Specimen preparation

Pith to bark specimens for analysis utilising the SilviScan-3 system were obtained from the northern radius within each sample disc. A fixed sampling axis was used across all sites in order to remove a potential source of variability within the dataset. In order to obtain specimens from sample discs taken from the 40 - 50 year old trees, each was first cut into three sections, an approximately 50 mm wide section along the north - south axis containing the pith, and two off cut sections containing the remainder of the east and west axis. A further cut was then made to divide the north - south section about the pith, ensuring that the pith was maintained within the northern half, as shown in Figure 3-7. Subsequently all but the northern section were marked such that growth ring numbers within them could be identified

and were stored for potential future use. Further cuts were made on the northern section to extract a specimen measuring approximately 15 x 15 mm longitudinally and tangentially, spanning from the pith to the bark. The offcuts from these specimens were kept to aid in the investigation of any anomalous results that may have been generated during the analysis. The 36 pith to bark specimens prepared for the first round of analysis are shown in Figure 3-8. Preliminary specimens from trees with ages > 50 years were removed from the upper end of each northern radius of the longitudinal sections extracted from the sample trees.

Following removal from the sample discs, all specimens were immersed in acetone for two 24 hour cycles. The primary objective of this was to ensure a rapid and even drying of all specimens which still had relatively high moisture contents. A secondary benefit of placing the specimens in acetone was the removal of any resins or extractives, although all specimens were subject to soxhlet extraction prior to analysis as described later in this section, to ensure the complete removal of any such material. Following the removal from acetone, all specimens were stored in an environmentally controlled room at 65 % relative humidity and 20 ºC prior to transportation to the Innventia Wood and Fibre Measurement Centre, where the remainder of the preparation was conducted.

Figure 3-8: 15 x 15 mm preliminary SilviScan-3 specimens

The final specimen size for testing using the SilviScan-3 system is 2 mm in the tangential direction and 7 mm in the longitudinal direction. In preparation for final sawing to these dimensions, all specimens were glued to a specimen holder along one of its radial-tangential edges with the use of a polyvinyl acetate based glue. Gluing was conducted ensuring that the pith was centrally located on the specimen holder and all specimens were left for a period of eight hours after gluing. An example of a specimen attached to a holder is shown in Figure 3-9.

Figure 3-9: Specimen glued to a specimen holder

The cutting of specimens to the final test dimensions was conducted in two stages utilising two custom built circular saws. Each saw contained a set of twin circular blades, with the first set to make a 2 mm cut and the second 7 mm. Prior to commencing sawing, the specimen on its holder was placed onto a metal sledge containing two pneumatic clamps to hold it securely in position. Upon activation of the saw, the metal sledge moved beneath the saw blades at a constant speed, stopping at the end of the process to allow for the removal of the specimen before resetting to the original position. Following the completion of the first stage of sawing, a pith to bark specimen measuring 15 mm longitudinally and 2 mm in the tangential direction was obtained. Prior to commencing the second stage of sawing, each specimen was broken

away from its holder and had any remaining pith and bark removed, before being placed laterally in a metal clamp designed to pass between the second set of circular saw blades. Each specimen was placed into the clamp between two pieces of thin birch wood, so as to reduce the risk of damage to the specimen as a result of pressure applied by the clamp. The specimen was then cut utilising the second saw, after which the final test dimensions of 2 mm tangentially and 7 mm longitudinally were achieved. All specimens were then marked and placed in a specially designed holder. The specimen cutting process is displayed in Figure 3-10. Following the cutting of specimens to final test dimensions, all were subject to soxhlet extraction for a period of 12 hours. This was conducted in order to remove any naturally occurring extractives found within the cell lumens which may have had an effect on the results obtained. The soxhlet extractor used is shown in Figure 3-11 and consists of a pot at its base containing acetone with a chamber above into which the specimens were placed. During the extraction process the acetone was heated to reflux, allowing the vapour to travel into the chamber above causing the extractives to dissolve. Once the chamber was full of solvent it was siphoned off back into the lower pot and the process repeated.

(a) (b)

(c) (d)

(e) (f)

Figure 3-10: Specimen sawing process (a) Saw system (b) Specimen clamped to sledge (c) 2 x 15 mm specimen (d) Metal specimen clamp (e) Final 2 x 7 mm specimen (f) Finished specimens in holder

Figure 3-11: Soxhlet extraction process

The final stage in preparing specimens for analysis was the polishing of one of the radial- tangential surfaces of each. This was conducted to ensure accurate and reliable results during the first stage of the SilviScan-3 analysis, which involves the measurement of tracheid dimensions and growth ring characteristics with the use of a digital optical microscope, as described in Section 3.5.3.1. The presence of any marks or debris on the surface from the sawing process may have therefore had a detrimental impact on the accuracy of the results achieved. Polishing was undertaken using a specially constructed system that allowed each specimen to be clamped into position and passed over abrasive sheets for a preset number of passes, typically 100 - 250. During the process the abrasive sheets were continually moved to prevent any of the grit stones becoming lodged within the cell lumens. A series of finer sheets, typically from 1200 to 4000 grit, were used to produce the desired surface finish. The specimen holder and polishing system are shown in Figure 3-12.

(a) (b) Figure 3-12: (a) Specimen in holder for polishing (b) Polishing system

To ensure that that the individual tracheid cell walls would be visible during the first stage of the SilviScan-3 analysis, each specimen was checked for signs of debris or damage using a digital optical microscope at the end of a complete polishing cycle. On occasions where it was necessary to repeat the polishing process, the cycle was restarted from the coarsest abrasive sheet. Images showing a comparison of the surface finish before and after polishing are shown in Figure 3-13.

(a) (b)

Figure 3-13: (a) Specimen surface prior to polishing (b) Specimen surface following polishing Following the completion of all preparatory steps, specimens were allowed to equalise to the laboratory environmental conditions of 23 °C and 43 % relative humidity, giving a moisture content of approximately 8.5 %. During the preparation of the specimens two were damaged in the process of removing the bark, resulting in the loss of some growth rings. One of these specimens was obtained from site one, which had an age of 42 years, as a result of the damage the maximum cambial age that could be assessed across all younger specimens at breast height was 40 years. The second was from site five and had an age of 75 years, as a result of the

damage the maximum cambial age that could be assessed across all older specimens at breast height was 65 years.