J Wood Sci (2008) 54:300–307
DOI 10.1007/s10086-008-0950-z© The Japan Wood Research Society 2008

ORIGINAL ARTICLE

Toshiharu Enomae · Yoon-Hee Han · Akira Isogai

Z-Directional distribution of fiber orientation of Japanese and western
papers determined by confocal laser scanning microscopy

Received: October 5, 2007 / Accepted: January 24, 2008 / Published online: April 16, 2008

Abstract Confocal laser scanning microscopy was applied
to visualize inside fibers stained with a fluorescent dye, andthe fiber orientation distribution in the thickness direction
was determined by image analysis from optically sliced
images. Fiber orientation angle and anisotropy were determined by the fast Fourier transform method, which holds
advantages over the conventionally applied Hough transform with regard to evaluation of fiber width-weighted
contribution and intrafiber segmental contribution. An
orthogonally layered Japanese paper handmade by theflow-sheet forming method resulted in a clear change offiber orientation angle from 0° to 90°. Machine-made woodcontaining printing paper showed the highest anisotropy,
while copier paper showed a low anisotropy. Thick papers
like the wood-free paperboard and recycled packaging
board showed that the maximum measured thickness was
about 150 mm. The depth limit to detect fluorescence wasconsidered to depend on the apparent density of the paper
and the light absorption character of the fibers.
Key words Confocal laser scanning microscopy · Fiber orientation · Handmade paper · Washi · Fourier transform

Introduction
In the papermaking industry, the use of innovative technologies such as the lamination of paper with other novel
T. Enomae (*) · Y.-H. Han1 · A. Isogai
Paper Science Laboratory, Department of Biomaterial Sciences,
Graduate School of Agricultural and Life Sciences, The Universityof Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Tel. +81-3-5841-8199; Fax +81-3-5841-5271
e-mail: enomae@psl.fp.a.u-tokyo.ac.jp
Present address:
1
National Archives and Records Service, Daejeon 302-701, Republicof KoreaPart of this study was presented at the 54th Annual Meeting of theJapan Wood Research Society, Sapporo, Japan, August 2004

materials and embedding of electronic microchips will
become more common in the near future. These new typesof paper products will go outside the reach of the conventional planer sheet structure and must be dealt with as athree-dimensional bulk structure. Therefore, analyticaltechniques that consider the three-dimensional structure ofpaper are predicted to become increasingly important. For
this purpose, several techniques have been established.
Mechanical slicing of paper with a microtome to make crosssections is a reliable but time-consuming method. Thincross-sectional slices made so can be observed with an
optical microscope. Developmentally, consecutive smoothcross sections of epoxy-embedded coating layers after successive grinding and polishing were obtained and reconstructed as a three-dimensional representation to visualize
pigment particles and pores.1 Recently, the focused ion
beam has emerged in place of the microtome to make intact
cross sections.2
Fiber orientation is one of the major parameters that
dominate the physical properties of paper. For example,
curl occurs when paper is moistened. The curl axis is usuallyconsistent with the fiber orientation axis. However, fiber
orientation, per se, does not cause curl, but the two-sided
nature or transverse variation of components or structuralparameters including fiber orientation causes it. Thus, theway in which fiber orientation distributes in the transverse
direction (z-direction) is worth measuring as one of thepaper structural parameters to predict three-dimensional
deformation. Actually, a flow of a fiber suspension induces
fiber orientation on a forming screen, and its degree has a
distribution.3,4
According to a survey of traditional handmade paper
like washi (Japanese paper) and hanji (Korean paper), it
was found that bast fibers tend to be oriented in the case of
washi more remarkably than conventional machine-madepapers. This is because a fiber flow on a slanted forming
screen consisting of bamboo splints woven together withsilk threads, corresponding to a “forming wire” in themodern papermaking industry, during dehydration resultsin evident fiber orientation as observed for the flow sheet
forming. If this principle is used the other way, fiber orienta-

