Accepted Manuscript

Wood pulp fiber modification by layer-by-layer (LBL) self-

assembly of chitosan/carboxymethyl cellulose complex: Confocal
microscopy characterization

Ricardo Klaus Kramer, Francisco Eduardo Gontijo Guimarães,

Antonio Jose Felix Carvalho

PII: S0167-7322(18)31701-X
DOI: doi:10.1016/j.molliq.2018.10.055
Reference: MOLLIQ 9800
To appear in: Journal of Molecular Liquids
Received date: 31 March 2018
Revised date: 10 October 2018
Accepted date: 12 October 2018

Please cite this article as: Ricardo Klaus Kramer, Francisco Eduardo Gontijo Guimarães,
Antonio Jose Felix Carvalho , Wood pulp fiber modification by layer-by-layer (LBL)
self-assembly of chitosan/carboxymethyl cellulose complex: Confocal microscopy
characterization. Molliq (2018), doi:10.1016/j.molliq.2018.10.055

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 ACCEPTED MANUSCRIPT

Wood pulp fiber modification by layer-by-layer (LBL) self-

assembly of chitosan/carboxymethyl cellulose complex:
Confocal microscopy characterization
Ricardo Klaus Kramera, Francisco Eduardo Gontijo Guimarãesb, Antonio Jose
Felix Carvalhoa,∗
a
São Carlos School of Engineering, University of São Paulo, CEP 13566-590, São Carlos, São Paulo, Brazil
b
São Carlos Institute of Physics, University of São Paulo, São Carlos, São Paulo, Brazil

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Abstract

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Bleached cellulose wood pulp fibers from eucalyptus were modified by layer-by-layer (LbL)
self-assembly of chitosan/carboxymethylcellulose (CH/CMC) complex. Pulp fibers modified

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with 10, 20 and 30 bilayers of CH/CMC were prepared and characterized by scanning electron
microscopy (SEM) and by laser scanning confocal microscope (LSCM) using the two-photon
absorption technique. CH/CMC complex was also deposited onto glass and regenerated
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cellulose (cellophane) films in order to evaluate the depositions process. CH/CMC films with
up to 2,750 nm thickness deposited on glass and cellophane were easily observed by SEM,
however the films deposited onto a wood pulp fiber surface were visualized only using the
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LSCM technique since it is very difficult to observe the interface between CH/CMC and
cellulose by SEM. Using confocal microscopy allows a precise characterization of the
CH/CMC films, which were observed outside and inside the wood pulp fibers. This result
also shows that during LbL processing, the polyelectrolyte solution penetrates very fast inside
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the hollow fiber leading to a complete surface modification of the fiber. Fibers can be
considered as hollow tubes with an inner diameter of the order of 15-20 μm and external
diameter of the order of 25 μm with a cellulose wall of 5 μm.
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Keywords: Layer-by-layer; cellulose fiber modification; confocal microscopy; two-

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photon absorption.

∗Corresponding author: Antonio Jose Felix Carvalho, E-mail address: toni@sc.usp.br , Tel. + 55 16 33738679
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1. Introduction
Cellulose and chitin are undoubtedly the most abundant natural polymers on earth [1,2],
and the most relevant cellulose is mainly produced from wood; however annual plants such
as sugarcane are potential sources for high quality cellulose. Wood pulp has several
applications and the most important are chemical ones, such as carboxymethylcellulose and
the paper industry [3,4]. New advanced materials obtained from chemical pulps are becoming
increasingly important, such as motivating research on nanocelluloses in several areas [5,6].
Chitin, the second most abundant natural polymer [2] is produced from exo-skeleton of
crustaceans and can be converted into chitosan, a cationic polyelectrolyte that is very
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interesting due to its special properties such as high capability to form strong thin films and
its bactericide and fungicide properties [2].
Bleached wood pulp is a commodity material produced on a large scale mainly used in
the paper industry, which due to the expected decrease in the use of paper has been the focus
of research for developing new applications. Modifying cellulose pulp fiber using a green-
water based process can boost the use of cellulose pulp in new applications. Nanostructured
multilayer polyelectrolyte materials formed by layer-by-layer (LbL) self-assembly [7] is one
viable process to modify cellulose fibers and has been the focus of several studies in this field,
including nanocellulose and paper modification [8–10]. The LbL self-assembly technique uses
at least two oppositely charged polyelectrolytes, which are successively deposited in alternate

