously shorter than those of earlywood tracheids. The length of
the cast (h) is probably significantly affected by the abrupt changes of lumen diam-
eters (d) between early- and latewoods rather than the injection time (t).
Vessel-casts
Sapwood of Pterocarya rhoifolia was selected because the wood is semi-ring-po-
rous with vessels arranged in a radial pattern, in other words vessel diameter shows a
wide range and the frequency is adequately low for SEM observation. The resin-cast
shown in Figure 7 is predominantly of vessels, as expected from the formula: because
of the high viscosity (η) of melted LDPE resin, the depth (h) of the resin impregnated
into vessels which have a much wider diameter (d) than fibres, is expected to be much
greater. Furthermore, because pit membranes between vessels and fibres and also
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Fig. 5 & 6. Resin-casts of laser-incisions and of tracheids in contact with the incisions. – 5: In
Agathis sp. wood, most tracheids were not fully filled up to the tips with the resin and the casts
decreased gradually in length from top to bottom. – 6: In Douglas-fir (Pseudotsuga menziesii)
wood, tracheid-casts, most of which did not fill up to cell tips, were wide and long in earlywood
and narrow and short in latewood. — Fig. 7. LDPE resin-cast of Pterocarya rhoifolia wood
prepared by pushing LDPE into wood: panoramic view of a dissected piece.
between fibres have only microcavities (i.e. openings of very small diameter d), it is
expected that the amount of melted LDPE resin penetrated through them is not enough
to fill up pit cavities within an injection period. As a result, casts of vasicentric ele-
ments such as axial parenchyma cells, ray cells and adjacent fibres did not usually
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accompany the vessel-casts. This is quite different from styrene casts (Fujii 1993a),
where all element lumina and intercellular cavities within a sample were filled with
the resin, because the monomer has a very low viscosity. In cases that melted LDPE
was not injected for the whole sample depth, the vessel-casts were not completely
filled leaving hollows in the centre. This is possibly due to the high wettability, namely
low surface tension, of melted LDPE resin.
Resin-casts of vessels in the casts injected for 1 minute (Fig. 8) were much shorter
than those in the casts injected for 2 minutes (Fig. 9). Within each cast, wider vessels
showed conspicuously deeper penetration than narrower vessels (Fig. 7 & 8). These
results support the empirical formula on the correlation between the depth of resin (d)
and the impregnation period (t).
The high temperature (210–220 °C) applied to LDPE resin to decrease the viscosity
possibly affects cell wall materials. At temperatures higher than 180 °C, chemical
components and mechanical cell-wall properties can be gradually degraded resulting
in artefacts in the resin-casts. However, no serious artefact was observed, as far as
vessel wall sculpturing is concerned, except that some vasicentric elements were oc-
casionally only partly cast. Lee and Fujii (1996) have applied the LDPE-resin-casting
method at much milder conditions (at 165 °C for 15 min., evacuated at -75 kPa) to the
study of the vessel network in Machilus sapwood. The needed injection period of 15
min. was much longer than the 1 or 2 minutes at 210–220 °C for about 1 cm depth
injection. This is because of the higher viscosity (η) of LDPE at lower temperature
(Fig. 4).
Vessel network and wall sculpturing
Pterocarya rhoifolia Siebold et Zucc.
Vessel network, fusion of vessels and continuous multiples of vessels in the wood
of Pterocarya were clearly visible for the whole length of the casts, which was only
restricted by the depth of the visible field in SEM and also by the size of the sample
chamber (Fig. 7–9). Vessel elements in a radial multiple cohered irregularly to each
other, and the radially cohering elements had a mostly similar length and position
suggesting that they were derived from one fusiform cambium initial (Fig. 10). Here,
one series of vessel elements in the multiple decreased in element size upward and
terminated with a partial cast of a vessel element leaving dense intervessel pits on the
tangential wall of the opposite element (indicated by an arrow in Fig. 10). This is
supposed to represent a vessel ending. A vessel element in the right in Figure 11 also
seems to be terminal as it seems to have only one perforation to the element below.
