Journal of Biomedical Optics 9(2), 259–264 (March/April 2004)

Characterization of collagen orientation in humandermis by two-dimensional second-harmonic-generation
polarimetry
Takeshi Yasui
Osaka University
Graduate School of Engineering Science
Toyonaka, Osaka 560-8531 Japan

Yoshiyuki Tohno
Nara Medical University
Department of 1st Anatomy
Kashihara, Nara 634-8521, Japan

Tsutomu Araki
Osaka University
Graduate School of Engineering Science
Toyonaka, Osaka 560-8531 Japan

Abstract. We have proposed an optical probe that can be used tocharacterize the orientation of collagen fibers in human dermis. A
specific probing ability for collagen results from the use of secondharmonic-generation (SHG) light induced by collagen molecules inthe tissue. Based on the concept of collagen SHG light, a reflectiontype polarization measurement system (named SHG polarimetry) witha probe light spot of 15 ␮m in diameter has been constructed, and thehuman reticular dermis has been measured using this system. Resultant data exhibit that the reticular dermis possesses approximately
uniaxial orientation of the collagen fibers. Furthermore, we demonstrated a nondestructive measurement of the collagen orientation in
the papillary dermis across an epidermis layer. For distribution measurement of the collagen fiber orientation in the reticular dermis, wehave extended the SHG polarimetry to one- (1-D) and twodimensional (2-D) measurement. By the macroscopic 2-D SHG polarimetry, we have observed that the orientation angle and organization
degree of collagen fibers vary widely depending on the discrete probing positions in the reticular dermis. Furthermore, microscopic 1-D
SHG polarimetry indicated a swell of the orientation angle and a large
variance of the organization degree in the collagen fibers in the microscopic region. These results imply that the reticular dermis posses atangled structure of collagen fibers, which is highly consistent with theresult of the anatomical examination of the skin. The proposed
method will be a powerful tool for monitoring the microscopic distribution of the collagen fiber orientation in the human dermis. © 2004
Society of Photo-Optical Instrumentation Engineers. [DOI: 10.1117/1.1644116]

Keywords: dermis; collagen; second-harmonic-generation light; femtosecond laser;orientation; polarization.
Paper JBO-014007 received Mar. 27, 2003; revised manuscript received Aug. 19,
2003 and Nov. 14, 2003; accepted for publication Nov. 17, 2003.

1

Introduction

Skin, the largest organ of the human body, is composed of athree-layer structure: epidermis, dermis, and subcutaneous tissue. Among these three layers, dermis plays a role in maintaining tension, elasticity, and water content physically in theskin. In the dermis layer consisting of reticular dermis andpapillary dermis, a structural protein, collagen is abundant andcontributes to the structural and functional properties of thislayer. Since the structural change of collagen molecules andtheir aggregates 共microfibrils, fibrils, fibers, and bundles兲 inthe dermis provides important information about diseases afflicting tissues and organs such as skin cancer, collagen disease, or burns, there is a considerable need for an effective
diagnostic technique by which the collagen structural changes
in the dermis can be informed. Various methods that reveal
the structure and orientation of collagen have been reported:
electron microscopy,1 x-ray diffraction,2 microwave method,3mechanical examination, biochemical, and histological analyAddress all correspondence to Takeshi Yasui. Tel. +81-6-6850-6215; Fax: +816-6850-6212. E-mail: t-yasui@me.es.osaka-u.ac.jp

sis. However, these methods are invasive and/or destructive
because an anatomical tissue biopsy is required to complete
the measurements. Although the biopsy-based methods arereliable for differentiating between normal and abnormal collagen structure, they inflict a heavy burden on the patient.
Hence, there is a significant need for a noninvasive and nondestructive method as preliminary diagnosis to examine
whether tissue biopsy is necessary or not.
Optical methods are attractive for such preliminary diagnosis, because they are simple, fast, and harmless to the patient. Since skin is a superficial tissue, an optical probe can bedirectly applied. However, the probing light signal is strongly
scattered inside the tissue, and such the property makes theorigin of detected light ambiguous. Surface reflection of thelight is another obstacle. To overcome these problems, it isnecessary to extract a weak but meaningful, less-scattered
light from the mixture of the multiple-scattered light inside
the sample and the surface-reflected light. A polarization gating technique is one possible method that eliminates the1083-3668/2004/$15.00 © 2004 SPIE

