Determination of collagen fiber orientation inhuman tissue by use of polarization measurementof molecular second-harmonic-generation lightTakeshi Yasui, Yoshiyuki Tohno, and Tsutomu Araki

Based on the reflection-type polarization measurement of second-harmonic-generation 共SHG兲 light induced by collagen molecules, we are able to determine the collagen fiber orientation in human tissues
taken from a cadaver. The resulting SHG radar graph shows the direction of the absolute orientation andthe degree of organization of collagen fibers. To evaluate the probing sensitivity to the collagen orientation, we compared the proposed method with other polarimetric methods. Use of the proposed method
revealed characteristic orientation differences among collagen fibers and demonstrated significant inhomogeneity with respect to the distribution of collagen orientation in human dentin. The proposed
method provides a powerful research and diagnostic tool for examining the collagen orientation in humantissues. © 2004 Optical Society of America
OCIS codes:
120.5410, 140.7090, 170.3890, 170.4580, 170.6510.

1. Introduction

Collagen is an important structural protein in thehuman body, and its molecules form structural aggregates successively 共i.e., microfibrils, fibrils, fibers,
and bundles of different sizes兲 in biological tissues
and organs. Because collagen molecules and theiraggregates play a role in maintaining the mechanicalproperty of tissues in a way similar to that of reinforcing bars in a concrete building, the orientation of
the collagen molecule aggregates is closely related tothe structural and functional properties of the tissues
and organs. For this reason, there is considerable
need in the areas of biological research and clinicalmedicine for a reasonable method that can reveal thecollagen orientation in situ. For example, in skingrafting for severe burns, the collagen orientation of
the grafted dermis should be consistent with that ofthe dermis surrounding the burn. Even a small inconsistency in collagen orientation causes a differ-

T. Yasui 共t-yasui@me.es.osaka-u.ac.jp兲 and T. Araki are with the
Graduate School of Engineering Science, Osaka University, 1-3
Machikaneyama, Toyonaka, Osaka 560-8531, Japan. Y. Tohno iswith the First Department of Anatomy, Nara Medical University,
40 Shijo, Kashihara, Nara 634-8521, Japan.
Received 2 July 2003; revised manuscript received 13 February
2004; accepted 23 February 2004.
0003-6935兾04兾142861-07$15.00兾0© 2004 Optical Society of America

ence in the mechanical property at the boundary of
the two dermises, resulting in ugly wrinkles in theskin. However, in the case of conventional skingrafting surgery, an accurate determination of thecollagen orientation depends on the doctor’s experience and skill.
Several studies describe the methods for observing the collagen orientation: electron microscopy,1x-ray diffraction,2 microwave method,3 mechanicalexamination, and biochemical and histologicalanalyses. Because a tissue biopsy is required formeasurement purposes, these methods are invasive
and兾or destructive and place a heavy burden on thepatient. Optical probe methods offer the possibility of noninvasive and nondestructive diagnosis of
the structural changes in biological tissues. For
example, optical coherence tomography 共OCT兲 hasbeen effectively applied to the optical biopsy ofeyes,4 skin,5 and digestive organs.6 On the other
hand, recent advances in ultrashort pulse lasers
have opened the door to new optical probe methods
based on the optical nonlinear interaction between
the incident light field and the analytical substance:
two-photon fluorescence, 7 second-harmonicgeneration 共SHG兲 light,8 and coherent anti-Stokes
Raman scattering light.9 Among these probes, SHG
light is preferred for the diagnosis of collagen-richtissues,10 –14 because it readily provides direct information about the collagen structure in the tissues
without photobleaching, phototoxicity, and addi10 May 2004 兾 Vol. 43, No. 14 兾 APPLIED OPTICS

2861

tional staining with fluorochrome. The efficiencyof SHG light is sensitive to the collagen orientation
when the incident light is polarized,10 and hence thepolarization measurement of SHG light is effective
in probing the collagen orientation in the tissues.10,
13, 14

