A New Sledge Microtome to Combine Wood Anatomy and Tree-Ring Ecology

452 IAWAIAWA Journal Journal 36 (4), 36 2015: (4), 2015 452–459

A new sledge microtome to combine wood anatomy and tree-ring ecology

H. Gärtner1,*, S. Lucchinetti2, and F.H. Schweingruber1 1Swiss Federal Research Institute WSL, Landscape Dynamics/Dendroecology, Zürcherstrasse 111, 8903 Birmensdorf, Switzerland 2Schenkung Dapples, Mechanische Werkstatt, Flühgasse 80, 8008 Zürich, Switzerland *Corresponding author; e-mail: [email protected]

Abstract Based on the Reichert Om E microtome a more sophisticated, nevertheless solely mechanically operated microtome was developed enabling to section specimens of various forms and sizes. The materials used and the special construction of the microtome resulted in a higher stability of the moving parts whilst simultaneously reducing weight. As a result of the high stability of the sledge guidance, bigger sections can be cut enabling more detailed analyses of anatomical structures within plant stems, roots or branches as well as their variation back in time. Consequently, wood anatomical parameters can be integrated more easily in time-series analyses supporting the aims of a quantitative wood ecology. Keywords: Dendroecology, sectioning, time-series analyses. [In the online version of this paper Figures 1–5 are reproduced in colour.]

Introduction

For detailed wood anatomical studies within the scope of a changing climate, the preparation of sections is essential to strengthen the understanding of the form and func- tional evolution of the xylem in different climatic regions. Nevertheless, details about micro-technique, especially the application of microtomes have hardly been discussed in scientific publications (Bracegirdle 1978; Gärtner & Nievergelt 2010; Gärtner & Schweingruber 2013; Gärtner et al. 2014, 2015). Most wood anatomical analyses are done on small specimens, mostly blocks of about 1 cubic cm to cut transverse, radial and tangential sections. To analyse age-dependent or environmentally induced anatomical variations, e.g., along a cross section of a tree stem, the ability to section bigger specimens would be of importance, but most current microtomes are not capable to cut big sections. The inability of cutting bigger sections of more or less dense woody material is as- tonishing when thinking about the serial production of big sections (2 × 5 cm; thickness 50–100 µm) prepared by Herrmann Nördlinger at the end of the 19th century (Nördlinger 1852–1888). The technical base for preparing sections of that size does no longer exist (Bubner 2008). However, when focussing on an anatomically based, annually resolved reconstruction of environmental conditions of the past, large sections are required to

Downloaded from Brill.com10/06/2021 03:15:28AM via free access Gärtner et al. – Sledge microtome for tree-ring ecology 453 reduce the amount of sample preparation needed to an affordable minimum. Big sections will allow to expand wood anatomical research to a highly resolved, time related, ecologically based wood anatomy. With this, the ability of analyzing new parameters and developing new methodologies to understand the short- and long-term effects of environmental factors on woody plant bodies is given. A first step in this direction was the development of the core microtome (Gärtner & Nievergelt 2010) for cutting micro sections of entire increment cores (Gärtner et al. 2015; Ivanova et al. 2015) as well as the GSL1 microtome (Gärtner et al. 2014). As a further step towards combining wood anatomy and dendroecology, we present a new sledge microtome enabling to cut big specimens of all forms for the detailed analyses of structural variations within the respective plant part to be analyzed back in time.

Reichert OmE

sled (block)

sliding surfaces

microtome body (cast iron)

WSL-Lab-Microtome

microtome body (aluminum) Figure 1. Photograph of a common Reichert Om E microtome (upper image) used as a base for the development of the WSL-Lab-Microtome (lower image).

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The Lab-microtome Basic principles A basic requirement for cutting sections of wood, no matter if using rotation or sledge microtomes, is the stability of the device. This is the main reason why old (and also recent) microtomes are extremely heavy, mostly made of (cast) iron. The pure weight of the device guarantees stability during the sectioning procedure. One of the most practicable sledge microtomes for wood anatomical purposes (although no longer produced) is the Reichert sledge microtome Om E (Fig. 1). Its design proved to be applicable for small specimens ranging from low to high density. The bigger the

threaded rod to fix the stopper for the platform knife-holder block

opening for stabilizing pole

knife-holder platform

sled 1 sled 2

angled stop for automated sample uplift

linear guide 1 linear guide 2 stopper for the platform

a b c

Figure 2. Construction of the knife-holder platform. – a: Stabilizing pole in position; b and c: knife holder stabilized by the pole for cutting sections of high density woods.

