Anal. Chem. 2008, 80, 1552-1557
Discriminating the Indistinguishable Sapwood from Heartwood in Discolored Ancient Wood by Direct Molecular Mapping of Specific Extractives Using Time-of-Flight Secondary Ion Mass Spectrometry Kaori Saito,† Takumi Mitsutani,‡ Takanori Imai,† Yasuyuki Matsushita,† and Kazuhiko Fukushima*,†
Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya, 464-8601, Japan, and Nara National Research Institute for Cultural Properties, 2-9-1, Nijo-cho, Nara, 630-8577, Japan
A new method that can chemically discriminate the visually indistinguishable sapwood from heartwood in discolored woods is presented in this paper. Discriminating between sapwood and heartwood, which are normally recognized by color in cross sections of stems of tress, is important in dendrochronological dating, as well as in evaluating qualities of woods such as durability. In treering chronology, the felling date, which affects the construction date of architectures, can be estimated only in woods that have a recognizable sapwood/heartwood boundary. However, the felling date cannot be estimated in discolored woods because it has indistinguishable sapwood. Time-of-flight secondary ion mass spectrometry (TOF-SIMS) analysis of specific chemical substances retained for approximately 1300 years after felling demonstrated the presence of sapwood in a discolored ancient architectural wood of Hinoki cypress (Chamaecyparis obtusa). Direct molecular mapping by TOF-SIMS clearly indicated that the specific substances, hinokinin, hinokiresinol, hinokione, and hinokiol, started to accumulate at the sapwood/heartwood boundary where only hinokinin was localized and retained predominantly in ray parenchyma cells. The result allowed the determination of the felling date of the discolored wood. TOF-SIMS has shown to be useful for investigating the distribution of minute amounts of chemical components in woods. In dendrochronology, the presence of sapwood is essential to estimate felling dates in the case of woods without bark,1,2 such as ancient architectural materials (Figure 1a). The felling date of trees is estimated by comparing the tree-ring sequence with a master chronology and is important to verify the construction dates of historical wooden buildings derived from documentary * To whom correspondence should be addressed. Graduate School of Bioagricultural Sciences, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. Phone: +81-52-789-4159. Fax: +81-52-789-4163. E-mail:
[email protected] † Nagoya University. ‡ Nara National Research Institute for Cultural Properties. (1) Hughes, M. K.; Milsom, S. J.; Leggett, P. A. J. Archaeol. Sci. 1981, 8, 381390. (2) Millard, A. Archaeometry 2002, 44, 137-143.
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Figure 1. Most of the Horyuji Temple buildings are built with Hinoki cypress wood. (a) Photograph of the Gojunoto (the five-story pagoda, right) and Kondo (main hall, left) in the Horyuji Temple, Nara, Japan. The photograph was supplied by Shogakukan Inc., Tokyo, Japan. (b) A transverse section of the stem of a Hinoki cypress showing sapwood and heartwood. In the living tree, newly formed sapwood cells in the cambial zone just beneath the bark are pushed toward the inner core and are subsequently transformed into heartwood.
