Micron 41 (2010) 263267

Contents lists available at ScienceDirect

Micron
journal homepage: www.elsevier.com/locate/micron

Short communication

Visualising impregnated chitosan in Pinus radiata early wood cells using light and
scanning electron microscopy
Adya P. Singh, Tripti Singh *, Catherine L. Rickard
Scion, 49 Sala Street, Private Bag 3020, Rotorua 3046, New Zealand

A R T I C L E I N F O

A B S T R A C T

Article history:
Received 2 October 2009
Received in revised form 13 November 2009
Accepted 17 November 2009

Chitosan, a deacetylated product of an abundant naturally occurring biopolymer chitin, has been used in
a range of applications, particularly in food and health areas, as an antimicrobial agent. In the work
reported here Pinus radiata wood was impregnated with chitosan as an environmentally compatible
organic biocide (Eikenes et al., 2005a,b) to protect wood against wood deteriorating microorganisms and
to thus prolong the service life of wooden products. We developed sample preparation techniques
targeted to visualise impregnated chitosan within wood tissues using light microscope and eldemission scanning electron microscope (FE-SEM). Sections were viewed with the light microscope
without staining with a dye as well as after staining with the dye toluidine blue. Light microscopy was
also undertaken on sections that had been stained with 1% aqueous osmium tetroxide (OsO4). For SEM
observations, the sections were treated with OsO4 and then examined with the FE-SEM, rst in the
secondary electron imaging mode (SEI) and then in the backscattered electron imaging (BEI) mode,
imaging the same areas of a section in both SEI and BEI modes. The preparation techniques employed and
the combined use of light and scanning electron microscopy provided valuable complementary
information, revealing that chitosan had penetrated into the cavities (cell lumens, intercellular spaces) of
all sizes present within wood tissues and had also impregnated early wood cell walls. The information
obtained is discussed in relation to its importance in further development of chitosan formulations and
renement of impregnation technologies to optimise chitosan impregnation into and distribution within
wood tissues as well as in assessing chitosan efcacy.
2009 Elsevier Ltd. All rights reserved.

Keywords:
Backscattered electron imaging
Chitosan
Field-emission scanning electron
microscope
Light microscope
Osmium tetroxide
Pinus radiata wood

1. Introduction
Wood is a versatile biomaterial with uses in a wide range of
applications because of its high strength relative to weight, aesthetic
appeal and relative economic production. However, when exposed
to moist or humid conditions wood can be attacked by decay
microorganisms and needs protection for a desired service life of the
products made from it. The traditional protection methods are based
largely on the use of inorganic and synthetic chemicals, although
some low-toxic chemicals have been used over the last few decades
but the public concerns remain about the use of these chemicals.
Effort made in the last few years to nd alternative environmentally
compatible bio-based protection systems have resulted in rapid
progress being made in the exploration and use of natural products
derived from plants, microorganisms and other sources. Chitosan,
which is a deacetylation product of chitin, the second most abundant

* Corresponding author. Tel.: +64 7 343 5899; fax: +64 7 343 5507.
E-mail addresses: adya.singh@scionresearch.com (A.P. Singh),
tripti.singh@scionresearch.com (T. Singh), catherine.rickard@scionresearch.com
(C.L. Rickard).
0968-4328/$ see front matter 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.micron.2009.11.006

biopolymer on earth after cellulose, has been widely used in food,

pharmaceutical and medical applications (Liu et al., 1997; Rhoades
and Roller, 2000; Singla and Chawla, 2001; Ueno et al., 2001; Yang
and Hon, 2009) primarily because of its unique biological and widespectrum antimicrobial properties (Raafat et al., 2008 and references
therein). Combinations of chitosan and other organic or inorganic
materials have also been used effectively against bacteria and fungi
(Wang et al., 2004; Chen et al., 2005).
For the last few years attempts have been made to investigate the
potential of chitosan and its derivatives as organic biocides for wood
protection (Eikenes et al., 2005a,b; Guo et al., 2006; Vesentini et al.,
2007; Singh et al., 2008b; Chittenden and Singh, 2009) and also to
enhance wood properties, such as stiffness (Torr et al., 2006).
Uptake into and distribution within wood are among the factors
that affect the efcacy of a protection agent with which wood is
impregnated, and methods have to be developed that can detect
the presence of impregnating agents and also provide information
on whether the agents impregnate only cell lumens or can also
penetrate cell walls. We examined sections from chitosan
impregnated radiata pine (Pinus radiata) wood by light and
scanning electron microscopy to understand the pattern of
distribution of impregnated chitosan within early wood tissues.

