CORRELATIVE MICROSCOPY Light,Confocal and Scanning ...€¦ · (LM), fluorescence confocal laser...

4
BIOGRAPHY Adya Singh received his PhD in botany from the University of Arkansas in 1969 and did his post- doctoral studies at Simon Fraser University and Northern Arizona University. He is now a senior scientist at Ensis in New Zealand. He has published widely in botanical and wood science areas utilizing light, confocal, scanning electron and transmission electron microscopy tools. ABSTRACT A novel approach involving the combined use of light microscopy, confocal laser scan- ning microscopy and field-emission scan- ning electron microscopy was developed to examine the wood-adhesive interface in a commercial plywood manufactured by glu- ing Pinus radiata wood plies with a phenol- formaldehyde adhesive. The intricate nature of the penetration of adhesive into surface tissue layers of plies from the adhe- sive bond line was revealed. This correlative approach provided evidence of adhesive penetration into intact cell walls and cell wall regions damaged during the mechani- cal peeling of veneers (plies) in addition to the cell lumen, strengthening the current view that the mechanical interlocking of adhesive into wood is very important for adhesive performance. KEYWORDS light microscopy, confocal laser scanning microscopy, field-emission scanning electron microscopy, correlative microscopy, ply- wood, wood-adhesive interface AUTHOR DETAILS Adya P. Singh Senior Scientist Ensis - Wood Processing Private Bag 3020 Rotorua, New Zealand Tel: +64 7 343 5853 Email: [email protected] Microscopy and Analysis 22(3):5-8 (EU), 2008 C ORRELATIVE M ICROSCOPY INTRODUCTION Microscopical techniques which provide com- plementary information, such as correlated light, confocal and electron microscopy, have been very useful in our work on the character- ization of wood products at the cellular and tissue levels and the assessment of their in- service performance. The work presented in this article is part of an investigation aimed to understand interfacial interactions between the components of biomaterials based prod- ucts, and is based on a technical approach involving combined use of light microscopy (LM), fluorescence confocal laser scanning microscopy (CLSM) and then field-emission scanning electron microscopy (FE-SEM) to probe the interface between wood and adhe- sive in Pinus radiata plywood. Plywood is a wood based product that is strong, durable and versatile, being used in both interior and exterior applications from wall panel and furniture making to caravan and bus construction. Plywood is produced by gluing together a number of layers (plies) of wood with the grain directions at right angles to each other in successive plies. Thus in the cross-sections through two successive plies, the grain in one ply is cut transversely and in the other longitudinally, as can be seen in the illus- trations presented in this article. Commercially, adhesives have been used to produce a range of wood products of high value, such as laminated beams, plywood, and strand, chip and particle boards. The perfor- mance of adhesives depends on many factors, and the physical and chemical characteristics of both wood surfaces and adhesives deter- mine the quality of bonding. Wood-adhesive interactions that take place at the interface, i.e. the place of contact of adhesive with wood, involve chemical bonding as well as physical factors, such as mechanical interlock- ing (entanglement) of adhesive into wood tis- sues. Collectively, these interactions determine the bond strength. Understanding factors that determine the quality of adhesive bonds can potentially lead to the production of high per- formance wood products using minimal adhe- sive amounts needed to adequately bond wood surfaces [1], which can result in signifi- cant savings in production costs. Techniques for characterizing wood-adhe- sive interface are continually evolving. Microscopy has been used before to charac- terize the adhesive line [2-4], but the main aim has been to examine the depth of adhesive penetration into contact tissue layers. In this article we describe a novel microscopy approach that we have developed to probe the wood-adhesive interface in order to more clearly understand the physical nature of wood-adhesive interactions in a commercial plywood product which had been bonded with a phenol-formaldehyde adhesive. The technique involved correlative microscopy of 90-μm thick, microtome-cut sections through Light, Confocal and Scanning Electron Microscopy of Wood-Adhesive Interface Adya Singh, 1 Bernard Dawson, 1 Catherine Rickard, 1 Jacqueline Bond 2 and Anamika Singh 1 1. Ensis, Rotorua, New Zealand. 2. Scion, Rotorua, New Zealand Figure 1: Light micrograph showing adhesive penetration into rays from the adhesive line (arrow). The wood-adhesive interface is not well defined. Scale bar = 60 μm. MICROSCOPY AND ANALYSIS MAY 2008 5

Transcript of CORRELATIVE MICROSCOPY Light,Confocal and Scanning ...€¦ · (LM), fluorescence confocal laser...