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tion can be a physical basis to evaluate the effects of the
motions of a mould (a wooden frame holding the forming
screen) in the process of hand-making of cultural paper.5
Other than evaluation of hand-making processes, information on fiber orientation distribution of a repair paper in the
thickness direction should be useful to predict deformationof the repair paper over time after repairing.
Regardless of the sheet-forming tool (machine or hand),fiber orientation is one of the most important factors of
paper properties and its distribution in the thickness direction plays an important roll for deformation of the paperby both quick and slow environmental changes. To analyze
the fiber orientation distribution, a representative conventional technique is the tape-stripping method. In thismethod, an adhesive tape is stuck on a paper surface; when
the tape is ripped, a fiber layer with a certain thickness isremoved together with the tape. The thickness and fiber
orientation of the layer is measured to determine fiber
orientation parameters as a function of the depth fromthe paper surface. Abe and Sakamoto6 applied the tapestripping method to separate thin layers from a whole sheet
and measured Fraunhofer diffraction using a laser beam forfiber orientation. They confirmed that the angle-dependent
distribution of optical density of the diffraction pattern
recorded on the photographic film was in good agreementwith fiber orientation distribution measured by anothermethod and showed the characteristic fiber orientation distributions in the thickness direction for a shaking wiremachine and a high speed machine, respectively. As well,
Erikkila et al.7 applied the tape-stripping method to split a
sheet to 10 to 12 individual layers to determine fiber orientation angle and the anisotropy for each layer. The method
for establishing the magnitude and direction of fibers inimages taken with a video camera was image analysis usinga Sobel operator-based procedure. Although such local
filtering operator-based procedures are excellent to accomplish a quick operation, thick fibers are not weighted
depending on their fiber width because only edges aredetected sensitively by those filtering operators.
In the tape-stripping method, fiber breakage and pullout
is inevitable and there is no guarantee that the layer thickness is uniform. A novel technique employed as an alternative in the present work is confocal laser scanningmicroscopy. The confocal system provides optical (nonmechanical) slicing, which solves the problems typicalof mechanical layering, although sufficient precision isrestricted within reach of the emitted light from fibers.
Sliced layer images are obtained directly and are easily

subjected to image analysis. As an image analysis technique, Xu et al.8 applied the Hough transform to detect fiberedges in pioneering work to acquire the index and angle offiber orientation from confocal images. However, thispopular method includes several problems in determinationof fiber orientation. Only two edge lines are detected by the
Hough transform for one fiber whether the fiber is thick orthin as well as the Sobel operator. In addition, curved fibers
tend to be excluded from orientation calculation when thesegments constituting one fiber are short, although eachsegment contributes to its tangential direction depending
on its length. Therefore, in this work, a fast Fourier transform (FFT)-based algorithm was applied to determine fiber
orientation parameters. The validity of the FFT method toevaluate fiber orientation was proved by Yuhara et al.9 withthe result obtained by applying it to a soft X-ray microdiagram of a paper sheet. This technique was extended tothe three-dimensional fiber orientation of paper in our
research.

Experimental
SamplesFive paper samples were used. Table 1 lists these samples
with basis weight, thickness, and apparent density. The
orthogonally layered Japanese paper was manufactured by
Mr. Makoto Numata, a craftsman of Mino City, Gifu Prefecture. He produces the paper called sai by layering two
thin sheet plies orthogonally to give one finished sheet ofpaper with improved dimensional stability. The other paper
samples are commercially available machine-made papers.
Two test pieces measuring about 20 × 20 mm were takenfrom each sample. They were soaked in a 0.02% ethanolic
solution of acridine orange, which is a fluorescent dye tostain pulp fibers. Each test piece was placed on a slide glassand covered with a cover glass. Then a drop of aniline wasput on its corner or edge to allow it to penetrate the sample
spontaneously to the other end, leaving as few air bubbles
as possible. As an impregnation liquid in the case of pulp
fibers, aniline was selected because its refractive index is
1.58, which is close to that of pulp fibers ranging between
1.57 and 1.60. A light beam does not refract at an interface
between substances with equal refractive indices, but passes
straight. Even a porous material like paper appears transparent when such a liquid fills its inside pores. Note that