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layers on a substrate from their aqueous solution. The thickness of the obtained film is

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generally directly proportional to the number of deposited bilayers. Among the bilayers, other
materials can be inserted, such as nanoparticles, other polyelectrolytes or ionic substances of

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low molar mass capable of forming complex nanostructured structures [7,11,12].
When the material to be covered is very similar to the polyelectrolytes used in the LbL

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process, the characterization of the produced nanostructured materials is a challenge. One of
the mentioned films for cellulose fiber modification is the complex formed by the cationic
chitosan (CH) and anionic carboxymethylcellulose (CMC) using LbL self-assembly,
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producing an CH/CMC-complex nanostructured film with thickness ranging from a few
nanometers to microns [13][14].
These films can be characterized by scanning electron microscopy (SEM) when deposited on
flat substrates such as glass and silicon, however when deposited on particles and especially on
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cellulose fibers, this technique is not suitable for its characterization. One technique with the
potential to characterize composite systems made with materials having a similar structure is laser
scanning fluorescence confocal microscope (LSCM) that provides a powerful approach for
characterizing cellulosic fibers and nanofibers covered with other polysaccharides since the
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fluorescence of these materials can be very distinct [15].

Here, we describe the use of the two-photon absorption technique for the characterization
of bleached cellulose wood pulp fiber modified with chitosan/carboxymethylcellulose
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complex obtained by LbL self-assembly. Chitosan and carboxymethylcellulose form

intermacromolecular complexes due to the electrostatic interaction between two opposing
signal polyelectrolytes [16].The two-photon technique used consists of the absorption of two
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photons with half energy (and identical frequency) for sample excitation and subsequent
emission of a photon due to fluorescence [17]. This approach allows a significant
improvement in confocal images in a sub micro/nanometric range, improving the
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visualization of the different materials, cellulose and CH/CMC making the interface clear
between these materials. Experiments replacing wood pulp fiber by glass and cellulose film
(cellophane) substrates were also performed in order to attest the deposition of CH/CMC and
validate the method.

2. Materials and Methods

2.1. Materials
Never-dried bleached Kraft cellulose wood pulp from eucalyptus (hardwood) was kindly

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provided by Suzano Celulose e Papel S.A., Brazil. Chitosan with 59,000 g∙mol-1 molecular
weight, a deacetylation degree (DS) of 89.5% and a high viscosity sodium carboxymethyl
cellulose (η ~2,000 – 3,000 cps, corresponding approximately to a molecular weight of
350,000 g∙mol-1) and a sodium content of 6.5% with a degree of substitution of 0.7 were used.
For substrates for LbL deposition, microscopy glass slides and cellophane (xanthate
regenerated cellulose) films were used with 0.2 mm thickness supplied by Coopercel, Brazil.
Fluorescein isothiocyanate was supplied by Sigma-Aldrich.

2.2. Chitosan grafted with fluorescein isothiocyanate (FITC-CH)

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FITC-CH was prepared according the procedure proposed by Qaqish and Amiji [18].
Briefly, 25 mL of 1 mg/mL methanol solution of fluorescein isothiocyanate was slowly added

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to 50 mL of 1 g/L chitosan in 0.1 M acetic acid dried methanol solution under agitation. The

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mixture was kept agitated for 60 min. The product was precipitated by adding the solution
obtained from a 0.1 M solution of NaOH. The FITC/d-glucosamine residue ratio is of about
1:50.

2.3. LbL film preparation

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2.3.1. LbL films on glass and cellophane substrates.
The glass substrate was washed with Extran, followed by KOH 1 M methanol solution in
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an ultrasound bath for 5 min. After that, the glass substrates were washed in Milli-Q ultrapure
water and dried in air before use. Cellophane films were attached to a plastic holder and
washed with Milli-Q ultrapure water before using it to remove soluble contaminants. Chitosan
solution for film deposition was 1 g/L with 1wt% of glacial acetic acid. Before using the
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solution, it was stirred for 48 hours and filtered with regular qualitative filter paper using a
Büchner funnel with a vacuum. The pH of the solution was adjusted to 3 by adding glacial
acetic acid. CMC solution was 1 g/L prepared with Milli-Q water. The solution was stirred
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for 24 hours and filtered with regular qualitative filter paper using a Büchner funnel with a
vacuum. The pH was adjusted to 3 by adding glacial acetic acid. These solutions of chitosan
and CMC have 20 mM NaCl for ionic strength control. For film preparation, the glass or
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cellophane substrate was first immersed in the cationic polyelectrolyte solution (chitosan) for
5 minutes, removed and washed three times with Milli-Q water and then immersed in the
anionic polyelectrolyte solution (CMC) for 5 minutes, removed and washed again in Milli-Q
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water to remove any remaining polyelectrolyte. The process was repeated according to the
number of desired bilayers.

