Dynamic visualisation of a solvent-borne preservative in wood using confocal laser scanning microscopy

Recently, a rapid method of protecting mass timber has been developed involving a combination of incising and spraying the incised wood with a solvent-borne preservative. The preservative penetrates the wood longitudinally from incisions and a deep protective envelope is achieved in the species tested to-date. However, many questions remain about the mechanism involved in creating this protective envelope. For example, how long does it take for the envelope to be created? How does wood’s microstructure affect preservative penetration? To answer these questions, we developed a novel confocal laser scanning microscopy (CLSM) system that permits the dynamic visualisation of rate and penetration patterns of a fluorescent solvent-borne preservative into wood. We demonstrate the value of this system by investigating the speed and penetration pathways of the preservative into sugi sapwood. We found that initial axial through-penetration of the preservative to a depth of 10 mm occurs rapidly (< 60 s) via tracheids in the early-to-latewood transition zone and then spreads to adjacent earlywood areas. Complete penetration throughout wood’s microstructure occurred within the holding time for commercial treatment (10 days). In conclusion our CLSM system provides valuable information on the effect of wood’s microstructure on the dynamic longitudinal penetration of a solvent-borne preservative in wood, and it could potentially be used to optimise the treatment of different wood species and mass timber using the newly developed incising/spraying treatment method.

Recently, a rapid method of protecting large dimension timber and wood composites has been developed in Japan involving a combination of incising and spraying the incised wood with a solvent-borne preservative [1,2,3,4]. This ‘deep penetration treatment’ method is used commercially to treat glulam and solid wood sill plates. Treatment penetration in Scots pine (Pinus sylvestris L.) and Japanese larch (Larix kaempferi (Lamb.) Carr.) glulam and also sawn western hemlock (Tsuga heterophylla (Raf.) Sarg.) and Douglas fir (Pseudotsuga menziesii (Mirbel) Franco) is comparable to that achieved using pressure treatment [1,2,3,4]. The treatment process involves a unique incising method, employing flat-tipped rather than knife-shaped incising blades. The incised holes created by the flat-tipped blades act as small reservoirs after the incised wood is sprayed with a solvent-based wood preservative containing the fungicide cyproconazole and the synthetic pyrethroid insecticide bifenthrin. Preservative located in the holes mainly penetrates the wood tissues axially achieving penetration and retention requirements specified by performance standards. Because the treatment is carried out at atmospheric pressure, a pressure treatment cylinder is not required, overcoming one of the main obstacles to the preservative treatment of mass timber composites such as cross-laminated-timber (CLT) [5, 6]. Preliminary investigations of the suitability of ‘deep penetration treatment’ for the preservation of CLT have yielded promising results [7, 8]. In addition to its use as a pre-construction treatment, it is also attracting attention as a post-construction maintenance management technology.

The effectiveness of incising for increasing preservative penetration is well-known [9]. Preservative penetrates wood from radial or tangential surfaces via incisions, and lateral penetration between incisions occurs in the axial (transverse) direction whose permeability is orders of magnitude greater than radial or tangential permeability [9]. Nevertheless, axial permeability varies significantly between wood species and influences the penetration of preservatives between incisions [10,11,12]. For example, Keith and Chauret [10] examined how wood’s anatomical characteristics influenced the penetration of a chromated copper arsenate (CCA) preservative from single tooth or needle incisions in White spruce (Picea glauca (Moench) Voss) and Jack pine (Pinus banksiana Lamb). They used colorimetric indicators to assess preservative penetration in wood and found that longitudinal movement of preservative was greater in Jack pine compared to White spruce. Spear et al. [12] examined the longitudinal penetration of an aqueous dye from a single laser incision in Scots pine, southern pine (Pinus spp.), Sitka spruce (Picea sitchensis Bong Carr.) and European beech (Fagus sylvatica L.) and found significant differences in the longitudinal penetration of the dye in the different species. In addition to the use of indicators and dyes to assess penetration of preservatives from incisions into surrounding wood, a direct-scan X-ray technique has also been used to assess the distribution of chromated copper arsenate around incisions in Douglas-fir heartwood [13]. Both Keith and Chauret [10] and Spear et al. [12] noted pronounced effects of wood microstructure on fluid penetration from incisions with implications for optimising incising pattern and pressure treatment of different wood species.