Table 1. Paper samples used in this study
Paper
Orthogonally layered Japanese paper
Wood-containing D-grade printing paper
Copier paper, 100% recycled
Wood-free paperboard
Packaging paperboard, 100% recycled

Basis weight (g/m2)

Thickness (μm)

Apparent density (kg/m3)

24
51
64
155
365

55
87
96
167
453

440
590
660
930
810

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Image analysis to determine fiber orientation properties

Detector

PinholeLens

Constructionof 3D image

Lens

In-focus
range

Confocal plane

Fig. 1. Acquisition of three-dimensional image by confocal laser scanning microscopy (CLSM)

Acquired optically sliced layer images were analyzed todetermine the angle and anisotropy with regard to fiber
orientation. Two-dimensional FFT was applied to determine those parameters. Fiber orientation can, in principle,
be defined as an angular distribution of a power spectrum as a result of the integration q(q) of a power spectrum
P(r, q) in polar coordinates, as shown in Eq. 1, where r
is the radius and q is the polar angle. In the actual calculation procedure adopted in this work, the images werebinarized using a dynamic threshold method of a simple
moving average. This was performed to correct the shadingof images taken under nonuniform illumination that would
frequently obscure the measurement of true fiber
orientation.
N 2

q (q ) = ∑ P (r, q )

(1)

r =0

aniline is toxic and care must be taken not to touch it orinhale its vapor.
Microscope conditions
A confocal laser scanning microscope (CLSM) LSM 510
with an upright body (Axioplan 2, Carl Zeiss, Germany)
was used. As Fig. 1 shows, functions characteristic of thistype of optical microscope to obtain three-dimensional(3D) images are optical slicing in the transverse direction
by the confocal system and the intense power of laser beams
to compensate for the resultant insufficient illumination.
The term “confocal” means collecting light exclusively froma single plane in focus with a pinhole that eliminates lightreflected from planes other than the focal plane. Laserbeams fall on the front of a sample, and then reflect andenter the detector to provide a fluorescence image. The
lenses used were a 10× objective and a 10× ocular to observe
the sample at 100× magnification.
Sample observation used laser irradiation at 488 nm andoptical filtering to restrict detection of light to fluorescenceat about 512 nm that was emitted by fibers stained withacridine orange. The dimensions of the acquired images
were 0.92 × 0.92 mm at a resolution of 512 × 512 pixels.
Confocal plane thickness was 5 mm for paper and 8 mm for
paperboard. The number of optical slices was 21 to 29 forone location and three to five locations were measured toaverage fiber orientation parameters at each depth.
Paper thickness measured by this CLSM method is comparable with that measured by the micrometer method,
because the clamping pressure that develops between acover glass and a slide glass is of the same order as thatexerted by the micrometer platen. The clamping pressure
P for the CLSM method depends on the surface tension gof the liquid and the distance d between the cover and slide
glasses. P is defined as 2g/d. g of aniline is 42.5 mN/m. If d
is assumed to be 100 mm, P is calculated to be 85 kPa, whichis equivalent to P specified in the standard10 for measuring
paper thickness (50 or 100 kPa).