As a noninvasive in vivo measurement of a thickliving tissue, the polarization measurement of SHG
light based on the reflection configuration 共called
SHG polarimetry兲 is attractive.15–18 Although thisconfiguration has been applied to the determinationof the collagen orientation in animal tissues,17, 18 few
reports deal with human tissues. When one considers the use of SHG polarimetry as the optical probe
combined with a biopsy technique for human tissues,
it is better to evaluate the proposed system’s sensitivity to the collagen orientation in human tissue
rather than in animal tissue. However, living human tissue is not readily available because of the
ethics guidelines governing its use. In a previous
study19 we examined whether there is an appreciable
difference between the SHG polarimetry of formalinfixed tissue and fresh tissue and confirmed that the
SHG polarimetry profile of the fixed sample was consistent with that of the unfixed sample. This result
implies the applicability of the SHG polarimetry toliving human tissues.
In the present paper, as a first step for future medical applications, we demonstrate the measurementof the collagen orientation in human cadaver tissues
by use of SHG polarimetry. The probing sensitivityof the SHG polarimetry to the collagen orientation isevaluated by comparison with that of other polarimetric methods. Furthermore, we discuss the distribution of the collagen orientation in human dentin in
the context of tooth aging.
2. Experimental Setup

Figure 1 shows the experimental setup for the SHG
polarimetry. An infrared pulse light with ultrahigh peak power and ultrashort pulse width is required to generate the collagen-induced SHG lightefficiently without causing thermal damage to thetissue. We used an 87-MHz Kerr-lens mode-locked
Ti:sapphire laser 共Avesta Project, Ltd., TiF-Kit-100兲
pumped by a 5-W frequency-doubled Nd:YVO4 laser
共Showa Optronics, JUNO5000兲 running at 532 nm.
The laser pulse has a duration of 60 fs and an
average power of 300 mW at 808 nm. After passing through an optical chopper 共OCP, chopping frequency of 3 kHz兲, ultrashort pulse light from thelaser is focused onto the sample through a lens 共L1,
focal length of 35 mm兲, resulting in an analyticalspot of 15-␮m diameter and 450-␮m depth in air.
Although SHG light that is induced in the sample
propagates mainly in the same direction as the incident light, a portion of SHG light is backscatteredinto the tissue and then collected via L1. Afterunnecessary laser light is eliminated with a harmonic separator 共HS, reflected wavelength of 400
nm兲 and a blue-pass filter 共F, pass wavelength of
300 –500 nm兲, the analytical SHG light is spectrally
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Fig. 1. Experimental setup: ML, mode-locked; M, mirror; L2,
lens; Ref., reference input; Sig., signal input. See text for otherdefinitions. For SHG polarimetry, polarizations of the incident
laser light and the detected SHG light are simultaneously rotatedwhile they are kept parallel to each other.

resolved with a monochromator 共resolution of 4 nm兲
and detected with a photomultiplier 共PMT兲 and alock-in amplifier.
For the polarization measurement of SHG light,
the polarization direction of the laser light was rotated every 3 deg with a mechanically rotatory halfwave plate 共␭兾2兲 after adjustment to the linear
polarization with a polarizer 共P兲. The polarization ofa detected SHG light was also rotated synchronouslywith the rotation of the laser light polarization bymeans of a mechanical rotatory analyzer 共A兲 in orderto maintain the polarization directions parallel toeach other. A depolarizer 共DP兲 was attached to therotatory analyzer to cancel the polarization dependence of the monochromator. The distribution of the
SHG light intensity was measured with respect to thepolarization angle. Although a rotation proceduresuch as this was equivalent to the rotation of thesample around the optical axis under fixed polarization optics, it was difficult to coincide the sample
rotating axis with the optical axis within the analytical spot. Hence we selected a polarization-rotating
setup. In this setup we also had to evaluate theinfluence of the anisotropy in the HS that is inducedby transmission and reflection of the polarizationrotating light. The polarization-rotating 800-nm
light is influenced by anisotropic difference of ⫾2.3%
in transmittance, whereas the polarization-rotating
400-nm light is subjected to anisotropy of ⫾2.8% inreflectance. However, we did not need to compensate
for the anisotropic difference in the practical polarimetry because the small difference did not so seriously
affect the results of SHG polarimetry. To achieve amore precise characterization of the collagen orientation with the proposed method, one must compensate the anisotropy by using a nonoriented SHG
material 共i.e., a powder of SHG crystal or opticallyactive material兲 whose generation efficiency of SHG
light is unchanged by the polarization-rotating light.
3. Samples