264

A.P. Singh et al. / Micron 41 (2010) 263267

2. Materials and methods

A 2% (w/v) chitosan stock solution was prepared using low
molecular weight chitosan (molecular weight range 50190 kDa,
SigmaAldrich Ltd., Milwaukee, WI, USA). The chitosan was
solubilised in 1% acetic acid and blended for 20 min at high speed
(Waring blender). Subsequently, the chitosan oligomer was
prepared by nitrous acid depolymerisation. End sealed sawn
radiata pine boards were treated with 1.5% (w/v) chitosan oligomer
using pressure (at 1400 kPa) by bethel process. Cross-sections from
mid-samples were oven dried (50 8C) after 2 days of air drying.
Small pieces cut from the impregnated timber were sectioned
by hand using single edge razor blades and also using a sliding
microtome. However, imaging was done only on hand-cut
sections, as microtome cutting produced some distortions to the
impregnated tissues, which were absent in the sections obtained
using hand-held razor blades.
2.1. Light microscopy (LM)
Sections cut transversely through chitosan impregnated
radiata pine wood at a thickness of 150200 mm were examined
and photographed with Zeiss Photomicroscope II. Some sections
were also stained with 0.05% aqueous toluidine blue prior to
examination with the photomicroscope. Light microscopy was
also undertaken on sections that had been stained with 1%
aqueous osmium tetroxide (OsO4) for 1030 min at room
temperature. Sections for scanning electron microscopy were
cut from the same blocks that had been used to produce sections
for light microscopy.
Fig. 2. Light micrograph of a section, that had been stained with toluidine blue,
shows excellent colour differentiation between chitosan (orange colour) and wood
cell walls (blue colour). Chitosan has lled cell lumens (arrows) and also
impregnated cell walls (arrowheads). Bar = 30 mm. (For interpretation of the
references to colour in this gure legend, the reader is referred to the web version of
the article.)

2.2. Scanning electron microscopy (SEM)

Additional hand-cut transverse sections from the chitosan
impregnated radiata pine wood were processed for examination
and imaging with SEM, using the protocol from an earlier work
(Singh et al., 2008a). The sections were treated with 1% aqueous
OsO4 for 2 h at room temperature, rinsed in distilled water and
then air-dried by clamping between two glass slides to prevent
them from curling. The sections were mounted on carbon discs
applied to stubs and then coated with chromium in a sputter coater
prior to examination with a JEOL 6700F eld-emission scanning
electron microscope (FE-SEM). The sections were rst imaged
under SEI (secondary electron imaging) mode and then under BEI
(backscattered electron imaging) mode. Thus, the same areas in a
section could be examined in the two different operational modes
to compare their usefulness for imaging chitosan material within
wood and understanding the pattern of its distribution within
tissues and individual cells.
Following settings were used to view the samples in the SEI
mode: working distance 15 mm, accelerating voltage 3 kV, emission
10 mA, probe current 7. The settings for the BSE mode were: working
distance 15 mm, accelerating voltage 15 kV, emission 20 mA, probe
current 9.
3. Results and discussion
Fig. 1. Light micrograph of a section that had not been stained with toluidine blue.
Deep orange coloured chitosan, which is distinguishable from the light orange
coloured wood cell walls, lls the lumen of some axial tracheids (arrows).
Bar = 30 mm. (For interpretation of the references to colour in this gure legend, the
reader is referred to the web version of the article.)