A two-dimensional Fourier transform inevitably produces irregular large powers only in the x and y axes in thefrequency domain when the gray levels of the left and rightedges and the top and bottom edges of the image differ
greatly, because FFT assumes infinite repetitions of theimage in those directions. This binarization process efficiently reduces this abnormally large power by assigning analmost identical number of pixels to black or white. In addition, this method of binarization can extract the edges ofout-of-focus fibers located below in-focus top fibers even incompletely out-of-focus images. The FFT was computedwith these binary images to obtain the power spectra.
When mean amplitude values are determined for eachvalue of angle q, each discrete frequency value of theFourier coefficients in the X-Y coordinates cannot be easily
converted to the exact corresponding position in the polar
coordinates. If one draws the radius of a given angle fromthe pole in the frequency domain, it would pass throughonly a few discrete positions. The interpolation was applied
using four adjacent amplitude values. The procedure isdetailed in the literature11 and is summarized in the following way. In the polar coordinates, the amplitude of theFourier coefficient was added in a radial direction from thepole and its mean A(θ ) was determined for each value ofangle q ranging from 0° to less than 180°. The closed curve
combining A(θ ) and A(θ + 180) = ( Aθ ) was approximated toan ellipse using FFT followed by inverse FFT after low-passfiltering. The maximum value a, value b in the direction
crosswise to a, and the angle a, that is, q when A(θ ) = b,were adopted as the lengths of the major axis, minor axis,
and the orientation angle for elliptic parameters, respectively. Consequently, the anisotropy and the fiber orientation degree are defined as a/b and a, respectively.
For one particular sample, representative values of orientation angle and anisotropy should be determined. Sliced
layer images at the same depth of the same sample werecollected from several locations within the same sample
sheet. The mean orientation angle and anisotropy aredefined as the means of several orientation angle and anisotropy values, respectively, calculated from every image at

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Depth from surface

Original slice image

Binary slice image(a) 25 μm

100 μm(b) 40 μm

Results and discussion
Figure 2 shows a set of optically sliced layer images forevery 15 mm in depth for the orthogonally layered Japanese
paper from the top side of the first ply to the screen side of
the second ply. In this context, the screen side corresponds
to the wire side in the modern papermaking industry. The
images shown are actually every third image from a seriesof 25 images taken for slices every 5 mm apart. The fibers in
the top two images (Fig. 2a, b) are oriented in the verticaldirection of the image while those in the bottom (screenside) two images (Fig. 2d, e) are oriented in the horizontal
direction. The central image (Fig. 2c) appears to include
equal numbers of fibers oriented in both the vertical andhorizontal directions. The orthogonally layered planes
appear to be located near the image shown in Fig. 2c. The
sliced layer images above that shown in Fig. 2a or belowthat shown in Fig. 2e gave very faint fiber shapes, probably
because leak fluorescence emitted by fibers in adjacentplanes was detected, even though there was practically nofibers in those actual confocal slices. The thickness of thissample was measured to be 55 mm with a micrometer, whichroughly corresponds to the difference in depth between
images shown in Fig 2a and Fig. 2e. For all of the sliced
layer images, fiber orientation angle and anisotropy were
calculated using FFT-based image analysis.
Figure 3 shows a calculated result of fiber orientation for
one location of the orthogonally layered Japanese paper.
The fiber orientation angle is consistently around 90° forthe top half (55 mm thick), and around 0° for the bottom
half (65 mm thick), which is consistent with the visual perception of the sliced layer images in Fig. 2. The thickness of
this orthogonally layered paper is 55 mm. Thus, outside thisthickness, light reflected from the top or bottom surfaces is(d) 70 μm

Orientation angle, degree(c) 55 μm

135

1.4

90

1.3

45

1.2

0

1.1

(a)

(b)

(c)

(d)

(e)

-45
(e) 85 μm

0

20

40

60

Anisotropy

the same depth. However, there was a problem in calculating the mean orientation angle. Orientation angle isexpressed a value between 0° and less than 180°. The angles
of 0° and slightly less than 180° represent the almost thesame orientation angle, but the mean angle is calculated to
be about 90°. In order to avoid this misleading result, theoverall calculation was devised. The overall orientation
angle and anisotropy is defined as a single value calculated
overall from accumulated amplitude values of Fourier coefficients at the same angle of every image at the same depth.
The difference in the orientation angle and anisotropy
made by the two calculation methods is shown in the
results.