We prepared four kinds of collagen-rich human tissue
samples: Achilles tendon 共from a cadaver兲, dermis
共from a cadaver), dentin 共extracted from a patient by
a dentist兲, and talus 共from a cadaver兲. Each tissue has
one of the following characteristic collagen fiber orientations: 共1兲 unaxial orientation, 共2兲 entangled orientation, 共3兲 biaxial orientation due to the meshstructure, or 共4兲 parallel-to-trabecule collagen distribution. Cadavers were given an injection of a mixture of 36% ethanol, 13% glycerin, 6% phenol, and 6%
formalin through the femoral artery. After a typicalanatomy class dissection had been carried out by
medical students, the tendon and skin were resected.
Once these tissue samples were washed with distilledwater, they were dried in air. We have confirmedthat these sample preparation procedures 共formalin
fixation and air-drying兲 do not affect the results of
SHG polarimetry.19 Two kinds of tendon specimens, each 2-mm thick, were prepared: one wassliced along the axial direction and the other acrossthe section. The skin was stripped off the cadaver’s
back, and the subcutaneous tissue was removed witha scalpel to expose the reticular dermis layer. The
resulting skin sheet sample, consisting of the epidermis and the exposed reticular dermis, was cut into a
44 mm ⫻ 55 mm square sheet of 1-mm thickness.
The dentin and talus were sliced to a 1-mm thickness
along the tooth axis and to a 2-mm thickness acrossthe section by use of a tooth cutter and a bone cutter,
respectively. A raw chicken skin was prepared as anative, wet biological tissue. We also prepared acollagen sponge for cell culture 共Koken, CS-35, type I,
from bovine Achilles tendon兲. The Achilles tendon
samples, which possess well-defined uniaxial collagen orientations, were used as the control.
4. Second-Harmonic-Generation Polarimetry
A. Relationship between Incident Laser Polarization and
Collagen Orientation

The collagen-induced SHG light is produced by the
specific structure of collagen, that is, by the absenceof a center of inversion symmetry on the scale of lightwavelength. The SHG phase-matching condition is
not achieved in the biological tissue owing to thesmall birefringence of the collagen fiber.20 Alternatively, the collagen-induced SHG light is in the nonphase-matching condition, in which SHG light fromeach collagen molecule is constructively interferedand hence coherently enhanced by multiple scattering within a coherent length in the tissue.11
The relation between the collagen orientation andthe laser polarization with respect to SHG light issummarized in Fig. 2. When an incident light isnormal to the cross section of the collagen fiber, thecollagen SHG light is not observed at any polarization angle. In an incidence configuration such as this,the cylindrical symmetry 共C⬁ symmetry兲 along the

Fig. 2. Relationship between collagen orientation and laser polarization with respect to the generation of SHG light.

longitudinal axis in collagen molecules makes any
laser polarization centrosymmetric with respect tothe collagen orientation, resulting in the prohibitionof SHG light. However, other configurations induce
SHG light because the relation between the collagenorientation and the laser polarization is noncentrosymmetric. In particular, when the laser polarization is parallel to the collagen orientation, a strong
SHG light is observed. The polarization direction of
the resultant SHG light is consistent with that of theincident laser light. On the other hand, SHG light isvery weak when the laser polarization is perpendicular to the collagen orientation. These polarization
dependences reflect the nonzero elements of thesecond-order susceptibility tensor 共␹共2兲兲 in collagenmolecules, the analytical model of which is discussed
elsewhere.10, 17
To confirm these relations, we measured the collagen SHG light on the human Achilles tendon, which
possesses a well-defined uniaxial orientation in thecollagen fiber. As expected, the resultant SHG light
output was strong when the polarization direction of
the incident light was parallel to the longitudinal
fiber direction and considerably weaker when the laser polarization and the collagen orientation wereperpendicular to each other 共ratio of SHG intensity
between the former and the latter configurations of
50:1兲. This result indicates that the SHG polarimetry is a probe specific to the collagen orientation.
B.

