ApplicAtion of 3D mAgnetic resonAnce microscopy to the ...titan.ijs.si/MRI/pdf/IAWA_J_32_pp_401-414.pdf · ApplicAtion of 3D mAgnetic resonAnce microscopy to the AnAtomy of wooDy

IAWA Journal, Vol. 32 (4), 2011: 401–414

ApplicAtion of 3D mAgnetic resonAnce microscopy to the AnAtomy of wooDy tissues

Primož Oven1, Maks Merela1,*, urša mikac2,3 and Igor Serša2,3

SUMMARY

We demonstrate the use of high-resolution three-dimensional magnetic resonance microscopy (3D MRM) for anatomical studies of woody tis-sues. Samples of normal and pathological structures in the branches of four tree species were imaged by 3D MRM immediately after removal from the tree, without additional tissue preparation. MRM data sets were displayed in 2D sections and in a 3D volume rendered mode. Good image contrasts between identical anatomical structures in different tree species suggest that 3D MRM may be a promising method in comparative wood anatomy. MRM can be used for general morphological observations, as well as for positioning and examination of a particular portion of tissue in the investigated object. This is demonstrated by examples of a nee-dle trace within the xylem in Norway spruce and by localisation of the protection zone at the base of a dead branch in beech. Visualisation of a wound scar in beech demonstrates the efficiency of 3D MRM in revealing the spatial dimensions of any moisture-related structural defects within wood. It can provide relatively high and isotropic resolution, thus ena-bling non-destructive and accurate determination of the moisture content of any wood structure. High-resolution 3D MRM has great potential in studying the anatomy of woody plants due to the non-destructive nature of the technique, the simplicity of tissue preparation and very versatile information retrieval by choosing an appropriate MRM method and its parameter set.Key words: Anatomy, Carpinus betulus, Fagus sylvatica, magnetic reso-nance microscopy, moisture content, MRI, needle trace, NMR, Picea abies, Quercus robur, wood, wounding.

INTRODUCTION

Only a few techniques enable the mapping of physical and chemical parameters of hygroscopic materials such as wood, tissues of living plants and other ligno-cellulosic materials non-destructively (Gil & Neto 1999). Imaging techniques based on nuclear magnetic resonance (NMR) are among the most versatile methods in this group. In

1) University of Ljubljana, Biotechnical Faculty, Department of Wood Science and Technology, Rožna dolina VIII /34, 1000 Ljubljana, Slovenia.

2) Jožef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia.3) EN-FIST Centre of Excellence, Dunajska 156, 1000 Ljubljana, Slovenia.*) Corresponding author [E-mail: [email protected]].