Journal of Biomedical Optics

䊉March/April 2004䊉Vol. 9 No. 2

259

Yasui, Tohno, and Araki

multiple-scattered light and/or surface-reflected light.4,5 This
technique utilizes the property that the scattering events reduce the degree of polarization. However, it is difficult tocharacterize the collagen structure in the dermis from the resultant spectroscopic information in the extracted signal. Optical coherence tomography 共OCT兲6 is another possible
method, and this method had been applied effectively to an
optical probe of eye,6 skin,7 or digestive organ.8 In OCT, surface reflection and internal reflection can be distinguished easily, because resultant information for depth structure develops
successively along the time axis. At the same time, unnecessary multiple-scattered light is effectively rejected by the coherence gate of light interference because the multiple scattering events reduce the coherency of light. Although OCT
can probe microscopic structure in the collagen-containing
tissues based on spatial variation in refractive index, it does
not necessarily provide direct knowledge of the collagen fiber
orientation in tissue. Polarization-sensitive OCT 共PS-OCT兲9probes intrinsic birefringence and formation birefringence ofthe biological tissue. Hence, the PS-OCT gives direct information about the collagen fiber orientation and thus can beapplied to the burn diagnosis of the skin.10 In addition to theselinear optical probes, recent advances of ultrashort pulse lasers have opened the door to new optical probe methods basedon the optical nonlinear effects in biological tissue: e.g., twophoton fluorescence,11 second-harmonic-generation 共SHG兲
light,12 and coherent anti-Stokes Raman scattering 共CARS兲
light.13 Among these linear and nonlinear optical probes, we
believe that SHG light is favorable to the diagnosis of thecollagen rich dermis. Because the SHG light efficiency andthe concentration of the collagen molecule are much higher
than those of other tissue components such as fat,14 the SHG
light in the dermis is mainly due to collagen molecules. Suchthe collagen SHG light is generated only into the dermis layer
because of no collagen content in the epidermis layer. In thissituation, it is easy to reject surface reflection and multiple
scattering of the incident laser light in the epidermis layer
because the wavelength of the SHG light is half the incident
laser wavelength. Hence, the collagen SHG light can give
direct information about the collagen structure in the dermis
without any gating technique. Moreover, the SHG light provides excellent three-dimensional resolution images and deep
penetration power, and there are no photobleaching, phototoxicity, and additional staining with fluorochrome. The efficiency of SHG light changes with respect to the collagen orientation when the incident light is polarized, and hence, thepolarization measurement of the SHG light is an effective toolthat probes collagen orientation in the tissues.15,16 In this situation, the polarization measurement of the SHG light based on
the reflection configuration 共we named SHG polarimetry兲17–19
is attractive when one achieves a noninvasive in vivo measurement of the collagen orientation in a thick living tissue.
However, there are few reports dealing with determination ofcollagen orientation in actual human tissues based on thatconcept. We previously demonstrated the measurement of collagen orientation in the several human tissues by the SHG
polarimetry, and presented remarkable differences among thetissues due to their characteristic collagen orientations.19
In this work, we present an optical probe method to study
the collagen orientation in human reticular dermis and papillary dermis, based on SHG polarimetry. Using this method,260

Journal of Biomedical Optics

䊉March/April 2004䊉Vol. 9 No. 2

Fig. 1 Experimental setup. M: mirror, PL: polarizer, ␭/2: half waveplate, HS: harmonic separator, L1 and L2: lenses, AN: analyzer, F:
blue-pass filter, DP: depolarizer, PMT: photomultiplier.

we also propose one-dimensional 共1-D兲 and two-dimensional
共2-D兲 SHG polarimetry to investigate the spatial distributionof collagen orientation in the reticular dermis.