80

100

1.0
120

Depth from top, μm

Fig. 2a–e. Original and binary confocal slice images of fibers in crosslapped Japanese paper sai

Fig. 3. Orientation angle (diamonds, left axis) and anisotropy (circles,
right axis) of fiber orientation for one location of cross-lapped Japanese
paper. Dotted lines marked a to e correspond to depths of slice images
shown in Fig. 2

135

1.4

90

1.3

45

1.2

0

1.1

Depth from surface

Anisotropy

Orientation angle, degree

304
Original slice image

Binary slice image(a) 25 μm

100 μm(b) 40 μm

-45
0

20

40

60

80

100

1.0
120

Depth from top, μm

135

1.4

90

1.3

45

1.2

0

1.1

-45

1.0
120

0

20

40
60
80 100Depth from top, μm(c) 55 μm(d) 70 μm

Anisotropy

Orientation angle, degree

Fig. 4. Mean orientation angle (diamonds, left axis) and mean anisotropy (circles, right axis) of fiber orientation for cross-lapped Japanese
paper. Bars show 95% confidence intervals

Fig. 5. Overall angle (diamonds, left axis) and anisotropy (squares,
right axis) of fiber orientation for cross-lapped Japanese paper

considered to be detected faintly. The confocal mechanism
does not guarantee complete limitation of light from outof-focus planes. Then indistinct virtual images are detected
about 20 mm above and below the paper surface layers.
Therefore, the paper is measured to be about 40 mm thicker
by the CLSM method than the thickness measured by the
micrometer method. Rough papers like Japanese paperwith fibers rising from the surface are measured to be about
10 mm thicker. The anisotropy is sensitive to the sheet structure change of orthogonal layering. At 55 mm from the top,
the anisotropy was lower than 1.1. With this calculation
process, anisotropies higher than 1.15 suggest strong fiber
orientation while those lower than 1.10 imply almost random(e) 85 μm

Fig. 6a–e. Original and binary confocal slice images of fibers in woodcontaining paperorientation. Thus, fiber orientation near the lapping plane
was estimated to be random due to the mixture of fibersaligned in cross directions. This sheet has two peaks ofanisotropy in the centers of the two respective plies.
Figure 4 shows the mean fiber orientation angle andanisotropy calculated from ten sets of sliced images for theorthogonally layered Japanese paper. Each set of images
was shifted in the depth direction so that the angle change
plane was between 35 and 40 mm in depth. There was no
difficulty during this arrangement because every set of thesliced layer images had the plane exhibiting a clear orientation angle change. Figure 4 confirms that this orthogonallylayered paper has the crosswise structure over the wholearea and that the CLSM technique is feasible to determine
the internal fiber orientation.

135

1.4

90

1.3

45

1.2

0

1.1

Depth from surface

Original slice image

Binary slice image(a) 25 μm

Anisotropy

Orientation angle, degree

305

100 μm(b) 40 μm

-45

1.0
0

50

100

150

Depth from top, μm(c) 55 μm

Fig. 7. Overall angle (diamonds, left axis) and anisotropy (squares,
right axis) of fiber orientation for wood-containing paper

Figure 5 shows an overall fiber orientation angle andanisotropy of the orthogonally layered Japanese paper. The
data for Fig. 4 are mean values of angle and anisotropy of
all images at each depth. Therefore, 95% confidence intervals can be calculated, as shown in Fig. 4, from all thevalues. However, the data for Fig. 5 are single values ofangle and anisotropy, and it is impossible to calculate 95%
confidence intervals. This overall calculation means thelarge-area sampling that covers the sum of the imaged areas
subjected to the analysis and permits determination of
the true orientation angle. Overall anisotropies tend to belower than the corresponding mean anisotropies. Overall
anisotropies higher than 1.10 suggest strong fiber orientation while those lower than 1.05 imply almost randomorientation.
Figure 6 shows a set of sliced layer images for woodcontaining printing paper. Among machine-made paper
samples used in this work, this wood-containing paper islikely to have the highest anisotropy. The fibers are observed
to be orientated clearly in the machine direction, namely,
the horizontal direction in the image. Figure 7 shows
the overall angle and anisotropy of fiber orientation for the
sample. The highest anisotropy reaches 1.30 across the
thickness. The sliced layer with this anisotropy peak occurs
toward the top surface, which is probably the wire side,
considering that the wire side of paper is formed earlier
than the top side, immediately after fibers contact the wire,causing more remarkable fiber orientation.
Figure 8 shows a set of sliced layer images for copierpaper. Generally, copier paper is designed for less fiber
orientation to prevent wrinkles that may occur aroundheating rolls that are installed in copying machines to fix
toner. For the sliced layer images, fiber orientation is notdiscernible by eye. Figure 9 shows that the anisotropy is(d) 70 μm(e) 85 μm