IAWA Journal, Vol. 32 (4), 2011402

general plant sciences, NMR has been employed in studies of metabolite distribution, water flow in the vascular system and in determining physical properties such as water diffusion and nuclear spin relaxation mechanisms in cellular compartments, morphology and the internal structure of various plant organs, taxonomic studies, hydraulic conduc-tivity of tissues and water transport at different levels, from cell-to-cell to long-distance transport (Van As & Schaafsma 1984; Glidewell et al. 1997, 1999, 2002; Ishida et al. 2000; Holbrook et al. 2001; Köckenberger 2001; Van As 2007). Wood is an ideal material for NMR studies, due to its high moisture content when green and its hygroscopic character when in use. The first magnetic resonance images of wood were used for macroscopic rather than microscopic examination of the scanned material. The structures revealed the shape of the sample, sapwood and heartwood, the presence of rays and also defects as varied as reaction wood, wet-wood, decay and gum spots, and knots and growth rings in healthy and affected trees were usually easily distinguished (Kucera 1986; Wang & Chang 1986; Chang et al. 1989; Flibotte et al. 1990; Torelli et al. 1990; Beravs et al. 1998; Coates et al. 1998; Bucur 2003a; Bucur 2003b; Morales et al. 2004; Merela et al. 2005; Johannessen et al. 2006). Magnetic resonance imaging has also been used for observation of anisotropic movement of water below and above the fibre saturation point (Hernandez & Caceres 2010; Meder et al. 2003), moisture movement and swelling of oriented strand boards (Van Houts et al. 2004), penetration of waterborne preservative in wood (Hall & Rajanayagam 1986; Dawson-Andoh et al. 2001), early detection of fungal infection in solid wood (Müller et al. 2001), for non-invasive dynamic studies of plant–pathogen interactions, and for studying the development of necrotic lesions in tree stems and the wound re-sponse of living tree tissues (Pearce et al. 1994, 1997; Barry et al. 2001; Kuroda et al. 2006; Oven et al. 2008). Most of these studies used 2D (in-plane) magnetic resonance imaging techniques, and the advantages of high-resolution 3D magnetic resonance microscopy (MRM) remained mostly unexploited in plant science. The aim of our research was to apply the 3D MRM technique for spatial visualisation of the morphology and anatomy of regular structures of living trees, various growth defects and traumatic structures, as well as to examine the variability and distribution of the moisture content in tissues. Our purpose was to demonstrate the potential of 3D MRM in the research of woody tissues rather than to analyse each of the selected topics in depth. In addition, light microscopy was applied for qualitative evaluation of the anatomical images obtained by magnetic resonance microscopy.

MATERIAL AND METHODS

Plant material Freshly sampled branches of Norway spruce (Picea abies), European hornbeam (Carpinus betulus), pedunculate oak (Quercus robur) and beech (Fagus sylvatica) were investigated. The dimensions of branches were limited by the size of the MRM radio frequency coil to a maximum diameter of 10 mm. Samples were either completely free of any damage, or traces of wounds, knots etc. could be seen by the naked eye on the surface of the bark. Approximately 80 cm long segments of branches were dissected, and cross sections were protected against desiccation with a dispersion coating Stipol-

403Oven, Merela, Mikac & Sersa — 3D Magnetic resonance microscopy

AF (Silvaprodukt, Slovenia). Images obtained by MRM were compared with those obtained by light microscopy of similar magnification. For this purpose, the material examined by MRM was dissected and prepared for light microscopy (LM). Samples were fixed in a formaldehyde, ethanol and acetic acid solution, sectioned by a sliding microtome (Leica SM 2000R, Nussloch, Germany) in the transverse plane at 20 µm, stained with safranin-red and astra-blue, mounted in Euparal and observed with a transmission light microscope (Nikon Eclipse E800, Japan).

MRM experiments MRM experiments were performed on a TecMag Apollo (Houston, Texas, USA) MRM spectrometer with a superconducting 2.35 T (100 MHz proton frequency) hori-zontal bore magnet (Oxford Instruments, Oxford, UK) equipped with field gradients and RF coils for MR microscopy (Bruker, Ettlingen, Germany). MRM imaging of the branch was done using a 10 mm diameter saddle-shaped RF coil. MRM image protons of the water in the specimen ensured that both parameters that control image contrast, i.e., echo time (TE) and repetition rate (TR), met the following conditions: TE << T2 and TR >> T1, where T2 and T1 are spin-spin and spin-lattice proton relaxation times, intrinsic properties of the sample. Due to hardware limitations of typical MRM scanners, TE is limited to a few ms and does not therefore enable imaging of rigid molecules, the protons of which have a T2 much below that limit. In our study, 3D high-resolution MRM based on the 3D spin-echo sequence (Fig. 1) was applied. The image contrast parameters were TE/TR = 2.4/600 ms, while the wood samples studied had the shortest T2 values in the range of 15 ms and the longest T1 values up to 500 ms, so that the acquired images corresponded reasonably well to the

Figure 1. 3D spin-echo MRM pulse sequence. Protons in watery samples are first excited by radio frequency (RF) pulses, then spatial information is encoded in a precession of protons by the use of three orthogonal gradients (GR, GP1 and GP2) and, finally, the NMR signal is acquired dur-ing the acquisition period (AQ). Echo time and repetition rate (TE/TR) are the two sequence parameters that control image contrast. The MRM image corresponds to the moisture content in the sample when TE is short and TR long.