records.3 The felling date is the year of formation of the outermost final annual ring just beneath the bark when the tree was felled (Figure 1b). Where no bark is present, the key to determine the felling date is the presence of sapwood, which is a relatively narrow outer zone just beneath the bark. In the case of wood with incomplete sapwood, the felling date ranges can be calculated using the statistical average number of sapwood rings.1,2 However, the felling dates in discolored old woods could not be determined, because there are no established methods for distinguishing sapwood from heartwood. It is difficult to discriminate between (3) Wight, G. D.; Grissino-Mayer, H. D. Tree-Ring Res. 2004, 60, 91-99. 10.1021/ac7021162 CCC: $40.75
© 2008 American Chemical Society Published on Web 01/31/2008
sapwood and heartwood based on color or morphological differences in the wood cell structures of old samples that are discolored or degraded, and sapwood may sometimes be incorrectly identified and hence felling dates incorrectly calculated. Normally, sapwood is visually discriminated from heartwood, the darker-colored inner core, as shown in Figure 1b. The darker color of heartwood results from the accumulation of large amounts of extractives such as terpens, flavonoids, and lignan, which are soluble in organic or aqueous solvents.4 The changes observed during the transition from sapwood to heartwood include death of living cells, disappearance of stored starch, the formation of heartwood extractives and changes in moisture content,4 although the mechanism of heartwood formation is not fully understood. Sapwood is more susceptible to decay and insect attack than heartwood. The presence of extractives is often used to detect heartwood by employing techniques involving staining, ultraviolet absorption, or X-rays;4 however, the extractives responsible for these detections are ambiguous. The radial tendencies in the concentrations of specific extractives from sapwood to heartwood have been studied by solvent-extraction followed by gas chromatography (GC)5 or high-performance liquid chromatography (HPLC)6 and by direct measurement without extraction using Fourier transform near-infrared (FT-NIR) Raman spectroscopy.7 Time-of-flight secondary ion mass spectrometry (TOF-SIMS) is a mass spectrometric technique with high mass resolution and provides information regarding the chemical features of the surfaces of untreated solid samples.8 Upon TOF-SIMS bombardment using a pulsed primary ion beam, numerous secondary ion species, including elemental and molecular ions with a molecular weight of less than approximately m/z 1000, are produced. A significant advantage of TOF-SIMS over the other techniques is its ability to detect molecular or fragment ions of interest directly without extraction of the target substance and to subsequently map the targeted ions at any position on the sample surface. The TOF-SIMS imaging analysis, which allows the location of all or any selected secondary ion in the obtained spectrum, is now widely applied to the imaging of biological samples at a cellular level with submicrometer spatial resolution.9,10 There are still a few studies that apply TOF-SIMS molecular imaging for plant tissues,11 although dynamic SIMS using a continuous primary ion beam has been applied almost exclusively for the mapping of inorganic ions in plant tissues.12-14 TOF-SIMS is less destructive under the static SIMS condition with a much lower primary ion (4) Hills, W. E. In Heartwood and Tree Exudates; Timell, T. E., Ed.; Springer Series in Wood Science; Springer-Verlag: Berlin, Heidelberg, 1987. (5) Ekeberg, D.; Flæte, P. O.; Eikenes, M.; Fongen, M.; Naess-Andresen, C. F. J. Chromatogr., A 2006, 1109, 267-272. (6) Takaku, N.; Choi, D. H.; Mikame, K.; Okunishi, T.; Suzuki, S.; Ohashi, H.; Umezawa, T.; Shimada, M. J. Wood Sci. 2001, 47, 476-482. (7) Bergstro ¨m, B. Forestry 2003, 76, 45-53. (8) Vickerman, J. C. In ToF-SIMS. Surface Analysis by Mass Spectrometry; Vickerman, J. C., Briggs, D., Eds.; IM Publications and SurfaceSpectra Limited: West Sussex, U.K., 2001; pp 1-40. (9) Ostrowski, S. G.; Van Bell, C. T.; Winograd, N.; Ewing, A. G. Science 2004, 305, 71-73. (10) Altelaar, A. F. M.; van Minnen, J.; Jime´nez, C. R.; Heeren, R. M. A.; Piersma, S. R. Anal. Chem. 2005, 77, 735-741. (11) Imai, T.; Tanabe, K.; Kato, T.; Fukushima, K. Planta 2005, 221, 549-556. (12) Heard, P. J.; Feeney, K. A.; Allen, G. C.; Shewry, P. R. Plant J. 2001, 30, 237-245. (13) De´rue, C.; Gibouin, D.; Verdus, M. C.; Lefebvre, F.; Demarty, M.; Ripoll, C.; Thellier, M. Microsc. Res. Tech. 2002, 58, 104-110.