Fig. 8a–e. Original and binary confocal sliced layer images of copier
paper

lower for the copier paper than any other paper samples
used here.
Figures 10 and 11 show overall angle and anisotropy offiber orientation for the wood-free paperboard and recycled
packaging board, respectively. For the wood-free paperboard, the possible range of measured thickness was 152 mm
at the highest, although the actual thickness measured by
micrometer was 167 mm. Reflected light photographed in
the sliced layer images near the last image at 152 mm indepth is so faint that sliced layer images below this layer
would not provide accurate analysis. Paper with very highdensity like this wood-free paperboard absorbs and scatterslight reflected from a deep layer to a great extent. The
results for the two paperboard samples suggest that internal
structure analysis by CLSM is applicable for paper or paper-

90

1.3

45

1.2

0

1.1

-45

1.0
0

20 40

135

1.4

90

1.3

45

1.2

0

1.1

Anisotropy

Orientation angle, degree

90

1.3

45

1.2

0

1.1

0

Fig. 9. Overall angle (diamonds, left axis) and anisotropy (diamonds,
right axis) of fiber orientation for copier paper

0

1.4

-45

60 80 100 120 140

Depth from top, μm

-45

135

1.0
20 40 60 80 100 120 140 160
Depth from top, μm

Fig. 10. Overall angle (diamonds, left axis) and anisotropy (diamonds,
right axis) of fiber orientation for wood-free paperboard

board to a maximum depth of 150 mm, although the actual
depth depends on the apparent density. The packaging
board had a higher maximum value of anisotropy than thewood-free paperboard. Below the sliced layer at 64 mm
deep, the anisotropy decreased with the depth for the same
reason; namely, an insufficient amount of reflected light. In
particular, dark gray fibers used in recycled paperboards
tend to absorb emitted fluorescent light.

Anisotropy

1.4
Orientation angle, degree

135

Anisotropy

Orientation angle, degree

306

1.0
20 40 60 80 100 120 140 160
Depth from top, μm

Fig. 11. Overall angle (diamonds, left axis) and anisotropy (diamonds,
right axis) of fiber orientation for paperboard for paper container

stained with acridine orange. The fiber orientation distribution in the thickness direction was determined by applying
Fourier transform image analysis to every sliced layer imageof paper. Fiber orientation angle and anisotropy were
determined by the FFT method, which holds advantages
over the Hough transform with regard to evaluation of fiber
width-weighted contribution and intrafiber segmental contribution. Japanese handmade paper is known to have astrong orientation because flow of the fiber suspension orientates fibers during dehydration in flow-sheet forming. An
orthogonally layered Japanese paper, a model of a structurewith a clear change of fiber orientation at the orthogonallylayered plane, resulted in a clear change of fiber orientation
angle from 0° to 90° and two peaks of anisotropy in the twoplies, respectively. This result demonstrates the validity of
the CLSM method. For machine-made papers, the woodcontaining printing paper showed the highest anisotropy,
while the copier paper showed a low anisotropy presumably
because copier paper is designed to be free of wrinkles
during copying. Thick papers like the wood-free paperboard and recycled packaging board showed the possible
range of measured thickness was about 150 mm. Fluorescence emitted from fibers deeper than 150 mm is too faintto be used for FFT-based image analysis, although thedepth limit depends on the apparent density of the paper
and the light absorption character of the fibers.
Acknowledgments The authors thank Mr. Makoto Numata for preparing orthogonally layered Japanese paper sai.

Conclusions

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