Page 1: CORRELATIVE MICROSCOPY Light,Confocal and Scanning ...€¦ · (LM), fluorescence confocal laser scanning microscopy (CLSM) and then field-emission scanning electron microscopy (FE-SEM)

B I O G R A P H YAdya Singh received hisPhD in botany from theUniversity of Arkansas in1969 and did his post-doctoral studies atSimon Fraser Universityand Northern ArizonaUniversity. He is now a senior scientist atEnsis in New Zealand. He has publishedwidely in botanical and wood science areasutilizing light, confocal, scanning electronand transmission electron microscopy tools.

A B S T R A C TA novel approach involving the combineduse of light microscopy, confocal laser scan-ning microscopy and field-emission scan-ning electron microscopy was developed toexamine the wood-adhesive interface in acommercial plywood manufactured by glu-ing Pinus radiata wood plies with a phenol-formaldehyde adhesive. The intricatenature of the penetration of adhesive intosurface tissue layers of plies from the adhe-sive bond line was revealed. This correlativeapproach provided evidence of adhesivepenetration into intact cell walls and cellwall regions damaged during the mechani-cal peeling of veneers (plies) in addition tothe cell lumen, strengthening the currentview that the mechanical interlocking ofadhesive into wood is very important foradhesive performance.

K E Y W O R D Slight microscopy, confocal laser scanningmicroscopy, field-emission scanning electronmicroscopy, correlative microscopy, ply-wood, wood-adhesive interface

A U T H O R D E TA I L SAdya P. SinghSenior ScientistEnsis - Wood ProcessingPrivate Bag 3020Rotorua, New ZealandTel: +64 7 343 5853Email: [email protected]

Microscopy and Analysis 22(3):5-8 (EU),2008

CO R R E L A T I V E MI C R O S C O P Y

I N T R O D U C T I O NMicroscopical techniques which provide com-plementary information, such as correlatedlight, confocal and electron microscopy, havebeen very useful in our work on the character-ization of wood products at the cellular andtissue levels and the assessment of their in-service performance. The work presented inthis article is part of an investigation aimed tounderstand interfacial interactions betweenthe components of biomaterials based prod-ucts, and is based on a technical approachinvolving combined use of light microscopy(LM), fluorescence confocal laser scanningmicroscopy (CLSM) and then field-emissionscanning electron microscopy (FE-SEM) toprobe the interface between wood and adhe-sive in Pinus radiata plywood.

Plywood is a wood based product that isstrong, durable and versatile, being used inboth interior and exterior applications fromwall panel and furniture making to caravanand bus construction. Plywood is produced bygluing together a number of layers (plies) ofwood with the grain directions at right anglesto each other in successive plies. Thus in thecross-sections through two successive plies, thegrain in one ply is cut transversely and in theother longitudinally, as can be seen in the illus-trations presented in this article.

Commercially, adhesives have been used toproduce a range of wood products of highvalue, such as laminated beams, plywood, and

strand, chip and particle boards. The perfor-mance of adhesives depends on many factors,and the physical and chemical characteristicsof both wood surfaces and adhesives deter-mine the quality of bonding. Wood-adhesiveinteractions that take place at the interface,i.e. the place of contact of adhesive withwood, involve chemical bonding as well asphysical factors, such as mechanical interlock-ing (entanglement) of adhesive into wood tis-sues. Collectively, these interactions determinethe bond strength. Understanding factors thatdetermine the quality of adhesive bonds canpotentially lead to the production of high per-formance wood products using minimal adhe-sive amounts needed to adequately bondwood surfaces [1], which can result in signifi-cant savings in production costs.

Techniques for characterizing wood-adhe-sive interface are continually evolving.Microscopy has been used before to charac-terize the adhesive line [2-4], but the main aimhas been to examine the depth of adhesivepenetration into contact tissue layers. In thisarticle we describe a novel microscopyapproach that we have developed to probethe wood-adhesive interface in order to moreclearly understand the physical nature ofwood-adhesive interactions in a commercialplywood product which had been bondedwith a phenol-formaldehyde adhesive. Thetechnique involved correlative microscopy of90-µm thick, microtome-cut sections through

Light, Confocal and Scanning ElectronMicroscopy of Wood-Adhesive Interface Adya Singh,1 Bernard Dawson,1 Catherine Rickard,1 Jacqueline Bond 2 and Anamika Singh1

1. Ensis, Rotorua, New Zealand. 2. Scion, Rotorua, New Zealand

Figure 1: Light micrograph showing adhesive penetration into rays from the adhesive line (arrow). The wood-adhesive interface is not well defined. Scale bar = 60 µm.