2

Experimental Setup

Figure 1 shows the experimental setup for reflection-type
SHG polarimetry. Instrumentation and basic performance of
our SHG polarimetry are discussed elsewhere in detail.19 The
laser source is a Kerr-lens-mode-locked Ti:sapphire laser
power
共AVESTA TiF-kit-100, pulse width⫽60 fs,⫽300 mW, repetition rate⫽87 MHz, center wavelength⫽808 nm) . Ultrashort pulse light from the laser was focused
onto the sample through a lens 共L1, focal length⫽35 mm) ,resulting in a focused spot of 15 ␮m diameter in air. Although
the SHG light induced in the sample mainly propagates in thesame direction as the incident light, part of the SHG light isbackscattered into the tissue, and then collected with L1. Afterelimination of the unnecessary laser light using a harmonic
separator 共HS兲 共reflected wavelength⫽400 nm) and a bluepass filter 共F兲 共pass wavelength⫽300– 500 nm) , the probing
SHG light is spectrally resolved with a monochromator( resolution⫽4 nm) and detected with a photomultiplier
共PMT兲. For polarization measurement of SHG light, a polarizer 共PL兲, a half waveplate 共␭/2兲, an analyzer 共AN兲, and a
depolarizer 共DP兲 are inserted in the light path. The polarization direction of the laser light is rotated every 3 degrees by amechanically rotatory ␭/2 after adjusting to the linear polarization with the PL. Polarization of a detected SHG light isalso rotated synchronously with the rotation of the laser lightpolarization to keep their polarization directions parallel toeach other by a mechanically rotatory AN. The DP is attachedto the rotatory AN to cancel the polarization dependence ofthe monochromator. The HS also causes slight anisotropy intransmittance and reflectivity for the polarization-rotating
light. However, we did not consider such an effect in theCharacterization of collagen orientation in human dermis . . .

present work, because it is so small that the results of SHG
polarimetry are not affected appreciably. For further strict
qualification and quantification of the collagen orientation,such the anisotropy must be compensated. The describedpolarization-rotating procedure is equivalent to the rotation of
the sample around the optical axis under fixed polarization
optics. However, it was difficult to coincide the sample rotating axis and the optical axis within the probing spot. Hence,
we adopted the polarization-rotating setup. Distribution of the
SHG light intensity was measured with respect to the polarization angle. For imaging measurement of SHG light, a 2-D
moving stage that scans the sample position laterally was
adopted.

3

Samples

We prepared a human skin sample composed of epidermis anddermis 共taken from cadaver’s back兲, human Achilles tendon
共from cadaver兲, and a collagen sponge for cell culture 共Koken,
CS-35, type I, from bovine Achilles tendon兲 as the sample
specimens. Cadavers were treated by injecting a mixture of
36% ethanol, 13% glycerin, 6% phenol, and 6% formalinthrough the femoral artery. After an ordinary dissection by
medical students in an anatomy class was finished, the skin
was stripped off from the cadaver’s back. The skin sample
consisting of the epidermis and dermis was prepared by removing the subcutaneous tissues with a scalpel to expose thereticular dermis layer. The resulting skin sample, whose one
side is the epidermis and another side is the exposed reticulardermis, was cut into a square sheet of 44 mm⫻55 mm with 1
mm thick. The Achilles tendon, resected from the cadaver’s
leg, was sliced to 2 mm thick along the axial direction. Afterwashing these tissue samples with distilled water, they weredried in air. These three kinds of sample were put between
two glass slides to flatten the sample surface.
The Achilles tendon and collagen sponge, which possess
uniaxial and completely random orientations in collagen fiber,
respectively, were used as the control samples.

4

Results

4.1 Effects of Sample Preparation Procedures on SHG
Polarimetry
Before determination of the collagen orientation in the humandermis, we have to investigate the effect of sample preparation procedures 共formalin fixing and air drying兲 on SHG polarimetry, because such formalin-fixed human tissue samples
have not been used generally in SHG methods.
We first evaluated effect of formalin fixing on the SHG
polarimetry by preserving a native dermis tissue in a chip
section of a raw chicken wing in the formalin-based fixation
medium 共36% ethanol, 13% glycerin, 6% phenol, and 6%
formalin兲. Intensity distribution of the detected SHG light wasmeasured as a function of polarization angle, and recorded as‘‘SHG radar graph’’ shown in Fig. 2, where 0 共or 180兲 and 90
共or 270兲 degrees mean horizontal and vertical polarizations,
respectively. The polarization angle of the major axis and the
profile of the SHG radar graph reveal the direction of theabsolute orientation and the organization degree of collagenfibers in the area of the light spot, respectively. Figures 2共a兲and 2共b兲 show SHG polarimetry before and 5 h after such

Fig. 2 Effects of sample preparation procedures on SHG polarimetry:(a) and (b) are SHG radar graphs before and 5 h after formalin fixation,(c) and (d) are those before and 13 h after air drying. Sample is anative dermis tissue in a chip section of a raw chicken wing.

