IAWA Journal, Vol. 21 (3), 2000: 347–359

THE LDPE RESIN-CASTING METHOD APPLIED TO

VESSEL CHARACTERISATION by Tomoyuki Fujii1 & 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

Downloaded from Brill.com10/04/2021 07:52:08AM via free access 348 IAWA Journal, Vol. 21 (3), 2000 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: 2 2 h = {d • γ L • cos θ + P • (d/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 (ηo) )

2 1011

1010

109 E' & E'' (dyne / cm 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. absorption

heat

Relative

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) 3 • 10 (Pa

o η

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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Downloaded from Brill.com10/04/2021 07:52:08AM via free access Fujii & Hatano — LPDE resin-casting 355 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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Fujii, T. 1993b. Application of resin casting method to wood anatomy. Plant Morphology 5: 3–17. [In Japanese with English summary.]. Fujii, T. 1994. Application of resin casting method to plant anatomy – Starting from an inter- est in the capillary structure of wood. Electron Microscopy 29: 64–67. [In Japanese with English summary.] Fujii, T. & Y. Hatano. 1995. Modification of resin-casting method with the application of ther- moplastic synthetic resin. Abstracts of the 40th Anniversary Conference of the Japan Wood Research Society: 608. [In Japanese with English summary.] Lee, S.G. & T. Fujii. 1996. Conductive function of intervessel pitting on a growth ring bound- ary of Machilus thumbergii Sieb. et Zucc. IAWA J. 17: 256–257. Mauseth, J.D. & T. Fujii. 1994. Resin-casting: A method for investigation apoplastic spaces. Amer. J. Bot. 81: 101–110. Muller, F.H. & J. Stauff. 1963. Rheological treatment of the rate of setting of adhesive joints. Kolloid-Zeitschrift & Zeitschrift für Polymere 192: 51–66. Nakajima, K., K. Ando, N. Hattori, S. Kitayama & T. Fujii. 1995. Comparison among shapes of holes and crack processed with laser, needle and knife incising using resin casting method. Abstracts of the 40th Anniversary Conference of the Japan Wood Research Society: 608. [In Japanese with English summary.] Stieber, J. 1981. A new method of examining vessels. Ann. Bot. 48: 411–414. Zimmerman, M.H. 1983. Xylem structure and the ascent of sap. Springer Verlag, Berlin.

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