2.3.2. Wood pulp fiber coating with chitosan/carboxymethylcellulose complex by LbL.

Chitosan and CMC solutions were prepared as described previously for the glass and
cellophane modification. The coating process started by mixing 50 mL of 1 g/L chitosan
aqueous solution (cationic polyelectrolyte) and 50 mL of a 0.5 wt% pulp suspension. The
pulp suspension was prepared by homogenizing the never-dried pulp in 20 mM NaCl solution
with an Ultra-Turrax IKA model T25 operating at 20,000 rpm for 2 min. The mixture was
then agitated for 5 min with the Ultra-Turrax and then the suspension was filtered and the

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fibers washed thoroughly three times in 50 mL of Milli-Q water each time to remove any
remaining chitosan. The ensuing fibers were dispersed in 50 mL of 1 g/L of CMC aqueous
solution (anionic polyelectrolyte) and this mixture was homogenized with the Ultra-Turrax
operating at 20,000 rpm for 5 min. The fibers were then centrifuged and washed as previously
done and the process was repeated to produce the desired number of bilayers.
This procedure was used both for non-modified chitosan and for chitosan grafted
fluorescein isothiocyanate.

2.4. Microscopy Characterization

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2.4.1. Scanning electron microscopy (SEM).
The images were obtained using a FEI inspect F50 electron microscope with a working

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distance of 10.0 mm and a voltage of 2 kV. The samples were sputter-coated with 20 nm gold.

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2.4.2. Confocal laser scanning microscopy (CLSM).
The images were taken with a Zeiss (inverted model LSM 780), with Ti: Sapphire LASER, a
40× objective lens, 1.2 NA and 0.28mm of work distance in water. Fibers were placed on a
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microscopy glass slide in a few drops of immersion water and analyzed directly in the confocal
microscopy.
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3. Results and discussion

Model films were prepared on flat surfaces such as glass slides and regenerated cellulose
(cellophane) and were characterized by scanning electron microscopy of the cross-section
Fig. 1 shows the scanning electron microscopy images of cross-sectional fractured layers of
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CH/CMC bilayers deposited on a glass substrate.

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Fig. 1. SEM image of glass coated with 20 bilayers of CH/CMC complex.

The thick layer of CH/CMC shows a regular profile with about 1200 nm thickness, which
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considering the fact that it is formed by 20 bilayers, has an average thickness of about 60 nm
per bilayer. This is in agreement with previously reported results for similar deposition
conditions [19]. It can be observed that the CH/CMC layer is very regular and compact
forming a continuous and adherent coat on the glass slide, since no signal of debonding was
observed.

Fig. 2a-c shows the SEM images of cross-section of fractured films of a cellophane
substrate coated with 10, 20 and 30 bilayers of chitosan/CMC, respectively. The thicknesses
of CH/CMC films for 10, 20 and 30 bilayers are respectively 780, 2034 and 2748 nm. The
relationship between the thickness and number of bilayers are presented in Fig. 2d, in which

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a linear correlation can be observed. The average thickness per bilayer is about 90 nm, and is

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higher than in glass, however of the same order and in accordance with previous work by us
and other researchers [19,20].

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3000
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Thinckness (nm)

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1000
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Number of bilayers

Fig. 2. SEM image of cellophane substrate coated with a) 10 bilayers, b) 20 bilayers and c) 30 bilayers
of CH/CMC and d) relation between number of bilayers and film thickness.

The observation of fragile fracture surfaces in Fig. 2 for the cellophane LbL coated films
shows that even for thicker films with approximately 3,000 nm, no deboning of the LbL layer
was observed showing evidence of a strong adhesion with cellulose. The fragile fracture
surface shows that the films are homogeneous and compact forming a solid barrier between

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the external surface and the cellophane surface.