Downloaded from Brill.com10/06/2021 03:15:28AM via free access Gärtner et al. – Sledge microtome for tree-ring ecology 455 sample and/or the higher the density, the higher the forces occurring while cutting. When a certain threshold is exceeded, the knife is lifted up and cutting sections is no longer possible. We cannot specify this threshold, because it varies depending on the respective combination of sample size and density. For instance, we realized that it is not possible to cut a continuous thin section (15 µm) of a larch stem sample (Larix decidua Mill.; sample size: 1 × 3 cm) without special treatment (e.g., boiling), if severe compression wood occurs within the sample.

Construction We aimed at developing a more sophisticated, nevertheless manually operated microtome while increasing stability and nevertheless reducing weight. Consequently, the body of the microtome was constructed as a frame consisting of four aluminum plates forming the base for all components of the microtome. The knife holder is placed in the centre of an aluminum platform, which is mounted on two small sleds running on linear guides (Fig. 2). The central axis of the two guides are fixed parallel (spacing: 7 cm) on the top facing the aluminum plate of the microtome frame. The ball-bearing guided sleds only allow a two-dimensional movement (forward/backward). The ball bearings do have hardly any internal play (± 1µm) providing maximum stability of the knife holder in all directions other than along the guides while cutting. The knife holder (Fig. 3) used for the Lab-microtome was initially designed for the core microtome (Gärtner & Nievergelt 2010). Basically it is constructed the same way as the old proven holders in the past, but made of aluminum. The knife (or removable

Figure 3. A: Side view of the mounted knife holder of the Lab-Microtome; 1: Knife-holder block; 2: Bar to adjust the vertical angle (arrows) of the knife; 3: Spherical head fixing the horizontal position of the knife-holder block on the platform. The position can be changed by opening and closing the head (see small arrows); 4: Two screws are used to fix the position of the knife (or 5: removable blade holder) in the block; 6: Knife-holder platform (compare Fig. 2). – B: Core holders of three different sizes (10, 5 and 2 mm diameter, length 6 cm) were developed to be placed in the sample holder of the microtome.

Downloaded from Brill.com10/06/2021 03:15:28AM via free access 456 IAWA Journal 36 (4), 2015 blade holder) is horizontally and vertically adjustable to adapt its position depending on the size and density of the sample. When sectioning samples of high density, the knife holder can be additionally fixed with a small pole. This pole is placed 3.5 cm left of the threaded rod fixing the knife-holder block on the platform (Fig. 2). At this position, the pole stabilizes the knife holder and prevents it from being turned sideward around the threaded rod (compare Fig. 2a–c). The sample holder is placed within a frame that allows a fine correction of the orientation of the sample when it is basically fixed in the clamps to allow a precise cutting of transverse, tangential or radial sections. The frame is set on a pole which is placed into a mount fixed on an aluminum plate which is guided by two sleds running along parallel linear guides. The precise upward movement of the platform and with this of the sample is realized by a micrometer screw connecting the platform and a hand wheel allowing for upward movements of the sample in steps of 1 µm. The semi-automatic uplift system is constructed in the same way as it was done for the Reichert OM E.

Sectioning procedure The basic sectioning procedure is comparable for all samples. After adjusting the optimal orientation of the sample in the holder (e.g., perpendicular to the longitudinal axis for transverse sections), the sample is fixed tight to prevent it from moving during sectioning. Depending on the size and density of the material, the vertical angle of the blade needs to be adjusted (Fig. 3A). The more dense the material is, the steeper the angle of the knife needs to be. The exact position has to be adapted for each specimen, in general an angle of about 20° (indicated on the side of the blade holder; Fig. 3A) is applicable for most softwoods. Although it is common practice to place the entire sample in water for several minutes before sectioning to soften the material, at least for bigger samples only the part to be

Figure 4. Photograph of a 10-mm core fixed in the core holder of the microtome. An 800-µm- thick section (small image, section fixed between two glass slides) was cut from the core using a stable microtome blade.