Fig. 8–12. LDPE resin-cast of Pterocarya rhoifolia wood prepared by pushing LDPE into
wood. – 8 & 9: Lateral view of the casts prepared under a given pressure. – 8: One-minute in-
jected. – 9: Two-minutes injected. – 10: Solitary vessel and two vessels in a radial multiple
with an initiation or termination of a vessel (arrow). – 11: A terminal element of a vessel in a
radial multiple. – 12: Detail of radial walls of vessel elements covered with dense intervessel
pits and a perforation plate.
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Fig. 13–16. LDPE casts of Ilex macropoda wood. – 13: Panoramic view of a dissected piece.
– 14: Part of vessel-casts in a radial multiple of three. – 15: Three vessel-casts with various pit
distributions on lateral surfaces. – 16: Casts of vessels in a radial multiple and fibre tracheids.
Note helical thickenings on vessel elements and fibre tracheids, and intervessel pits and perfo-
rations.
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Fig. 17–20. LDPE cast of Acer pictum wood. – 17: Panoramic view. – 18: Vessels in a radial
multiple with some vasicentric axial parenchyma cells. – 19: Vessels in a radial multiple of
five but lacking casts of one series of vessel elements. – 20: Detail of the same multiple as
shown in Fig. 19, but viewed from another angle. Note imperfect cast of a terminal or initial
element of a vessel connected with intervessel pits.
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Casts of intervessel pits were well illustrated by LDPE resin and the flat surfaces
originated from pit membranes (Fig. 12) suggest that the resin hardly penetrates through
them.
Ilex macropoda Miq.
In LDPE casts of Ilex wood, vessels were so crowded because the wood is diffuse-
porous with densely distributed narrow vessels that the three-dimensional network of
vessels was not clear without further dissection (Fig. 13). Vessel elements in a radial
multiple had almost the same length and were located at the same longitudinal level.
They were continuous longitudinally through perforations in each vessel, which re-
sembled pit-casts in their appearance, and were connected laterally with dense
intervessel pits (Fig. 14 & 15). The vessel-pit-casts had round to oblong flat surfaces
and were characteristic for the opposite and partial uniseriate pit arrangement (Fig.
14–16). Helical and annular thickenings on the walls of vessel elements and fibre
tracheids were faithfully represented by LDPE-casts (Fig. 16). Interestingly, most heli-
cal thickenings appeared to run continuously across the perforations, suggesting that
they had developed not independently in each cell but under unified control.
Acer pictum Thunb.
In the LDPE cast of Acer pictum wood, vessel-casts had wide spaces between each
other due to low vessel density. Vessels were solitary or in radial multiples of two to
more than four, especially in latewood (Fig. 17). Vessel-casts occasionally were ac-
companied by some vasicentric elements such as paratracheal axial parenchyma cells
(Fig. 18). In this case, on the lateral surfaces of the two vessels comprising a radial
multiple, imperfect casts of very short vessels without perforations to the vessels in
the multiple were also attached showing dense intervessel or vessel-tracheid pits.
These imperfect casts of vasicentric elements may have arisen as an artefact of ther-
mal decomposition during resin injection, namely partial breakdown of pit membranes.
In casts of vessel multiples, individual vessel element series were sometimes ʻmiss-
ingʼ or only incompletely cast, although the presence of intervessel pits on the adjoin-
ing elements indicated their original presence (Fig. 19 & 20). This may indicate that
vessels are quite short in radial multiples and that terminal elements connected through
dense intervessel pitting with the other elements of the multiple prevent the filling of
all elements in the vessel multiple. Zimmermann (1983) described similar phenom-
ena in the wood of Cedrela fissilis, and also inferred that all vessel ends are in contact
with other vessels through pits. Our results support that conclusion.
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