In this study we describe the development of a confocal imaging system for visualising and quantifying the rapid axial penetration of a solvent-borne preservative into wood. The system was designed to provide information that is particularly relevant to preservatives applied to incised wood using non-pressure methods such as rapid spraying. The specific objective of the study is to evaluate whether the system can quantify the rate of axial penetration of a solvent-borne preservative into sugi (Cryptomeria japonica D.Don) sapwood and provide information on the effects of its microstructure on preservative penetration.

Development of confocal laser scanning imaging system

The system we developed to visualise and quantify the axial penetration of preservatives into wood is unique, although confocal microscopy of wood sections and surfaces cut from wood blocks has been used to image the location of dyes, polymerised furfuryl alcohol polymers and coatings within wood’s microstructure [14,15,16,17]. The system we developed consisted of:

1. (1)

   Solvent-borne preservative used in Japan for ‘deep penetration treatment’ of incised glulam and solid wood sill plates, mentioned above (Sanpreser OP Ace, Xyence, Tokyo, Japan) containing the fluorescent stain Nile red (9-(Diethylamino)−5H-benzo[a]phenoxazin-5-one), (144–08811, FUJIFILM, Tokyo, Japan) at 30 μmol/L concentration. Nile red was selected as the fluorescent stain because previous research has shown it has good specificity for hydrocarbon solvents [18]. Furthermore, it does not fluoresce in the presence of water, a feature that has led to its widespread use as a detection agent for micro-plastics in water [19].

2. (2)

   Confocal Laser Scanning Microscope (CLSM; TCS SP8, Leica Microsystems GmbH, Wetzlar, Germany) with fully integrated infrared (IR) excitation lasers, high efficiency detectors, and infrared-optimised optical transmission. The CLSM was modified by removing the overhead transmission detector and adding an electronic dispenser (Tofutty MSIC06-01, Icomes Lab Co., Ltd., Morioka, Japan) above the x–y stage, which dispensed a 30 μl droplet of the fluorescent solvent-borne preservative onto the adaxial (upper) face of a sugi sapwood block simply placed on a 24 ×ばつ 32 mm glass cover slip (Micro cover glass, Muto Pure Chemicals Co, Ltd) located on the opening of x–y stage above an HCX PL Fluotar 5 ×ばつ (magnification) ×ばつ 0.15 (numerical aperture) (dry) objective lens (Leica Microsystems GmbH, Wetzlar, Germany) of the CLSM (Fig. 1). High magnification imaging employed a HCX PL APO 50 ×ばつ (magnification) ×ばつ 0.90 (numerical aperture) (dry) objective lens (Leica Microsystems GmbH, Wetzlar, Germany). Images of the preservative reaching the abaxial (bottom) surface of the specimen on the microscope stage were recorded by the CLSM using two types of photo-multiplier tubes detecting fluorescence created by the lasers at excitation wavelengths of 488 nm and 552 nm. Wood cell walls and the solvent-borne preservative were visualised separately using fluorescence at 500 nm and 600 nm, respectively.

3. (3)

   Imaging and data processing employed a Hewlett–Packard HP Z420 work station with an Intel Xeon CPU E5-1620, operating at 3.6 GHz with 8 GB of RAM and 64-bit operating system. The integrated Leica LAS X software was used for image capture and analysis and to create time lapse movies during image acquisition. The imaging conditions are as follows: image size, 1024 ×ばつ 1024 pixels; scan rate, 400 Hz; scan times, 1. LAS X software was also used to assemble large widefield images of abaxial (lower) surfaces.