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Figure 5. – A: Reproduction of “Nördlinger” sections of exposed roots (thickness 60 µm); A1 and A4: Fagus sylvatica; A2 and A3: Larix decidua. – B: Examples for the range of possible sample sizes to be sectioned with the Lab-Microtome (cross sections; scanned slides and related micro photos); B1: Acer campestre, stem, sample size 1.2 × 8.5 cm; B2: Fagus sylvatica, exposed root, sample size 3.3 × 2.4 cm; B3: Pinus sylvestris, stem, sample size 1.1 × 6.5 cm; B4: Piper nigrum, stem, sample size 0.5 × 0.5 cm; B5: Cycas circinalis, leaf, enrolled, sample size 0.1 × 0.3 cm.

Downloaded from Brill.com10/06/2021 03:15:28AM via free access 458 IAWA Journal 36 (4), 2015 sectioned should be water-saturated. The problem is the fact that the entire sample gets softened when it is completely water-saturated and, as a result, bigger samples tend to be deformed (= pressed upward) in the centre while cutting because of the relatively high forces always occurring while the knife cuts off a section. In extreme cases, the sample is lifted up in the central part to such an amount, that the knife gets stuck in the sample. A solution for this problem is to fix the sample dry in the holder. Then just moisten the surface of the sample with water by using a brush, wait for a moment until it is soaked in the upper part of the sample and start cutting. Make sure, that the part to be sectioned is always wet when cutting. The way a sample needs to be fixed in the holder always depends on the specimen. The construction allows disks of branches, roots or small stems up to a diameter of 2.5 cm to be fixed directly in the clamps of the holder. The same is true for rectangular samples (inner size of sample holder: 2.5 × 6 cm). Special clamps were developed to allow cutting 5-mm and 10-mm increment cores up to a length of 6 cm, but also micro cores taken using, e.g., a Trephor device (Rossi et al. 2006) having a 2-mm diameter (Fig. 3B). Moreover, the ability to fix 10-mm cores enables preparing cores for isotopic analysis. The surface can be prepared to analyze the common ring structure and micro sections can be taken to further analyze the anatomical structure without contaminating the remaining sample. Furthermore, when stabilizing the blade with the pole, 800 µm sections can be cut from the core (Fig. 4) for “en bloc” cellulose extraction before the annual rings are separated from the section for isotopic analyses (e.g., Schollän et al. 2014). This procedure also re- quires to fix the core dry in the holder. Only the surface needs to be wetted for trimming the core to a certain amount and then soak the surface until roughly a 1-mm layer is soaked before the micro sections can be cut. The stability of the construction even enables cutting the so-called “Nördlinger” sections (Fig. 5), if the samples are fixed dry in the holder.

Conclusion A big drawback in sectioning woods until nowadays was the fact that samples were restricted to a relatively small size. The enhanced stability of the Lab-microtome helps resolving this problem and enables cutting a wide range of specimens, from petioles to long radii sampled from tree trunks (Fig. 5).

References Bracegirdle B. 1978. A history of microtechnique. The evolution of the microtome and the development of tissue preparation. Heinemann, London. Bubner B. 2008. The wood cross sections of Hermann Nördlinger (1818–1897). IAWA J. 29: 439–457. Gärtner H, Banzer L, Schneider L, Schweingruber FH & Bast A. 2015. Preparing micro sections of entire (dry) conifer increment cores for wood anatomical time-series analyses. Dendrochronologia 34: 19–23. Gärtner H, Lucchinetti S & Schweingruber FH. 2014. New perspectives for wood anatomical analysis in Dendrosciences: the GSL1-microtome. Dendrochronologia 32: 47–51.

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Gärtner H & Nievergelt D. 2010. The core-microtome. A new tool for surface preparation on cores and time series analysis of varying cell parameters. Dendrochronologia 28: 85–92. Gärtner H & Schweingruber FH. 2013. Microscopic preparation techniques for plant stem analysis. Verlag Dr. Kessel, Remagen. Ivanova A, Dolezal J, Gärtner H & Schweingruber FH. 2015. Forty centimeter long transverse and radial sections cut from fresh increment cores. IAWA J. 36: 460–463 (this issue). Nördlinger H. 1852–1888. Querschnitte von hundert Holzarten, Vol. I –XI. Cotta, Stuttgart. Rossi S, Anfodillo T & Mendardi R. 2006. Trephor: a new tool for sampling microcores from tree stems. IAWA J. 27: 89–97. Schollän K, Heinrich I & Helle G. 2014. UV-laser-based microscopic dissection of tree rings – a novel sampling tool for δ13C and δ18O studies. New Phytol. 201: 1045–1055.

Accepted: 10 August 2015

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