Comparison with Other Polarimetric Methods

To evaluate SHG polarimetry, we compared the probing sensitivity to the collagen orientation with otherpolarimetric methods: 共a兲 reflected fundamental
light, 共b兲 transmitted fundamental light, and 共c兲 twophoton excitation autofluorescence 共TPEAF兲. Experiments using these methods employ the same
experimental setup as the SHG polarimetry. Vertically and horizontally directed human Achilles tendons were used as the standard samples. Figure 3
shows the resultant radar graphs of the four methods
with respect to the distribution of the detected lightintensity as a function of polarization angle, where 0
共or 180兲 and 90 共or 270兲 deg indicate the horizontal
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Fig. 3. Comparison of the SHG polarimetry with other polarimetric methods: 共a兲 reflected fundamental light, 共b兲 transmitted fundamental light, 共c兲 TPEAF, and 共d兲 SHG. The human Achilles
tendon is used as a sample with an uniaxial collagen orientation.
Gray arrows in each graph indicate the actual direction of thecollagen orientation.

and vertical polarizations, respectively. The grayarrows in each graph indicate the actual direction of
the collagen orientation.
In the polarimetry of the reflected light, the bluepass filter was removed, and a feeble fundamental
light reflected at the HS was used as an analyticallight. We moved the half-wave plate to the position
between the HS and the lens 共L1兲 to eliminate thelarge anisotropy in the HS 共⫾31%兲 induced by reflection of the polarization-rotating 800-nm light. Furthermore, we have to consider a difference in thesampling volume between the linear 共reflected andtransmitted fundamental light兲 and the nonlinear optical methods 共SHG and TPEAF兲. The latter canlimit the probing volume in the vicinity of the focusedlight spot owing to nonlinear optical processes,
whereas the former probes a larger volume in front of
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and behind the focused spot. Multiple scattering inthe sample deteriorates the focusing condition, resulting in a further increase in the sampling volume.
The performance of the four polarimetric methods
can be compared quantitatively by use of an equivalent sampling volume. To realize the comparison,
we introduced confocal configuration with a 50-␮m
diameter pinhole in front of the analyzer, resulting ina transverse spot of 53 ␮m and an axial spot of 414
␮m in air. The resultant radar graph showed a circular shape and no correlation between the graphshape and the collagen orientation 关see Fig. 3共a兲兴.
Although this result may reflect an inherent birefringence 共0.003兲 in the collagen fiber20 and兾or surface
scattering in the sample, it is too small to detect anappreciable difference between the two graph profiles
with the present method.
In the polarimetry of the transmitted light, we useda similar confocal configuration to adjust the sampling volume. As a result, we observed similarcrossed shapes in the vertical and horizontal orientations 关see Fig. 3共b兲兴. This profile was oriented at
45 deg by rotating the sample to the same degree 共notshown兲, resulting in a possible relation between thepolarization direction and the collagen orientation.
However, it is difficult to distinguish between thevertical and the horizontal orientations by use of thismethod, in which birefringence, absorption anisotropy, and兾or forwardscattering in the sample could
cause such a profile. Considering the very small birefringence in the collagen fiber and the low absorption in the infrared region, we conclude that thecrossed profiles are due mainly to forward-scattering
light process in the tissue.
In the TPEAF polarimetry, the center wavelengthof the incident laser was changed to 730 nm to produce an efficient two-photon excitation. The TPEAF
with a 425-nm emission maximum was observed
from the human Achilles tendon at an excitation
wavelength of 730 nm. The resultant TPEAF radargraph indicated that the major axis of the ellipsoid
profile was consistent with the collagen orientation关see Fig. 3共c兲兴. However, it was less sensitive to thecollagen orientation because of a small difference between the major and the minor axes of the ellipsoid.
We believe that this property is due to the possible
polarization dependence of the fluorescence quantum
efficiency in the two-photon excited fluorophores.
The major components of the human Achilles tendon
are collagen and the elastin fibers. Both components show a blue autofluorescence under UV excitation. Possible fluorophores of the collagen and elastin
are modified amino acids and pyridinoline aggregates, respectively. We observed weak fluorescencewith an emission peak at 385 nm when irradiating
the collagen powder with a UV line of 335 nm. On
the other hand, the elastin powder and elastin fiber inhuman skin exhibit a 480-nm emission maximum.21
However, when we observed the fluorescence of the
mixture of collagen and elastin, the fluorescence of
these components could not be distinguished fromeach other because of the spectral overlap of emis-sion. From these results, we conclude that the
TPEAF from the human Achilles tendon is caused by
the mixture of collagen and elastin. Therefore the
TPEAF polarimetry does not necessarily provide direct information about the collagen orientation, but it
does provide indirect information about the local environments of collagen and elastin.
In the SHG polarimetry we observed a typicalfigure-eight shape with a marked axis parallel to thecollagen orientation 关see Fig. 3共d兲兴. This shape ischaracteristic of the uniaxial fiber orientation. Thus
the SHG radar graph reveals the direction of theabsolute orientation and the degree of the organization from the polarization angle of the major axis andthe profile shape, respectively. The SHG process iscaused by the second nonlinear polarization 共P2兲 asfollows:
P 2 ⫽ ␹ 共2兲E 2,(1)

