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). Downloaded from Brill.com10/04/2021 07:52:08AM via free access Fujii & Hatano — LPDE resin-casting 349 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, Downloaded from Brill.com10/04/2021 07:52:08AM via free access 350 IAWA Journal, Vol. 21 (3), 2000 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.