fixation, respectively. As a result, the fixation process indicates almost unchanged in SHG polarimetry while the SHG
intensity was slightly increased 共not shown兲. Since formalin
fixing induces crosslinking of collagen in tissues, this result
may imply that the crosslinking does not affect collagen orientation essentially but contributes efficiency of SHG light.
We next investigated the effect of air drying on SHG polarimetry by leaving the earlier formalin-fixed chicken dermis
sample in air for a long time. The SHG polarimetry before
and 13 h after the air drying were shown in Figs. 2共c兲 and2共d兲, respectively. One can confirm that SHG polarimetry wasalmost unchanged during such drying process. However, theSHG intensity was decreased to about a fourth 共not shown兲.
We think that the decrease of the SHG intensity results inchange of a linear optical property, i.e., scattering efficiency,
rather than that of efficiency of SHG radiation in the tissues.
From these two preliminary results, we conclude that the
sample preparation procedures used in this paper do not affect
the SHG polarimetry even if the SHG light intensity is influenced by them.

4.2 Comparison of Collagen Orientation Among
SamplesResults of SHG polarimetry for the samples are shown in Fig.
3. Arrows in each polarimetry indicate the direction of thecollagen orientation expected from the SHG radar graph. The
human Achilles tendon indicates a typical ‘‘figure-eight’’ profile whose major axis is parallel to the collagen orientation asshown in Fig. 3共a兲. In general, collagen SHG light is strongly
outputted with the same polarization as the incident light,
when the polarization direction of the incident light was parallel to the longitudinal direction of the collagen fiber. However, the SHG light almost disappears if the laser polarization
Journal of Biomedical Optics

䊉March/April 2004䊉Vol. 9 No. 2

261

Yasui, Tohno, and Araki

SHG light,20 this SHG light was originated from the collagenmolecule in the dermis interacted with the laser light passing
through the epidermis. The resulting SHG radar graph in Fig.
3共d兲 indicates a similar shape to that of the collagen sponge in
Fig. 3共b兲 but with eccentric characteristics. Also, one can confirm a distinct difference from that of the reticular dermis in
Fig. 3共c兲. Such a difference might be due to the difference ofthe collagen orientation between reticular and papillary dermis, because we probed a boundary between the epidermis
and the dermis, that is, a papillary dermis rich layer. Although
it is also necessary to consider the effects of multiple scattering into the epidermis layer, i.e., decrease of spatial resolution
and depolarization of the laser light, the present result implies
a possibility of nondestructive, in vivo monitoring of the dermis collagen in clinical medicine.
Here, to quantify the collagen orientation in the tissue, let
us lead two parameters, ␪ maj and PA, obtained from the SHG
radar graph. The ␪ maj value is defined as a polarization angleof the major axis in the SHG radar graph. For organization
degree of the collagen orientation, we define a polarization
anisotropy of the SHG light 共PA兲 as,
Fig. 3 SHG polarimetry profiles for tissue samples: (a) human Achilles
tendon, (b) collagen sponge, (c) human reticular dermis, and (d) human papillary dermis. Arrows in each graph indicate expected direction of collagen orientation.

is perpendicular to the collagen orientation, because the second optical nonlinear property along this direction is considerably low. Hence, such a figure-eight profile is characteristicof a uniaxial fiber orientation.19 The oval profile of the collagen sponge implies completely random orientation of thecollagen fibers 关Fig. 3共b兲兴, because this results in that SHG
intensity is approximately equal for all polarization angles.
We note here that small asymmetry in the SHG radar graph isdue to imperfect optical alignment in the experimental setup
because the SHG polarimetry is very sensitive to small alignment errors 共for example, incomplete normal incidence of the
SHG light to the analyzer兲. For the dermis, we studied thecollagen orientation by two different approaches. In the first
approach, the laser light was incident from a subcutis side of
the skin sheet sample, i.e., bare reticular dermis, and the SHG
light from it was measured. Although the resulting profile of
the reticular dermis is similar to that of the Achilles tendon, its
figure-eight profile is lacking a node 关Fig. 3共c兲兴. This result
indicates that the orientation of the collagen fiber in the reticular dermis is not completely uniaxial such as the Achilles
tendon, but nearly uniaxial. In general, anatomical findings ofthe reticular dermis indicate that it possesses tangled structureof collagen fibers in every direction. We think that such atangled degree of the collagen fiber within the probing area of
15 ␮m makes the imperfect figure-eight profile in the SHG
polarimetry. The second approach is more challenging, that is,
probing of the dermis collagen orientation across the epidermis layer. In this approach, the laser light is incident from theepidermis side. When the laser light was focused on the surface of the epidermis, the collagen SHG light was not observed. However, if the probing spot was moved to a deeper
region and reached the dermis layer, the SHG light signal
appeared. Since keratin in the epidermis does not radiate the262