The advances achieved in this work in the micro-imaging

technique for visualising chitosan in suitably prepared and stained
sections and combining LM with FE-SEM, involving correlative

A.P. Singh et al. / Micron 41 (2010) 263267

Fig. 3. Low magnication light micrograph of a section that had been stained with
osmium tetroxide. Cell walls around lumens that are lled with chitosan (black
coloured regions) appear darker (arrow) than those associated with empty lumens
(arrowhead). Bar = 60 mm.

viewing of the same section area under both SEI and BEI modes,
yielded valuable information. This enhances our understanding of
the distribution of impregnated chitosan in radiata pine wood
tissues. Collectively, the micrographs illustrated in Figs. 17
provide evidence that chitosan was present in cell lumens (cell
wall lined empty spaces) and small intercellular cavities. There are
also indications that chitosan had penetrated into cell walls.
It was possible to detect the presence of chitosan in cell lumens
in the light micrographs taken from unstained sections, as the
deep orange coloured chitosan was distinguishable from the light
orange coloured cell walls (Fig. 1). The staining of sections with
toluidine blue, a stain widely used to contrast lignied plant
tissues (OBrien and McCully, 1969), greatly enhanced the
differentiation between orangish chitosan and bluish cell walls,
making it possible to readily examine the pattern of chitosan
within wood tissues. Judging by the light orange appearance of the
cell walls of those cells that contained chitosan in their cell
lumens, it appears that cell walls were also inltrated with
chitosan (Fig. 2). It appears that the stain was partly or completely
excluded from impregnated cell wall regions, depending upon the
extent to which chitosan had penetrated into cell walls. In
comparison, the walls of those cells that did not contain chitosan
in their lumens displayed the usual bluish colour of lignied cell
walls, that is achieved following staining of sections with
toluidine blue (OBrien and McCully, 1969), and the staining of
cell walls was fairly uniform.
Stronger support for chitosan impregnation of cell walls came
from light microscopic observations of sections that had been
stained with OsO4, which is known to complex with chitosan,
involving chemical interaction with chitosans amino groups
(Huang et al., 2003). As observable in Figs. 3 and 4, cell lumens

265

Fig. 4. High magnication light micrograph of a section that had been stained with
osmium tetroxide. Cell walls around lumens that are lled with chitosan (black
coloured regions) appear darker and more sharply dened (arrow) than those
associated with empty lumens (arrowhead). Bar = 30 mm.

contain black deposits, interpreted here to represent chitosan

material, and allowing some variability in the colour intensity the
cell walls around chitosan-lled lumens appear darker and more
sharply dened than those associated with empty lumens.

Fig. 5. Scanning electron micrograph of a section taken in the secondary electron

imaging mode. Chitosan (arrows) is poorly differentiated from wood cell walls
(arrowheads). Bar = 60 mm.

266

A.P. Singh et al. / Micron 41 (2010) 263267

Fig. 6. Scanning electron micrograph of the same section that appears in Fig. 5 was
taken in the backscattered electron imaging mode. The chitosan, which appears
intense bright and thus readily distinguishable from dull grayish cell walls, can be
seen in cell lumens of all sizes within wood tissues. Arrows identify large lumens
and arrowheads indicate very small size lumens of tracheid tips. Bar = 60 mm.

The scanning electron micrographs in Figs. 5 and 6 were taken

from the same area of a section, in the secondary electron imaging
(SEI) and backscattered electron imaging (BEI) modes respectively. A
comparison of Figs. 5 and 6 illustrates the usefulness of the
technique developed in relation to backscattered electron imaging
for enhancing visualisation of chitosan, and thereby more accurately
assessing the pattern of chitosan distribution within the impregnated wood tissues. In the images obtained using the SEI mode
chitosan was poorly differentiated from cell walls because of a
similar contrast (Fig. 5). In the images produced in the BEI mode
chitosan was readily detected, as it appeared very bright compared
to the gray coloured cell walls (Fig. 6). In low magnication BEI
images it was possible to examine the distribution of chitosan within
relatively large areas of a section, which was a distinct advantage
over LM and which would be useful in studies aimed to quantify the
proportion of tissues lled with chitosan. In higher magnication BEI
images (Fig. 7) the presence of chitosan was more readily detectable
in the spaces within wood tissues, including the very small size
cavities as intercellular spaces.
It has been demonstrated that the combination of suitable light
and scanning electron microscopic techniques provided complementary information and thus a more complete understanding of
the pattern of chitosan distribution within radiata pine wood
tissues. Imaging with LM after staining of sections with toluidine
blue and osmium tetroxide revealed presence of chitosan in cell
lumens as well as cell walls. However, because of the limitation
with depth of focus and resolution it was possible to bring into
focus only relatively small areas of a section. Furthermore, the
presence of chitosan within small size spaces, such as intercellular
cavities, could not be detected. The use of FE-SEM in combination
with BEI proved valuable in resolving the presence of chitosan in
the very small size intercellular cavities and in the lumens of
tracheid tips, which are greatly reduced in size due to tapering of
tracheids at either ends during cell differentiation. Two main
factors contributed to imaging excellence achieved using the
technical approach adapted. One, high resolution imaging capa-