Fig. 3 (a) and (b) shows the morphology of the neat cellulosic pulp and a 3 bilayer LbL
modified cellulose fiber, respectively. The fibers showed a thickness of approximately 15 to
20 µm and it can be observed that differences in thickness or morphology cannot be identified
due to fiber modification by CH/CMC film. For 3 bilayers, a thickness of approximately 270
nm is expected, which is low if compared to the fiber diameter.

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Fig. 3. SEM images of cellulose pulp (a) untreated (b) coated by 3 bilayers of CH/CMC.
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The images in Fig. 3b show that scanning electron microscopy is not effective for pulp fiber
characterization making it impossible to determine the presence of the LbL film and to estimate its
thickness.
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Fig. 4 (a) shows the confocal plane of the untreated fiber, showing the fiber walls and their
bulk, with a total thickness of about 20 µm. Fig. 4 (b) shows the emission spectrum of the
fiber, taken in the point scanning mode, represented by a circle in the fiber wall in Fig. 4 (a).
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The maximum emission occurs at about 440 nm and is associated to the other constituents of
cellulose pulp (hemicelluloses and residual lignin) since cellulose itself does not present an
emission at the wavelength interval shown. In Fig. 4 (c) the hollow fiber profile is shown,
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observed by the cross section indicated by the arrow in Fig. 4(a). The fiber wall was
approximately 5.8 µm and the fiber external diameter was about 20 µm thick.
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(b)
80

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Intensity
40

20

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400 450 500 550 600 650 700

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Emission wavelength (nm)

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120 (c)

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Distance (µm)
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Fig. 4. (a) Confocal microscopy image of the neat wood pulp cellulose fiber without coating in
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aqueous media; (b) cellulose emission spectrum taken in the circle marked in image and (c)
confocal cut profile of the fiber (arrow shown in the image).
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Fig. 5a shows the confocal microscopy image of the 2 bilayer CH/CMC coated fiber. The
coating or the interface cannot be identified since the emissions of cellulose and chitosan
overlap. This is because both of them occur at 440 nm. Fig. 5b shows the cellulose and
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chitosan spectra, showing the signal overlap.

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(a)
(b)
200

150
Chitosan

Intensity
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Cellulose

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Distance (um)

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Fig. 5. a) Confocal microscopy image of the fiber coated with 2 bilayer of CH/CMC in aqueous media
and (b) confocal cut of the covered fiber. Note: The colors on the chart are illustrative and do not
match the wavelength emitted by each substrate.

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Since confocal microscopy was not able to identify the CH/CMC from the fiber in a coated
fiber, a fluorescent chitosan was used to prepare the self-assembly CH/CMC films. For this
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purpose, a chitosan modified with fluorescein isothiocyanate was used. Fig. 6 a shows the
cellulose fiber without chitosan and Fig 6b-c shows the confocal images of LbL modified
fiber with isothiocyanate modified chitosan (FITC-CH).
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Fig. 6. a) Confocal microscopy image of the non modified fiber and (b-c) confocal images of the
FITC-CH modified cellulose fiber.

FITC emission occurs at about 520 nm, while cellulose emission occurs at 440 nm,
however due to the very high intensity of light emission from FITC cellulose from FITC-CH
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cannot be identified. Although these results have shown that the FITC-CH used does not
identify the components of the hybrid material due to emission saturation, it is possible that
the use of smaller amounts of fluorescein in CH modification allows its use in LbL films. We
decided to use another confocal microscopy technique for a fiber modified with the non-
modified chitosan. The aim was to use a technique which can distinguish cellulose from
chitosan and carboxymethylcellulose, the two-photon fluorescent emission microscopy. This
technique has been successfully used to identify cellulose and chitosan, allowing the
identification of each one, even in combination due to the different signals each one emits
[21,22].
Fig. 7 shows images using the two-photon emission technique for a cellulose wood pulp

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fiber collated with 2 bilayers of CH/CMC film.