Experimentation

A sugi log, 40 cm in diameter (over-bark) at breast height, harvested from the Kasuya Research Forest of Kyushu University in Fukuoka Prefecture, Japan (latitude: 33.63829, longitude: 130.518235) was air dried under cover for 3 years. A 30 cm long sapwood stick with its long axis in the fibre direction was sawn from the log using a band saw. The stick was cross-cut to produce thirteen specimens with nominal dimensions of 8 mm (radial) ×ばつ 8 mm (tangential) ×ばつ 10 mm (longitudinal). Specimens were conditioned at 20 °C and 50% relative humidity to achieve a moisture content of 10%. The actual longitudinal dimensions of the specimens measured with a digital caliper varied from 9.46 to 11.28 mm. The time taken for the preservative to first appear in cells (tracheids) located at the abaxial (bottom) surface was measured, when a droplet of solvent-borne preservative was placed on to the opposing adaxial (upper) transverse surface.

The growth-ring boundary was positioned either parallel or perpendicular to the upper surface. Average density and growth ring widths of the 13 specimens used in this research were 0.40 g/cm³ and 1.55 mm, respectively. Both top and bottom surfaces of all dry specimens were lightly moistened with distilled water applied with a bristle brush. Both surfaces were then smoothed using a sliding sledge microtome (Yamato-TU213) containing a Yamato 24 cm (long) ×ばつ 4.4 cm (wide) blade holder and a Feather S35 disposable microtome blade. A new blade was used for each specimen. Specimens were stored at 20 °C and 50% relative humidity for more than 24 h before they were placed on the stage of the CLSM. Once a preservative droplet was dispensed on the upper surface of a specimen, a timer triggered capture of images of the abaxial (lower) surface of the specimen above the objective lens. Images were captured every 3 s over a period of 10 min. The time taken for the preservative to penetrate through each specimen was calculated using captured time-lapse images. Specimens were conditioned at 20 °C and 50% relative humidity for 10 days as this is the holding time for glulam and solid wood sill plates that are treated commercially in Japan [1, 3, 7]. Specimens were then reimaged to observe the distribution of the preservative.

Rate of longitudinal penetration of solvent-borne preservative

The relationship between the height of the 13 specimens and the time taken for preservative to be detected in tracheid lumens at the imaged abaxial (lower) surface is shown in Fig. 2. The preservative completely penetrated all specimens, but there was large variation in the detection time (min = 3 s; Max = 36 s). There was no correlation between the height of samples and detection time (R² = 0.0037).

Dynamic visualisation of penetration pathways using confocal laser scanning microscopy (CLSM)

Images of the abaxial (lower) surface of specimens where through-penetration of the preservative occurred after 3 s (Fig. 2i), 21 s (Fig. 2ii) and 36 s (Fig. 2iii) are used as examples of the effects of wood microstructure on longitudinal penetration of the solvent-borne preservative. Figure 3 and Additional file 1: Figure S1 show the distribution of the preservative in the specimen, where the preservative reached the abaxial (lower) surface after only 3 s. In these figures, green represents autofluorescence (490–520 nm) of lignin in wood cell walls, and red indicates fluorescence (580–630 nm) of Nile red dissolved in the solvent-borne preservative. Two growth rings are present in the sample. Red fluorescence was observed in the transition zone from earlywood to latewood in the lower growth ring after only 3 s (Fig. 3b, arrowed) indicating that the solvent-borne preservative had rapidly penetrated parts of this zone. Subsequently after 3, 7 and 10 min, increasing red fluorescence was detected in the earlywood-to-latewood transition zones of both growth rings (Fig. 3c–e).

Figure 4 and Additional file 2: Figure S2 show the dynamic visualisations carried out on specimens, where the preservative reached the abaxial (lower) surface after 21 s. The preservative was initially detected (after 21 s) in first-formed earlywood tracheids. Thereafter, the preservative appeared in the transition zone between the earlywood and latewood (Fig. 4b, c), and in the first-formed earlywood (Fig. 4b, c).

Figure 5 and Additional file 3: Figure S3 show the dynamic visualisations carried out on the least permeable specimen where through-penetration of the specimen by the preservative took 36 s. In accord with the more permeable specimens (Figs. 3, Additional file 1: Figure S1 and 4, Additional file 2: Figure S2), the preservative was mainly detected in the transition zone between earlywood and latewood.

Overall, dynamic visualisation of the through-penetration of the solvent-borne preservative into sugi sapwood showed that penetration in the axial direction mainly occurred in the transition zone between earlywood and latewood, and at a rate of approximately 1 cm within 1 min in every specimen. Penetration into surrounding cells occurred after 10 min.