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moisture content of the wood samples. Image resolution parameters were the following: field of view (FOV), 25 × 12.5 × 12.5 mm3; imaging matrix (image size), 256 × 128 × 128 pixels. The spatial resolution was 100 µm isotropic and the total imaging time was 22 h; 8 signal averages were used. We reconstructed the 3D magnetic resonance micrographs by the 3D Fourier trans-form of the corresponding k-space domain signals (Hall & Rajanayagam 1986). The sample can be displayed either in the form of sequential planar (2D) images or a single volume rendered (3D) image. MRM data were processed and analysed by the computer software NTNMR 2.3.1, ImageJ 1.41 (NIH, USA) and Amira 3.0 (Mercury, Berlin, Germany).

Moisture content determination In addition to the wood material designated for MRM examination, parallel sam-ples were collected for moisture content (MC) determination. The samples were first weighed in a green condition and thereafter dried to a constant mass at 103 ± 2 °C. MC was calculated according to the equation MC(%) = (mgreen – m0)/m0, where mgreen denotes the weight of green wood and m0 the weight of oven-dry wood. Gravimetrically determined MC values were used for determining the MC of individual tissues from MR micrographs, according to the following procedure. The average pixel intensity value (PIV) was acquired and calculated from several successive MRM cross sections of the investigated sample. The correction factor was the quotient of the gravimetrically deter-mined MC and the average PIV. The product of the quotient and the pixel intensity gave the MC.

RESULTS AND DISCUSSION

The technical advantage of 3D MRM in comparison with standard 2D (in-plane) mag-netic resonance imaging and traditional light microscopy is the ability to display the object in either a sequence of planar images or in a volume rendered three-dimensional image (Fig. 2 & 3). Figure 2 shows every second, successive virtual longitudinal section through a beech branch, starting from tangential to radial and again to tangential slices through the branch. The 3D MR acquired image data set enabled spatial reconstruction of the imaged object by the computer volume rendering procedure, as demonstrated in Figure 3. Comparing the advantages of serial virtual and mechanical sectioning of woody tissues, the latter is not only time-consuming but can also lead to artefacts due to the irregular thickness of the sections and the manual stacking of the series of images when a spatial image of the object is reconstructed. As in advanced microscopic techniques (Kitin et al. 2003), computer-assisted 3D MRI reconstruction is faster and more precise than the 3D reconstruction that can be achieved by mechanical sectioning. Compared with planar images, volume rendered 3D MR images offer additional information on the spatial arrangement of a water-containing wood structure. Volume rendered images of an oak branch revealed typical displaced rings (cf. Schweingruber et al. 2006, p. 129), which are seen as longitudinal indentures on the surface of the outer bright cylinder and star-shaped pith in the middle of the rotated structure (Fig. 3).


Figure 3 shows that the ring-porous arrangement of vessels and broad radial xylem rays that are typical of pedunculate oak are not visible in 3D MR images. In contrast, radial oriented complexes of vessels and vasicentric tracheids displayed a high MR signal (Fig. 3), reflecting their vascular function. It has been reported that earlywood vessels represent an efficient water transport system at the beginning of the growing season, characterised by an optimal soil water table (Tyree & Zimmermann 2002). In dry summer months, large vessels become dysfunctional due to the stress exceeding the tensile strength of water columns in these conducting elements. Thereafter, the smaller vessels of latewood (20–30 µm) take over the role of translocation of water in ring-porous wood species. In European hornbeam with diffuse-porous wood, planar (2D) proton density MR micrographs clearly revealed the topography of tissues displayed by bright field micro-scopy (Fig. 4A, C). The tissues with high MC yielded a high MR signal and therefore appear bright in the MR micrographs. The pith, the cambial zone region with current xylem increment and the inner bark have higher MR signals than other structures (Fig. 4A). The signal of the outer bark is lower than the signals of other tissues. Informa-tion obtained by MRM is slightly different from that obtained by light microscopy, i.e., growth rings are not discernible; regions containing aggregate xylem rays exhibit only a low MR signal and can be seen as dark, radially oriented wedge-shaped areas between regions containing the axial vascular system (Fig. 4A). The vascular system, morphologically organised in a diffuse-porous pattern in European hornbeam, appears functionally structured in a continuous radially oriented pattern in MR images. Our observation, made on European hornbeam and pedunculate oak, is in agreement with observations demonstrating the existence of intervessel pit fields between early- and latewood vessels of adjacent growth rings (Tyree & Zimmermann 2002; Kitin et al.