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CH/CMC layer

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(a) (b)
100

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chitosan
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Intensity

emission
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cellulose cellulose
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emission emission

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Distance (µm)
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Fig. 7. a) Confocal microscopy image of a 2 bilayer CH/CMC coated fiber (~0.2 μm) using the two-
photon emission technique and b) confocal profile along the fiber section showing the cellulose and
chitosan emissions.
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Fig. 7b shows the profile of cellulose and chitosan from CH/CMC layer emission. The
chitosan line profile shows that CH/CMC layers lie inside and outside the hollow cellulose
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fiber, corresponding to 4 emission peaks; one inside and one outside of the two walls of the
fiber profile. The internal adsorption of chitosan shows that chitosan and CMC
polyelectrolytes penetrate into the inner part of the fiber forming a CH/CMC self assembly in
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the internal fiber wall, resulting in a complete surface coating of cellulose wood pulp fiber.

4. Conclusion

Confocal two-photon absorption laser scanning microscopy (CLSM) was successfully used to
characterize wood pulp cellulose fiber coated with chitosan/carboxymethylcellulose adopting a
layer-by-layer self-assembly method. Scanning electron microscopy was also used, however it
was successful only for the characterization of similar films on flat substrates such as glass and
regenerated cellulose (cellophane). The thin film of chitosan/carboxymethylcellulose complex
was observed both outside and inside the fiber walls showing that polyelectrolyte solutions used

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in LbL penetrate very fast into the fiber probably by its pores or damaged portions. Confocal
microscopy proved to be an efficient way to characterize wood pulp fiber modified with a
polysaccharides complex. The efficiency of fiber modification using the L-b-L method was also
confirmed.

Acknowledgements
The authors gratefully acknowledge CNPq and Capes for the financial support, Suzano
Celulose e Papel S.A for providing refined “never-dried” cellulose fibers, as well as Debora T.
Balogh (IFSC-USP, São Carlos, SP, Brazil) for the support in chemical modification of fibers.
This study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível

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Superior - Brasil (CAPES) - Finance Code 001.

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References

RI
[1] A. Gandini, M.N. Belgacem, The State of the Art, in: A. Gandini, M.N. Belgacem (Eds.), Monomers, Polym.

SC
Compos. from Renew. Resour., Elsevier, Amsterdam, 2008: pp. 1–16.
[2] C. Peniche, W.M. Argüelles-Monal, F.M. Goycoolea, Chitin and Chitosan: Major Sources, Properties and
Applications, in: A. Gandini, M.N. Belgacem (Eds.), Monomers, Polym. Compos. from Renew. Resour.,
Elsevier, Amsterdam, 2008: pp. 517–542.
NU
[3] D. Klemm, B. Heublein, H.-P. Fink, A. Bohn, Cellulose: Fascinating Biopolymer and Sustainable Raw
Material, Angew. Chemie Int. Ed. 44 (2005) 3358–3393. doi:10.1002/anie.200460587.
[4] D. Fengel, G. Wegener, Wood—chemistry, ultrastructure, reactions, Walter de Gruyter, Berlin and New York,
1984: pp. 414-481, 482-525.
[5] D. Klemm, F. Kramer, S. Moritz, T. Lindstr??m, M. Ankerfors, D. Gray, A. Dorris, Nanocelluloses: A new
MA

family of nature-based materials, Angew. Chemie - Int. Ed. 50 (2011) 5438–5466.

doi:10.1002/anie.201001273.
[6] O. Nechyporchuk, M.N. Belgacem, J. Bras, Production of cellulose nanofibrils: A review of recent advances,
Ind. Crops Prod. 93 (2016) 2–25. doi:10.1016/j.indcrop.2016.02.016.
[7] G. Decher, Fuzzy Nanoassemblies: Toward Layered Polymeric Multicomposites, Science (80-. ). 277 (1997)
ED

1232–1237. doi:10.1126/science.277.5330.1232.
[8] Z. Zheng, J. McDonald, R. Khillan, Y. Su, T. Shutava, G. Grozdits, Y.M. Lvov, Layer-by-layer nanocoating of
lignocellulose fibers for enhanced paper properties, J. Nanosci. Nanotechnol. 6 (2006) 624–632. doi:Doi
PT

10.1166/Jnn.2006.081.
[9] H. Orelma, I. Filpponen, L.-S. Johansson, J. Laine, O.J. Rojas, Modification of Cellulose Films by Adsorption
of CMC and Chitosan for Controlled Attachment of Biomolecules, Biomacromolecules. 12 (2011) 4311–4318.
doi:10.1021/bm201236a.
CE

[10] H. Li, S. Fu, L. Peng, H. Zhan, Surface modification of cellulose fibers with layer-by-layer self-assembly of
lignosulfonate and polyelectrolyte: effects on fibers wetting properties and paper strength, Cellulose. 19 (2012)
533–546. doi:10.1007/s10570-011-9639-3.
[11] Q. Wang, P.J. Hauser, Developing a novel UV protection process for cotton based on layer-by-layer self-
AC

assembly, Carbohydr. Polym. 81 (2010) 491–496. doi:10.1016/j.carbpol.2010.03.008.