Distribution of the solvent-borne preservative after conditioning

Figure 6 shows images of three different specimens (see Figs. 3, 4, 5), 10 min and 10 days after a drop of solvent-borne preservative, was applied to their upper surfaces. Each of the images in Fig. 6a–c are composed of multiple separate images making it possible to see the entire abaxial (lower) surface of each sugi sapwood specimen. Figure 6d–f shows the same surface after 10 days. After 10 min (Fig. 6a–c), the solvent-borne preservative was only found in central abaxial area of the specimen directly opposite the adaxial surface. Ten (10) days later it was detected across the entire abaxial surface (Fig. 6d–f). High magnification images of a specimen after 10 days of conditioning at 20 °C and 50% relative humidity are shown in Fig. 7. It is clear that the solvent-borne preservative is present in the ray parenchyma and the cell lumens of latewood tracheids (Fig. 7). The feint outlines of tracheids can also be seen suggesting that the preservative penetrated cell walls. Overall, these results indicate that the solvent-borne preservative initially penetrated the transition zone at the earlywood and latewood boundary, but the majority of penetration occurred during the 10 day conditioning period.

The laser confocal system described here has been able to visualise the location of a solvent-borne preservative within the microstructure of sugi sapwood specimens, following application of a droplet of preservative on to a transverse adaxial (upper) surface, through penetration of the specimen by the preservative and its arrival within tracheid lumens at the opposite abaxial transverse surface. Fluorescence of the solvent-borne preservative contrasted with fluorescence of wood cell walls providing insights into the tissues that are most permeable to the preservative (tracheids in the earlywood/latewood transition zone). The system was also able to quantify the initial rate of penetration of the preservative, which varied significantly between specimens (Fig. 2) possibly because of variation in the extent of aspiration of the bordered pits which permit fluid flow from tracheid to tracheid. The results indicate that the most critical period of the treatment process for achieving full penetration of specimens is the conditioning period following application of the preservative. We compared preservative penetration in specimens after 10 min and 10 days, but in theory it is possible to image specimens continuously over a period of 10 days to obtain a more precise estimate of the time taken for full penetration of the preservative to occur. It is also possible to use the system to image the through-penetration of sugi sapwood from radial and tangential surfaces (unpublished results) to compare the rates of penetration in the longitudinal, tangential and radial directions, respectively. Similar research could be performed on sapwood and heartwood in different wood species to better understand the limitations imposed by wood microstructure on penetration of the solvent-borne preservative into the woods used for the manufacture of mass timber composites. These studies could be complemented by techniques such as 4D X-ray micro-CT to observe the movement of the solvent-borne preservative through wood specimens before it is detected at the abaxial (lower) surfaces [20].

It is difficult to compare our results with those in existing literature because there have been no comparable studies. However, Shibui et al. [4] examined the axial penetration rate of ‘our’ solvent-borne preservative into Japanese larch (Larix kaempferi (Lamb.) Carr.) heartwood. They found that the preservative was able to penetrate a 2 cm long specimen ‘in about 10 s’, a rate which is comparable to that observed here. However, such a rapid rate of preservative penetration in Japanese larch heartwood only occurred, where axial resin canals connected the adaxial (upper) and abaxial (lower) surfaces [4]. Similarly, Spear et al. [12] found that resin canals played a significant role in transporting fluid from a laser incision over relatively long distances both longitudinally and radially in Scots pine and southern pine sapwood. Sugi lacks resin canals, but rapid axial preservative penetration of the solvent-borne preservative occurred via tracheids in the transition zone between earlywood and latewood. This finding accords with observations of the greater penetration of water-borne wood preservatives in latewood in sugi sapwood, which is thought to be due to the lower percentage of aspirated pits in latewood compared to earlywood [4, 21] resulting in pits that are open, permitting flow in latewood tracheids. Nevertheless, after the conditioning period it appeared as though there was complete penetration of both latewood and earlywood in sugi sapwood.