where ␹共2兲 is the second-order susceptibility tensor
and E is the electric field vector. In a microscopic
region of a molecular order, ␹共2兲 is modified with thefirst hyperpolarizability ␤ as follows22:
␹ 共2兲 ⫽ N s具␤典,(2)where Ns is density of the molecules and ⬍␤⬎ is anaverage of the molecule direction with regard to ␤.
Hence the collagen-induced SHG light depends on
both the amount and the orientation of collagen molecules. It is important to note that the SHG polarimetry is effective in extracting the angular
distribution of ⬍␤⬎ that is related to the collagenorientation. Hence, the SHG polarimetry offers a sensitive probe for the detailed distribution of the collagen orientation.
A comparison of the four kinds of probing lights
by use of simple polarization optics indicated that
SHG light was more sensitive to the collagen orientation than the other lights were. We emphasize
that, although the other probing lights 共autofluorescence and reflected and transmitted fundamental light兲 show interesting results when they areapplied to other methods of comparison, they aresubstantially influenced by both collagen fibers andother tissue components 共e.g., elastin and NADH
for autofluorescence兲, resulting in contaminateddata in the actual tissue. In contrast, collagen is
SHG-only source in the tissues, giving it a specificsensitivity to the collagen orientation. Hence we
can conclude that SHG light offers a superior performance in determining the collagen orientation.
C. Comparison of the Collagen Orientation in BiologicalTissues

We compared SHG radar graphs among several tissue samples to evaluate the performance of SHG polarimetry. The figure-eight shape in the human
Achilles tendon is characteristic of the uniaxial orientation of collagen fibers, as shown in Fig. 3共d兲. The
resultant profiles of six other tissue samples areshown in Fig. 4. The gray arrows in each graphFig. 4. SHG radar graphs for human tissue samples: 共a兲 humandermis, 共b兲 human dentin, 共c兲 and 共d兲 human talus, 共e兲 chicken skin,and 共f 兲 collagen sponge. Gray arrows in each graph indicate theexpected direction of the collagen orientation.