[12] H. Li, S. Fu, L. Peng, Surface modification of cellulose fibers by layer-by-layer self-assembly of
lignosulfonates and TiO2 nanoparticles: Effect on photocatalytic abilities and paper properties, Fibers Polym.
14 (2013) 1794–1802. doi:10.1007/s12221-013-1794-8.
[13] Q. Xing, S.R. Eadula, Y.M. Lvov, Cellulose fiber-enzyme composites fabricated through layer-by-layer
nanoassembly, Biomacromolecules. 8 (2007) 1987–1991. doi:10.1021/bm070125x.
[14] T. Wu, R. Farnood, Cellulose fibre networks reinforced with carboxymethyl cellulose/chitosan complex layer-
by-layer, Carbohydr. Polym. 114 (2014) 500–505. doi:10.1016/j.carbpol.2014.08.053.
[15] A.T. Horvath, A.E. Horvath, T. Lindström, L. Wågberg, Diffusion of cationic polyelectrolytes into cellulosic
fibers, Langmuir. 24 (2008) 10797–10806. doi:10.1021/la800669d.
[16] C. Rosca, M.I. Popa, G. Lisa, G.C. Chitanu, Interaction of chitosan with natural or synthetic anionic
polyelectrolytes. 1. The chitosan–carboxymethylcellulose complex, Carbohydr. Polym. 62 (2005) 35–41.
doi:10.1016/j.carbpol.2005.07.004.
10
 ACCEPTED MANUSCRIPT

[17] W. Denk, J. Strickler, W. Webb, Two-photon laser scanning fluorescence microscopy, Science (80-. ). 248
(1990) 73–76. doi:10.1126/science.2321027.
[18] R. Qaqish, M. Amiji, Synthesis of a fluorescent chitosan derivative and its application for the study of
chitosan–mucin interactions, Carbohydr. Polym. 38 (1999) 99–107. doi:10.1016/S0144-8617(98)00109-X.
[19] D.C. Venanzi, F.J. Pavinatto, D.T. Balogh, O.N. Oliveira Junior, A.J.F. Carvalho, Self-standing lbl thin films
of chitosan/carboxymethylcellulose complexes, (2008) 22690.
[20] T. Radeva, K. Kamburova, I. Petkanchin, Formation of polyelectrolyte multilayers from polysaccharides at
low ionic strength, J. Colloid Interface Sci. 298 (2006) 59–65. doi:10.1016/j.jcis.2005.12.010.
[21] H. Li, L. Peng, Antimicrobial and antioxidant surface modification of cellulose fibers using layer-by-layer
deposition of chitosan and lignosulfonates, Carbohydr. Polym. 124 (2015) 35–42.
doi:10.1016/j.carbpol.2015.01.071.
[22] K. Tømmeraas, S.P. Strand, W. Tian, L. Kenne, K.M. Vårum, Preparation and characterisation of fluorescent
chitosans using 9-anthraldehyde as fluorophore, Carbohydr. Res. 336 (2001) 291–296. doi:10.1016/S0008-

T
6215(01)00275-0.

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Graphical Abstracts

Wood pulp modification by LBL self-

assembly of chitosan/carboxymethyl

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CH/CMC layer

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100 (b)

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chitosan
80 emission Cellulose fiber
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60 chitosan
Intensity

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cellulose cellulose
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Highlights

 Chitosan/carboxymethylcellulose self-assembly over wood pulp cellulose fibers

 Carbohydrate complex films characterization by Laser Scanning Confocal
Microscope
 Layer-by-Layer thin films of carbohydrates onto cellulose fiber; detection method
 Two photon excitation microscopy of chitosan/carboxymethylcellulose films

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