Figure 3. Volume rendered images of an oak branch. The images provide an insight into the 3D structure of the branch from an arbitrary viewpoint; the branch is rotated around the vertical axis. Brighter regions correspond to tissues with higher moisture content and reveal a spatial water distribution in the branch. The outer cylinder corresponds to earlywood of the forming growing ring, while the central structure is the pith. Arrow = radial complexes of conducting tissues in wood; * = displaced rings. — Scale bar = 5 mm.


Figure 4. Comparison of virtual MR cross sections and micrographs obtained by optical micros-copy in bright field: (A) and (C) European hornbeam (Carpinus betulus) and (B) and (D) Norway spruce (Picea abies). Mark C = callus, * = compression wood. Position of wood having a very low MR signal is demarcated by a white line in B and D. — Scale bars = 2 mm.

Figure 5. A: 3D MRM, virtual cross section. Wound-wood is growing over the exposed and dehydrated wood. – B: Macro-image of a wound scar on a young stem of beech. – C: LM, cross section of a closed wound. w-w = wound-wood; * = tension wood; RZ = reaction zone. — Scale bars = 2 mm.


2003). Examination of beech xylem by 3D MRM (Fig. 5A) revealed a different func-tional pattern of the anatomically diffuse-porous organised xylem vascular system. In agreement with Merela et al. (2005), concentric layers of earlywood vessels were prominent in MRM images, indicating that earlywood vessels of the growth ring in beech may be predominantly involved in upward water transport. Spruce bark has a relatively strong signal, whereby structures can clearly be dis-cerned. Dark spots in the bark indicate the presence of sclereids, which have no MR signal (Fig. 4B, D). In contrast to the hornbeam specimen, the spruce specimen pith had no MR signal. Growth rings are clearly visible in MRM due to differences be-tween the MR signal of earlywood (high MR signal) and that of latewood (lower MR signal). Multiseriate rays containing radial resin canals were not visible by MRM in spruce. The position of compression wood can be identified by light microscopy and MRM, due to its low proton density signal, which can be ascribed to the lower mois-ture content of compression wood (Fig. 4B, D). Due to its anatomical characteristics (Timell 1986), compression wood has a lower moisture content than normal wood. In the study of Tarmian et al. (2009) the MC in the latter wood ranged from 88 to 124%, whereas the MC of compression wood was in the range of 39–57% in white spruce. The consecutive T2 relaxation times of compression wood are lower than the T2 relaxation times of normal wood (Araujo et al. 1992). Compression wood is usually classi-fied morphologically on the basis of its severity. This study, however, indicates that the vascular function of morphologically different compression wood may be comparable (Fig. 4B, D). Even in the cases of anatomically less apparent compression wood, its vascular contribution appears small, i. e., it exhibits lower specific hydraulic conductivity (Mayr et al. 2006). Our study made on small branches further indicated that compres-sion wood is quite variable in occurrence: it is usually formed toward the end of the growth ring but cases in which it occurs in the middle or at the beginning of the annual ring are not unusual (Fig. 4B, D). Figure 5A indicates the relatively low MRsignal of tension wood (Fig. 5C) in beech, which is in agreement with Tarmian et al. (2009) reporting a lower MC of tension wood (68–75%) as compared to normal wood (82–91%) in beech. Figure 4B, D and 5 demonstrate the morphological and water-related response of living tissues to physical disturbance. Wounding of trees and the resulting response of tissues is a common phenomenon in trees. In general, the response of living tissues of trees to wounding usually results in changes associated with an alteration of tissues formed be- fore wounding and the formation of new structures after wounding (Shigo 1991; Pearce 2000; Schwarze & Baum 2000; Dujesiefken & Liese 2008; Oven et al. 2008; Shortle et al. 2010). Figure 4B and D show the changes in wounded Norway spruce, while Figure 5 shows the planar MRM and LM of a wounded beech branch. Wound-wood already formed over damaged xylem (Fig 4B, D) or beginning to overgrow the wound (Fig. 5) revealed a similar MR signal to that of normal wood. However, tissue formed after wounding, referred to as callus, exhibits a lower MR signal in both species. This find- ing indicates that callus, formed immediately after wounding, does not have a moisture-related function in post-wounding reactions but rather represents physical protection of the tissues formed after wounding. This enables restoration of vascular and meristematic continuity, as well as the storage and vascular function of tissues (Pearce 1996).