The purpose of developing our CLSM system was to better understand the mechanisms involved in the penetration of preservative into wood using the newly developed deep penetration (incising/spraying) treatment process, and ultimately to use such knowledge to optimise the process for different wood species and composites. Capillary wicking possibly modified by gravitational and evaporative effects may be the dominant mechanism involved in the initial rapid penetration of specimens by the solvent-borne preservative [22]. Diffusion may be the mechanism responsible for the complete penetration of the wood by the preservative [23]. Wood cell walls fluoresced red after the 10 day holding period suggesting that the solvent-borne preservative diffused into cell walls. However, another possible explanation for our observation that cell walls were penetrated by the preservative is that through penetration of the preservative led to its spread across the abaxial (lower) transverse surface and absorption by sectioned cell walls of tracheids. Further research is necessary to determine which effect led to the penetration of wood cell walls by the solvent-borne preservative. The solvent-borne preservative contained a zinc tracer for colorometric detection of preservative penetration in commercially treated timber composites. Hence, scanning electron microscopy and energy dispersive analysis of X-rays (EDX) or synchrotron X-ray fluorescence microscopy of this zinc tracer in specimens conditioned for different periods of time could be used to determine the rate at which preservatives diffuses into cell walls and the areas, where the preservative is concentrated [24].

We developed a novel confocal laser scanning microscopy (CLSM) system that was able to quantify the rate and penetration patterns of a fluorescent solvent-borne preservative in small wood blocks. We demonstrated the value of this system by investigating the speed and axial penetration pathways of the preservative in sugi sapwood. Our results show that rapid initial axial penetration of small specimens is limited to highly permeable tissue, and complete penetration occurs more slowly possibly via diffusion. Hence, we conclude that a holding period following initial application of the solvent-borne preservative is essential to obtain complete penetration of the preservative in treated wood. In theory our system could be used to obtain information of the effect of wood microstructure and processing parameters, such as drying on longitudinal penetration of solvent-borne preservatives into incised wood, and ultimately help to optimise ‘deep penetration (incising/spraying) treatment’ of wood composites such as CLT that are used in mass timber construction.

The data sets used and/or analysed during the current study are available from the corresponding author upon reasonable request.

CLSM:

Confocal laser scanning microscope

CLT:

Cross laminated timber

CCA:

Chromated copper arsenate

IR:

Infrared

EDX:

Energy dispersive analysis of X-rays

We thank Xyence Co., Ltd. for providing the solvent-borne wood preservative used for their ‘deep penetration treatment’.

This work was supported by JSPS KAKENHI Grant Numbers JP23H02281, JP23 K26974.

1. Hiroki Sakagami

   Present address: Department of Forest Sciences, Faculty of Agriculture, Iwate University, Ueda 3-18-8, Morioka, Iwate, 020-8550, Japan

Authors and Affiliations

1. Department of Agro‐Environmental Sciences, Faculty of Agriculture, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819‐0395, Japan

   Hiroki Sakagami

2. Department of Agro-Environmental Sciences, Graduate School of Bioresource and Environmental Sciences, Kyushu University, 744 Motooka, Nishi-ku, Fukuoka, 819-0395, Japan

   Zhang Yuchi

3. Forest Products Research Institute, Hokkaido Research Organization, 1-10 Nishi-Kagura, Asahikawa, Hokkaido, 071-0198, Japan

   Teruhisa Miyauchi

4. Department of Wood Science, University of British Columbia, 2900 2424 Main Mall, Vancouver, BC, V6 T 1Z4, Canada

   Philip D. Evans

5. Department of Wood Improvement, Forestry and Forest Products Research Institute, 1 Matsunosato, Tsukuba, Ibaraki, 305-8687, Japan

   Hiroshi Matsunaga

Contributions

HM and HS conceptualised the study and conducted preliminary experiments. HS and TM designed experiments. ZY performed the experiments under the supervision of HS. HS and HM analysed experimental data, and HM wrote the first draft of the manuscript. PE interpreted data and revised the first draft of the manuscript. All the authors read and approved the final manuscript. HM acquired funding and managed the research project.

Corresponding author

Correspondence to Hiroshi Matsunaga.

Competing interests

The authors declare that they have no competing interests.

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