dose density than dynamic SIMS8 and allows the imaging of molecular ions of a higher molecular weight as well as inorganic ions. In this report, we apply the TOF-SIMS technique to the analysis of the distribution of specific substances in woods in order to clearly discriminate the indistinguishable sapwood from the heartwood in a discolored wood. In the TOF-SIMS analysis, we can examine each annual ring of sample surfaces displayed on the video monitor during measurement. The method allows us to identify the sapwood/heartwood boundary where the specific substances start to accumulate in a discolored wood by the mapping of molecular ions of the substances. The ancient wood sample used here is derived from the Horyuji Temple in Japan, a World Cultural Heritage site, and is known to be the oldest surviving wooden building in the world (Figure 1a). The coniferous Hinoki wood is an important building material that has been widely used in Japan since ancient times.15 EXPERIMENTAL SECTION Plant Material. For the experiment, two trees of the Hinoki cypress (Chamaecyparis obtusa) were used. A 233-year-old Hinok tree was felled in the autumn of 2001 in Nagano, Japan. A 5 cmthick stem disk sample was obtained and stored after it was naturally dried at room temperature. A 31-year-old Hinoki tree was cut in the summer of 2005 in Nagoya University Forest, Inabu, Japan. A 5 cm-thick stem disk sample was obtained, and it was immediately frozen in liquid nitrogen after cutting and preserved at -80 °C until use. The architectural material derived from the Horyuji Temple is preserved in the National Museum of Japanese History, Sakura, Japan. This sample is a part of transverse section (approximately 3 cm-thick, 12 cm × 12 cm) of stem of Hinoki cypress. The sapwood and heartwood were unrecognizable due to discoloration. The tree-ring sequence of the wood sample was cross-dated with a master chronology for Chamaecyparis obtusa in Japan,16,17 producing a high t-value (t ) 7.1, overlap period ) 170 years, year of the last ring ) 705 A.D.). A 100-µm-thick transverse section (approximately 1 cm × 1 cm) was prepared from all the samples by using a sliding microtome. The sections were dried naturally at room temperature before mounting on the TOF-SIMS sample holder. Standard purified compounds of hinokinin, hinokiresinol, hinokione, and hinokiol were supplied by courtesy of Prof. H. Ohashi and Prof. K. Takahashi. These compounds in the powder or oil form were pressed onto indium foil and placed in the TOFSIMS sample holder. TOF-SIMS Analysis. All TOF-SIMS measurements were performed on a TRIFT III spectrometer (ULVAC-PHI, Japan) equipped with an Au liquid metal ion gun. Positive and negative ion spectra within the m/z 0-1850 mass range were obtained (14) Mangabeira, P. A.; Gavrilov, K. L; de Almeida, A. A. F.; Oliveira, A. H.; Severo, M. I.; Rosa, T. S.; Silva, D. D.; Labejof, L.; Escaig, F.; Levi-Setti, R.; Mielke, M. S.; Loustalot, F. G.; Galle, P. Appl. Surf. Sci. 2006, 252, 3488-3501. (15) Iwamoto, J. In Forestry and the Forest Industry in Japan; Iwai, Y, Ed.; University of British Columbia Press: Vancouver, Canada, 2002; pp 3-9. (16) Tanaka, M.; Mitsutani, T.; Sato, T. In Dendrochronology in Japan (in Japanese with English summary); Nara National Cultural Properties Research Institute, No. 48: Dohosha, Kyoto, Japan, 1990; pp 3-195. (17) Mitsutani, T.; Tanaka, M. In Proceedings of the International Dendrochronological Symposium, Ystad, South Sweden, September 3-9, 1990; pp 3-9.
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Figure 2. Difference in TOF-SIMS spectra between sapwood and heartwood in Hinoki cypress. Positive spectra obtained from (a) sapwood and (b) heartwood. The figure also shows the structures of extractives that are specific to the heartwood of Hinoki cypress. Mw: molecular weight.
using a 22 keV Au1+ beam operated at a current of 0.01 pA, with a pulse width of 10 ns (