MICROSCOPY AND ANALYSIS MAY 2008 5

Page 2: CORRELATIVE MICROSCOPY Light,Confocal and Scanning ...€¦ · (LM), fluorescence confocal laser scanning microscopy (CLSM) and then field-emission scanning electron microscopy (FE-SEM)

the wood-adhesive interface, after appropri-ate treatments of sections. The same sectionswere sequentially examined by LM, CLSM andFE-SEM. This is a new approach in wood-adhe-sive research which has provided valuable newinformation, particularly on the intricatenature of the adhesive penetration pathwayswithin the surface tissues of the bonded woodplies.

M AT E R I A L S A N D M E T H O D SThe correlative microscopy approach under-taken in our work involved the developmentof specific staining and imaging techniques,which enabled the same sections to be exam-ined sequentially by LM, CLSM and FE-SEMand gather information that was not possibleto obtain using any one of these microscopetypes alone.

Light and Confocal MicroscopyThe sections cut through the wood-adhesiveinterface using a sliding microtome at a thick-ness of 90 µm were stained with aqueous tolu-idine blue stain and mounted in glycerol on aglass slide prior to examination with LM. Afterviewing and photographing with LM, thesame sections were examined with a LeicaTCS/NT CLSM under fluorescence mode at exci-tation wavelengths of 488 and 568 nm andemission wavelengths of 530 and 600 nm, asdescribed in an earlier work [5].

Scanning Electron MicroscopyThe toluidine blue stained sections, which hadbeen examined with LM and subsequentlywith CLSM, were removed from the glass slidesby immersing in water in a Petri dish and thenprocessed for the FE-SEM work according to amethod described earlier [6]. Briefly, the sec-tions were treated with 1% aqueous OsO4

(osmium tetroxide) for 2 hours at room tem-perature, washed in water, briefly rinsed inethanol, clamped between two glass slidesand air-dried.

The sections were then mounted on carbontapes applied to stubs, coated with chromiumin a sputter coater and examined with a JEOL6700F FE-SEM.

First, the sections were viewed in the sec-ondary electron imaging (SEI) mode at anaccelerating voltage of 3 kV, emission 10 µA,probe current 7 and working distance 15 mm,and then in the backscattered electron imag-ing (BEI) mode at an accelerating voltage of 15kV, emission 20 µA, probe current 9 and work-ing distance 15 mm. For a comparison, the sec-tions that had not been treated with OsO4

were also examined in both SEI and BEI modes.

R E S U LT S A N D D I S C U S S I O NIn the light microscope the toluidine bluestain, which imparts a bluish green colour tolignified wood cell walls, proved effective inclearly differentiating wood cell walls fromthe phenol-formaldehyde adhesive, whichkept its original brownish colour. Lightmicroscopy proved useful in following ways:

1. It provided evidence that the adhesivehad penetrated into the lumens of rays andalso of axial tracheids up to four cells deepfrom the adhesive line (Figures 1, 2).

2. Light microscopy indicated that the adhe-sive may also have penetrated into the cellwalls particularly of tracheids in contact withand in vicinity to the adhesive line, as the cellwalls in these tissue regions displayed anorangeish and not the bluish green colour typ-ical of the cell walls of tissues that were distantfrom the adhesive line and out of direct reachof the adhesive.

3. Light microscopy enabled a large numberof sections to be examined in a relatively shorttime, and thus large segments of the wood-adhesive interface. However, the inability oflight microscopy to bring particularly the dis-tant wood tissues in the same focal plane asthe wood-adhesive interface in a section was asevere limitation of this type of microscopy.

The combination of toluidine blue stainingof sections and use of suitable operating con-ditions of CLSM enabled the adhesive to besharply differentiated from wood cell wallsbased on bright contrasting colours of theadhesive (appearing greenish) and wood cell

Figure 2: Light micrograph showing a higher magnification view of the wood-adhesive interface. The adhesive penetration into tracheids is up to several cellsdeep from the adhesive line (arrow). Scale bar = 35 µm.

MICROSCOPY AND ANALYSIS MAY 20086

Figure 3: Confocal fluorescence micrograph ofthe adhesive line (greenish) betweentwo plies, showing deep penetrationof adhesive into the rays. The adhesivehas also penetrated into tracheids inthe surface layers of the glued plies allalong the adhesive line as well as intocracks within the tissues (arrow). Scalebar = 50 µm.