indicate the expected direction of the collagen orientation. There are characteristic differences among
the samples owing to their corresponding collagenstructures. The profile of the human dermis is similar to that of the human Achilles tendon; however,its figure-eight profile lacks a node 关see Fig. 4共a兲兴.
This result indicates that, unlike the fibers in the
Achilles tendon, the orientation of collagen fibers in
the human dermis is nearly, rather than completely,
uniaxial. In general the anatomical results from thehuman dermis indicate that it possesses a tangled
structure of collagen fibers that extends in every direction. We believe this that tangled structure ofcollagen fibers within the probing volume causes theimperfect figure-eight profile observed in the SHG
polarimetry. The collagen fibers that exist in theform of a mesh structure in the dentin tissue result ina biaxial profile 关see Fig. 4共b兲兴. However, the shapeof the biaxial profile was distorted when it was measured from the dentin root toward the dentin crown
共discussed in Section 5兲. In the human talus, we observed incompletely crossed biaxial shapes on different analytical spots 关see Figs. 4共c兲 and 4共d兲兴. The
acute angles between the two orientation axes 共grayarrows兲 were similar to each other 共⫽70 deg兲; however, the ratio of the length of the two orientation
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axes was significantly different, depending on theanalytical spot: 2.2:1, as shown in Fig. 4共c兲, and1.3:1, as shown in Fig. 4共d兲. Such a difference may
reflect the position-dependent distribution of the mechanical strength of the talus to the mechanical load
against the talus. The result of the chicken skinsample reveals a distorted random collagen orientation because the specific axis is not apparent 关see Fig.
4共e兲兴. The oval profile from the collagen sponge implies a completely random orientation of the collagenfibers 关see Fig. 4共f 兲兴. This observed diversity in thecollagen orientation plays an important role in determining the original property of each tissue.
5. Distribution of the Collagen Orientation

An investigation of the localization or inhomogeneity of the collagen orientation provides important
information for the study of tissue morphology andphysiology. Here, as a fundamental study of the
distribution of the collagen orientation, we measured the collagen orientation on the human dentin
section at different analytical spots. Figure 5共a兲shows a sketch of a cross section of a human tooth,on which four analytical spots are indicated, together with a growth line on the dentin tissue.
The growth line, likened to the annual ring of a tree,
indicates the age of the dentin tissue, which grows
toward the crown of the tooth. By paying attention
to this growth line and by examining the four corresponding SHG radar graphs 关see Fig. 5共b兲兴, wewere able to formulate an interesting hypothesis.
The resultant graphs for spots 共1兲 and 共2兲 are almost
identical to each other. These two spots are considered to be on the same growth line and hence areformed simultaneously. On the other hand, thegraphs for spots 共3兲 and 共4兲 are on different growthlines and show profiles that are different from thosefor 共1兲 and 共2兲. The spots that are on the same
growth line may show the same metabolic characteristics. From a comparison of these graphs, wenote that the shape of the radar graph is distorted
from the root toward the crown, indicating that theorientation of the dentin collagen is disordered during tooth aging. Such a gradual disordering of thecollagen orientation might be related to the metabolism of the dentin tissue. The results of nanosecond fluorescence measurement suggest that the
aging mechanism for the crown dentin differs fromthat for the root dentin.23 However, further examination of different types of teeth is necessary inorder to confirm our hypothesis.
6. Conclusion

We have proposed a method, based on reflectiontype SHG polarimetry, for determining the collagenorientation in human tissues taken from a cadaver.
The resultant SHG radar graphs reveal the direction of the absolute orientation and the degree oforganization of collagen fibers in the tissues.
From a comparison among the possible polarization
measurements, we confirmed that the SHG polarimetry shows an excellent probing sensitivity to
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Fig. 5. Distribution of the collagen orientation in human dentin.
共a兲 Sketch of a cross section of a tooth. The analytical spots andgrowth line in the dentin tissue are shown. 共b兲 Results of the SHG
polarimetry on four analytical spots.

the collagen orientation. The apparent differencein the collagen orientation among several humantissues was demonstrated: a figure-eight shape forthe human Achilles tendon and dermis, a biaxial
shape for the dentin and talus, and an oval shapefor the collagen sponge. Furthermore, we observed a position-dependent change in the collagenorientation in human dentin. From these findings
we conclude that the proposed measurement system is a powerful tool for the biological research andclinical diagnosis of the change in the collagen fiber
orientation in human tissues. In the near future
we will apply the proposed measurement system toliving human tissues.
This work was supported in part by grants-in-aid
for scientific research 15656069 and 15650094 from
the Ministry of Education, Culture, Sports, Science,
and Technology of Japan. We thank M. Hashimotofor many insightful discussions and K. Shimabayashi

and H. Kawaguchi for their technical support in our
experiments.
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