Fig. 7. Scanning electron micrograph of a section taken at high magnication in the

backscattered electron imaging mode. Chitosan (*) is present within intercellular
spaces (arrowheads) of those tracheids that are lled with this material. Chitosan is
absent (arrows) from the intercellular spaces that are present among empty
tracheids. Bar = 15 mm.

bility of the FE-SEM itself. Two, intense brightness of the chitosan

material under BSE imaging mode, resulting from the chemical
interaction of the heavy metal stain OsO4 with chitosan, involving
its amino groups (Huang et al., 2003), and consequent enhancement in the output of backscattered electron signals. Biological
objects are poor emitters of backscattered electron signals, and
enhancement of signal yield requires sample treatment with high
atomic number stains and additives, such as heavy metals
(Dichiara et al., 1980; Ushiki and Fujita, 1986). OsO4 has been
used widely as a xative to preserve cellular components of both
animal and plant tissues, particularly for observations with
transmission electron microscope. OsO4 has also been used as a
heavy metal stain alone and in combination with other heavy
metals and additives for backscattered imaging of biological
samples in combination with SEM (Harris et al., 1999). BSE imaging
is also providing valuable information in assessing the performance of agents used for the enhancement of desirable properties
and also the service life of products made from wood (Singh et al.,
2007, 2008a). For example, penetration of a protective coating into
very small size (nano-size) cell wall cracks present in the surface
tissue layers of a textured plywood product was revealed by a
technique employing a combination of OsO4, which reacted with
the coating, and FE-SEM-BSE imaging (Singh et al., 2007). The new
information obtained in this study proved valuable in understanding in-service performance of the applied coating based on its cell
lumen and cell wall penetration characteristics.
In conclusion, the novel sample preparation techniques
developed and the combined use of LM and SEM to examine

A.P. Singh et al. / Micron 41 (2010) 263267

chitosan impregnated radiata pine wood provided information on

chitosan distribution within wood tissues that could not have been
achieved if either the LM or the SEM had been used alone. For
optimal protection of wood against wood deteriorating microorganisms it is important that bioactive agents in use not only ll
cell lumens and cavities but also impregnate cell walls. This can
enhance the adhesion of the bioactive agent present in the cell
lumen with that impregnating cell wall, establishing a tight seal
against the invading microorganisms. Thus, wood protection is
based on bioactivity of the impregnated agent as well as physical
protection of cell walls from it.
Acknowledgments
We thank Kourosh Nasheri and Colleen Chittenden for their
input into chitosan treatment of wood samples.
References
Chen, S., Wu, G., Zeng, H., 2005. Preparation of high microbial activity thiourea
chitosanAg+ complex. Carbohydr. Polym. 60, 3338.
Chittenden, C., Singh, T., 2009. In vitro evaluation of combination of Trichoderma
harzianum and chitosan for the control of sapstain fungi. Biol. Control 50, 262
266.
Dichiara, J.F., Rowley, P.P., Ogilvia, R.W., 1980. Backscattered electron imaging (BEI)
of parafn sections stained with heavy metal histopathologic stains, with
observations on some variables encountered in BEI. Scanning Electron Microsc.
3, 181188.
Eikenes, M., Alfredsen, G., Christensen, B., Militz, H., Solheim, H., 2005a. Comparison
of chitosan with different molecular weights as possible wood preservative. J.
Wood Sci. 51, 387394.
Eikenes, M., Alfredsen, G., Larnoy, E., Militz, H., Kreber, B., Chittenden, C., 2005b.
Chitosan for Wood ProtectionState of the Art. The International Research
Group on Wood Protection. Document No. IRG/WP-30378. IRG Secretariat,
Stockholm.
Guo, Z., Chen, R., Xing, R., Liu, S., Yu, H., Wang, P., Li, C., Li, P., 2006. Novel derivatives
of chitosan and their antifungal activities in vitro. Carbohydr. Res. 341, 351354.