Comparison of 3D MRM and LM images of wounded wood in spruce (Fig. 4B, D) and beech (Fig. 5) revealed a response that can be summarised in the following water-related morphological features. Superficial wounding of xylem triggered dehydration of the compromised tissue, as illustrated by MRM in spruce (Fig. 4B). On the other hand, LM of this sample indicated that no apparent anatomical response was associ-ated with the traumatic event. It has been reported, however, that resinosis, aspiration of bordered pits and accumulation of phenolic deposits accompanied the response of xylem to wounding and infection in conifers (Blanchette 1992). Examination of wounded xylem in beech by MRM revealed a very narrow dehydrated area, sharply delineated from sound functional wood (Fig. 5). MRM did not reveal that this dehy-dration was delineated from the sound sapwood by a conspicuous reaction zone. On the other hand, LM did not reveal the moisture-related changes discerned by MRM. The response of beech to superficial wounding can be interpreted in terms of the formation of protection wood (Grosser et al. 1991). Although the 3D MR image did not reveal differences between the superficial desiccation zone, discoloration zone and marginal zone of the protection wood (cf. Grosser et al. 1991), this technique demon-strated that dysfunctional xylem tissue was sharply walled-off of the sound wood. Not only tissue visualisation but also information on moisture content (MC) can be obtained by 3D MRM (Fig. 6). The dark core in MR and LM images in Norway spruce (Fig. 4) can be considered to be heartwood, with a low MR signal due to low MC (Fig. 6). The planar MR image clearly discerns this secondary change of wood, which cannot be identified in the LM micrograph. The central part of the Norway spruce tissue was relatively dry, with a moisture content of approximately 20–40%. Within wood, differences in MC can easily be obtained from a MR image, even for particular layers of the growth ring: latewood or earlywood. The MC of earlywood was 125–150%, latewood was drier, with a MC of 60 to 90%. The last-formed growth ring, including the cambial region, had a MC higher than 150%. The MC of bark was on average 50–70% (Fig. 6).

Figure 6. Radial moisture profile of a green sample of spruce (branch diameter is 8 mm) obtained from a corresponding 3D MR micrograph.