Page 3: CORRELATIVE MICROSCOPY Light,Confocal and Scanning ...€¦ · (LM), fluorescence confocal laser scanning microscopy (CLSM) and then field-emission scanning electron microscopy (FE-SEM)

CO R R E L A T I V E MI C R O S C O P Y

walls (appearing reddish). It was also possibleto visualize adhesive penetration into woodtissues more clearly than with LM, as theunique capabilities of CLSM in optical section-ing through an object and then obtaining acomposite image of sequential sectionsthrough a considerable depth enabled largetissue areas to be brought in the same focalplane as the adhesive line, which was not pos-sible with light microscopy. Furthermore, acombination of superior colour differentia-tion between the adhesive (greenish) andwood cell walls (reddish) and image sharpnessmade it possible to visualize wood-adhesiveinterface more clearly than was possible withlight microscopy, which greatly enhanced thevalue of information obtainable on wood-adhesive interactions.

In Figure 3, the entire view in field is in thesame focal plane and thus sharply defined,enabling the intricate pathways of adhesivepenetration into wood tissues of the pliesflanking the adhesive line to be clearlyresolved. The adhesive has penetrated intothe lumens of ray tissues and axial tracheids,as well as into cracks present within the plyface exposed to the adhesive line that arelikely to have formed during the peeling ofveneers from logs. The light green toorangeish colour (not reddish) of the surfacetissues embedded in the adhesive suggeststhat the adhesive has also penetrated woodcell walls. Penetration of the adhesive into cellwalls is more obvious in Figure 4, a highermagnification view of a wood-adhesiveregion shown in Figure 3.

Comparison of tracheids which are farthestfrom the adhesive line, where cell walls arereddish coloured with the middle lamellashowing greatest brightness and thus clearlydistinguishable from the secondary wall, withthose nearest the adhesive line, where cellwalls appear light green, suggests that theadhesive may have completely penetrated cellwalls in addition to penetrating cell lumens. Inthe transition zone, cell walls appear to beonly partly impregnated with the adhesive, asthe patchiness of cell wall colours (cell wallsnot uniformly coloured) would suggest.

Figure 4 also displays an intricate pattern ofadhesive distribution within the tissues near-est the adhesive line, which appear to havebeen damaged probably during veneer peel-ing, with some cells severely compressed andcracks within cell walls present. The adhesivehas penetrated all accessible spaces and is pre-sent in cell lumens, cell wall cracks and cellwalls, thus creating a wood-adhesive polymercomposite in the wood-adhesive interfaceregion.

SEM added another important dimension tovisualizing adhesive penetration into woodtissues. Use of FE-SEM in our work in combi-nation with BEI imaging provided remarkablyhigh definition of the wood-adhesive inter-face, made possible by the high resolutioncapability of this instrument and the enhance-ment of differentiation between the adhesiveand wood cell walls based on a special tech-nique that we developed to increase the con-trast of the adhesive relative to cell walls.

The technique involved treating the tolui-dine blue-stained sections (which we hadexamined with light microscopy and subse-quently with CLSM) with OsO4 prior to exami-nation with the FE-SEM in combination withBEI. BEI, which enables contrast differentiationbetween two or more components in a com-posite material to be obtained based onatomic number differences, has been widelyused to examine both biological and non-bio-logical objects [7,8]. Higher atomic numbercomponents appear brighter than loweratomic number components under BEIbecause of greater yield of backscattered elec-

trons. For this reason, differentiation betweenthe components differing in their atomic num-ber is based on brightness intensities, withhigher atomic number components appearingbrighter than the lower atomic number components.

External applications, as stains or tracers, ofhigh atomic number substances have alsobeen employed to enhance the yield ofbackscattered signals and thus contrast differ-entiation based on atomic number differences[9,10]. Osmium is a high atomic number sub-stance, and therefore it enhances the emissionof backscattered electrons from the materials

MICROSCOPY AND ANALYSIS MAY 2008 7

Figure 4: Confocal fluorescence micrograph showing an enlarged view of an area of the adhesive line in Figure 3. In addition to filling the lumens and the dam-aged tissue regions the adhesive appears to have penetrated cell walls, particularly in the surface tissue layers of the upper ply (arrow pointing to lightgreen coloured cell walls). Scale bar = 25 µm.

Figure 5: FE-SEM micrograph taken in secondary electron imaging (SEI) mode. The contrast of the adhesive is similar to that of wood cell walls and thus theadhesive is poorly differentiated from wood tissues. Scale bar = 50 µm.

Page 4: CORRELATIVE MICROSCOPY Light,Confocal and Scanning ...€¦ · (LM), fluorescence confocal laser scanning microscopy (CLSM) and then field-emission scanning electron microscopy (FE-SEM)

with which it can physically or chemicallyreact, such as the phenol-formaldehyde adhe-sive used in our work, thus increasing theirbrightness under SEM-BEI viewing.