267

Harris, L.G., ap Gwynn, I., Richards, R.G., 1999. Contrast optimisation for backscattered electron imaging of resin embedded cells. Scanning Microsc. 13, 71
81.
Huang, K., Lie, H.-W., Dou, X., Huang, M.-Y., Jiang, Y.-Y., 2003. Silica-supported
chitosanosmium tetroxide complex catalyzed vicinal hydroxylation of olens
using hexacyanoferrate (III) ion as a cooxidant. Polym. Adv. Technol. 14, 364
368.
Liu, L.-S., Liu, S.-Q., Liu, S.Y., Froix, Ng.M., Ohno, T., Heller, J., 1997. Controlled release
of interleukin-2 for tumour immunotherapy using alginate/chitosan porous
microspheres. J. Control. Release 43, 6574.
OBrien, T.P., McCully, M.E., 1969. Plant Structure and Development. The Macmillan
Company/Collier-Macmillan, Limited, London.
Raafat, D., von Bergen, K., Haas, A., Sahl, H.-G., 2008. Insights into the mode of action
of chitosan as an antibacterial compound. Appl. Environ. Microbiol. 74, 3764
3773.
Rhoades, J., Roller, S., 2000. Antimicrobial actions of degraded and native chitosan
against spoilage organisms in laboratory media and foods. Appl. Environ.
Microbiol. 66, 8086.
Singh, A.P., Dawson, B.S.W., Rickard, C.L., Singh, A., 2008a. Light, confocal and
scanning electron microscopy of wood-adhesive interface. Microsc. Anal. 22,
58.
Singh, A.P., Ratz, A., Dawson, B.S.W., 2007. A novel method for high-resolution
imaging of coating distribution within a rough-textured plywood surface. J.
Coat. Technol. Res. 4, 207210.
Singh, T., Vesentini, D., Singh, A.P., Daniel, G., 2008b. Effect of chitosan on physiological, morphological and ultrastructural characteristics of wood-degrading
fungi. Int. Biodeter. Biodegrad. 62, 116124.
Singla, A.K., Chawla, M., 2001. Chitosan: some pharmaceutical and biological
aspectsan update. J. Pharmacol. 53, 10471067.
Torr, K.M., Singh, A.P., Franich, R.A., 2006. Improving stiffness of lignocellulosics
through cell wall modication with chitosan-melamine co-polymers. NZ. J. For.
Sci. 36, 8798.
Ueno, H., Mori, T., Fujinada, T., 2001. Topical formulations and wound healing
applications of chitosan. Adv. Drug. Deliv. Rev. 52, 105115.
Ushiki, T., Fujita, T., 1986. Backscattered electron imaging. Its applications to
biological specimens stained with heavy metals. Arch. Histol. Jpn. 49, 139154.
Vesentini, D., Steward, D., Singh, A.P., Ball, R., Daniel, G., Franich, R., 2007. Chitosanmediated changes in cell wall composition, morphology and ultrastructure of
two wood-inhabiting fungi. Mycol. Res. 111, 875890.
Wang, X., Du, Y., Liu, H., 2004. Preparation, characterisation and antimicrobial
activity of chitosanZn complex. Carbohydr. Polym. 56, 2126.
Yang, H.-C., Hon, M.-H., 2009. The effect of the molecular weight of chitosan
nanoparticles and its application on drug delivery. Microchem. J. 92, 8791.