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With 3D MRM, in vivo, non-destructive and accurate MC determination at any position within the sample was enabled. Since the spatial resolution of our imaging technique was 100 µm isotropic, the MC could be defined for any wood structure of this size. This is a unique feature of the MRM method, which can only be achieved by a few other non-destructive techniques for the examination of wood properties (cf. Fromm et al. 2001; Holbrook et al. 2001; Bucur 2003a). Figure 7 illustrates the way in which a small branch is attached to the stem. The basis of this phenomenon is described by Shigo (1985). The morphology of the stem at the position of the branch attachment, including the branch collar and bark ridge (Fig. 7C) is demonstrated by 3D MRM. Dieback of the branch usually results in a more or less apparent, dead branch stub, visualised in Figure 7A and B. The location and shape of the protection zone surrounding the base of the dead branch tissue and the amount of wood belonging to the branch collar can be clearly visualised in planar longitudinal MR micrographs (Fig. 7B). The branch stub is very dry and does not give any MR signal (Fig. 7C). Figure 8A shows a volume rendered image of a Norway spruce branch with clearly visible growth ring boundaries, as well as the image of needles (Fig. 8B). When inves- tigating a structure such as a needle trace (Pensa et al. 2004), many difficulties can be experienced in sampling the material by serial physical sectioning. However, when sectioning is done virtually during the processing and display of MRM data, the posi-tion of a needle trace becomes quite clear. It can appear approximately 1.5 to 2 mm

Figure 7. Anatomy of branch attachment to the stem in beech. (A) LM longitudinal section of a branch, (B) virtual radial section and (C) volume rendered MRM image of the same branch. PZ = protection zone. — Scale bar = 5 mm.

Figure 8. Segment of spruce branch visualised in (A) 3D and (B) in-plane MR mode. – A: Attach-ment of the needles to the surface of the xylem cylinder. – B: Position of a needle trace extending to the pith (arrows). Partial necrosis of the needle is also visualised. — Scale bar = 5 mm.


lower than the position seen on the surface of the bark by the naked eye. Traces of needles have received particular attention in the Needle Trace Method (NTM) (Kurkela & Jalkanen 1990), which is based on examination of the length and location of needle traces embedded in stem-wood. Non-destructive examination of needle traces by 3D MRM may be an easier and faster tool for obtaining needle trace data. The results of our study demonstrate the possible application of 3D MR microscopy and subsequent manipulation of 3D data in the study of normal and pathological tree structures. Although the resolution achievable by optical microscopy is better than that obtained by MRM, the latter possesses unique imaging advantages, which can be sum-marised as follows. After sampling the green material, the only requirement for speci-men preparation was protection of the tissues against desiccation. Chemical fixation of tissues, physical sectioning, staining and mounting of sections (traditional procedures of specimen preparation for optical microscopy) are not needed in MRM, since any virtual section can be obtained from the acquired MRM data. The non-destructiveness of MRM, 3D manipulation of data and additional information on structure-related MC make high-resolution 3D MRM a powerful xylotomical tool, despite the better resolu-tion of optical microscopy. The spatial resolution of the presented MR micrographs was 100 µm, isotropic. However, as has been reported (Ciobanu et al. 2002), this can still be considerably improved. Rapid and repeated data acquisition in any desired plane, with the option of selecting MR signal regions that most accurately describe the tissue of interest from a range of gray-scale values, makes MRM a powerful tool in interpreting histological material (Glidewell et al. 1999). It is a non-invasive method that enables monitoring of several physiological processes, repeatedly, at the same position as in vivo (cf. Van As 2007; Oven et al. 2008). A comparison between light microscopy and MRM indicated that the latter can yield a different MR signal from identical ana-tomical structures in different tree species, due to their different moisture content, which is a promising feature for application of 3D MRM in wood anatomy. This conclusion is supported by findings that different structure of the hardwoods manifested on spin-spin relaxation T2 values (Almeida et al. 2007). The 3D MRM technique reveals not only the different wood structures but also provides information on the complexity of spatial arrangement of any normal structure or growth defect as well as on the distribution of the moisture content within the tissue.

ACKNOWLEDGEMENTS

This work was supported by the Slovenian Research Agency of the Ministry of Higher Education, Science and Technology of the Republic of Slovenia within research program P4-0015 and national project J1-7042. The authors thank Martin Zupančič and Ana Sepe for technical help.

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