The micrograph in Figure 6, which was takenwith the FE-SEM in the BEI mode illustrates thevalue that BEI adds to imaging osmium tetrox-ide-reacted phenol-formaldehyde adhesive. Incomparison to the rather poor differentiationbetween wood cell walls and the adhesiveachieved when imaged with the FE-SEM in thesecondary electron imaging (SEI) mode, whichis the standard imaging mode in the SEM work(Figure 5), excellent differentiation betweenwood cell walls and the adhesive was achievedwhen the same sections were subsequentlyexamined in the BEI mode (Figure 6) for thereason mentioned above. This enabled adhe-sive distribution within wood tissues to beclearly visualized, as shown in Figures 6 and 7.

In Figure 6, a low magnification FE-SEM-BEIimage, the pathway of adhesive penetrationinto the damaged surface tissues of a ply isclearly visible. The adhesive is present withinthe lumens of tracheids and also in the dam-aged tissue regions containing cracks.

The rather intricate pattern of adhesive dis-tribution within surface wood tissues is observ-able in the high resolution image shown inFigure 7, which is a high magnification view ofa region of the wood-adhesive interface in Fig-ure 6, The presence of adhesive is detectableeven in very small dimension cell wall cracks,which light microscopy and CLSM could notresolve.

C O N C L U S I O N SThe unique imaging approach that weemployed, involving sequential viewing of thesame sections taken through wood-adhesiveinterface, by correlative LM, CLSM and FE-SEM-BEI microscopy, provided new fundamen-tal information on wood-adhesive interaction,which forms the basis for understanding whythe mechanical interlocking of adhesive intowood tissues is considered important in adhe-sive performance.

Effective interlocking of wood tissues by anadhesive involving adhesive penetration intoall accessible micro- and nano-pores along theadhesive line is even more crucial for optimaladhesive performance where mechanicallyweakened wood surfaces, such as in woodplies produced commercially by mechanicalpeeling of logs, have to be stabilized andstrengthened.

R E F E R E N C E S 1. Bekhta, P. and Marutzky, R. Reduction of glue consumption

in the plywood production by using previously compressedveneer. Holz Roh Werkst. 65:87-88, 2007.

2. Singh, A. P. et al. The effect of planing on the microscopicstructure of Pinus radiata wood cells in relation to pene-tration of PVA glue. Holz Roh Werkst. 60:333-341, 2002.

3. Smith, M. J. et al. Wood-thermoplastic adhesive interface -method of characterization and results. Intern. J. AdhesionAdhesives 22:197-204, 2002.

4. Kamke, F. A. and Lee, J. N. Adhesive penetration in wood - areview. Wood Fiber Sci. 39:205-220, 2007.

5. Singh, A. P. and Dawson, B. S. W. Confocal microscope - avaluable tool for examining wood-coating interface. J.

MICROSCOPY AND ANALYSIS MAY 20088

Figure 6: FE-SEM micrograph of the same region of the section as in Figure 5 taken in backscattered electron imaging (BEI) mode. The adhesive is well differen-tiated from wood cell walls because of its much greater brightness as compared to cell walls. The adhesive has penetrated a few cells deep into axialtracheids, but more deeply into a cracked region (arrowhead). The adhesive is excluded from a surface tissue region containing highly compressed tracheids (arrow). Scale bar = 50 µm.

Figure 7: Higher magnification FE-SEM-BEI view of the compressed tissue region in Figure 6. The adhesive has penetrated the lumens of uncompressed surfacetracheids and cracks within damaged tissues, including very small dimension cell wall cracks (arrowheads) but is largely excluded from the lumens ofhighly compressed tracheids (arrow). Scale bar = 20 µm.

Coat. Technol. Research 1:235-237, 2004.6. Singh, A. P. et al. A novel method for high-resolution

imaging of coating distribution within a rough-texturedplywood surface. J. Coat. Technol. Research 4:207-210, 2007.

7. Harris, L. G. et al. Contrast optimization for backscatteredelectron imaging of resin embedded cells. ScanningMicroscopy 13:71-81, 1999.

8. Herzog, B. et al. Electron microprobe imaging for thecharacterization of polymer matrix composites. Composites:Part A 35:1075-1080, 2004.

9. DeNee, P. B. and Carpenter, R. L. Application of heavy metalstaining (OsO4)/backscattered electron imaging techniqueto the study of organic aerosols. In Proceedings of the 14thAnnual Conference of the Microbeam Analysis Society, SanAntonio, 8-10, 1979.

10. Schraufnagel, D. E. and Ganesan, D. P. Tracers in vascularcasting resins enhance backscattering brightness. ScanningMicrosc. 12:631-639, 1998.

©2008 John Wiley & Sons, Ltd