Journal of Biomedical Optics

䊉March/April 2004䊉Vol. 9 No. 2

PA⫽ 共 I 储 ⫺I⬜ 兲 / 共 I 储 ⫹I⬜ 兲 ,共1兲

where I 储 and I⬜ are the SHG intensity at the ␪ maj and thatperpendicular to the ␪ maj , respectively. Collagen orientationis unidirectional for PA⫽1, while it is isotopic directional forPA⫽0. The ␪ maj values for the Achilles tendon and reticulardermis are 6.5 and 131 degrees, respectively 共see Fig. 3兲.
Since the collagen sponge and papillary dermis did not indicate a clear major axis, the ␪ maj values for them were omitted.
On the other hand, the PA values were 0.89 for the Achilles
tendon, 0.13 for collagen sponge, 0.74 for reticular dermis,
and 0.15 for papillary dermis. One can see a significant difference of the PA value among the four specimens. Such a
difference is due to the collagen orientation. We can quantitatively expect the orientation conditions of collagen fibers fromthe sample dependent ␪ maj and PA values.

4.3 Macroscopic and Microscopic Lateral
Distribution of Collagen Orientation
Dermis exists as a sheet of collagen-rich tissue, and acts as acover and protector of the inner tissues and organs. Therefore,
the lateral distribution of the collagen orientation is interesting
when considering the structural and functional properties ofthe dermis. From this point of view, we extended SHG polarimetry to 1-D and 2-D measurement to further investigate thestructural orientation of collagen fibers in the dermis. Here,
we probed the exposed reticular dermis. The present SHG
polarimetry requires a measurement time of 1 min/spot because of the time required for the mechanical rotation of polarization optics, resulting in considerable time consumption
for precise two-dimensional measurement. The acquisition
time can be shortened dramatically by controlling the polarization with electro-optical devices instead of mechanicalones. In spite of the limitation of the present system, we will
demonstrate here the primitive measurement of 20 spots by
SHG polarimetry on the reticular dermis. Mapping of SHG
radar graphs is shown in Fig. 4, where the spot measurement
was made every 10 mm on the sample sheet of 40 mm⫻55 mm. Although each profile showed orientation profiles

Characterization of collagen orientation in human dermis . . .

Fig. 4 Macroscopic 2-D SHG polarimetry in reticular dermis. Measurement was done in spot areas at 10 mm intervals on dermis sheetof 55 mm⫻40 mm.

similar to Fig. 3共c兲, we found a slight difference in the degreeof organization, depending on the probing position. Furthermore, the angle of the major axis was not ordered. Such characteristic collagen distribution in the reticular dermis wascompletely different from those in the Achilles tendon andcollagen sponge that indicate uniform distribution of collagenorientation throughout 共not shown兲. We approximate that thereticular dermis has nearly uniaxial collagen fiber orientation
in a microscopic region of the scale of the probing spot 共15
␮m diameter兲, while such oriented portions are randomly distributed throughout the macroscopic region. This result partially indicates an entangled structure of the collagen fiber inthe reticular dermis.
Figure 4 gives us the intuitive findings of macroscopic anddiscrete lateral distribution of the collagen fiber orientation in
the reticular dermis. On the other hand, it is also interesting toprobe the continuous distribution of the collagen orientationin the microscopic region. Hence, we next interpolate a microscopic lateral distribution of the collagen orientation between the two discrete probing locations 关共A兲 and 共B兲兴 in Fig.
4 by microscopic 1-D SHG polarimetry. The probing lightspot was one-dimensionally moved stepwise in 100 ␮m intervals from 共A兲 to 共B兲 by operating the scanning stage. Microscopic distribution of the collagen orientation was evaluatedusing the ␪ maj and PA values as shown in Figs. 5共a兲 and 5共b兲,
respectively. These values in the reticular dermis are compared with those in the Achilles tendon. Furthermore, the position region from 0 to 1-mm in Figs. 5共a兲 and 5共b兲 is expanded by 10 ␮m step scanning in Figs. 5共c兲 and 5共d兲.
Statistics of these values in position ranges of 0–10 and 0–1
mm are summarized in Table 1. The ␪ maj value in the Achilles
tendon is almost constant in both ranges, indicating wellordered axial orientation in the collagen fiber in the micro-

Fig. 5 Microscopic lateral distribution of collagen orientation in reticular dermis: (a) ␪ maj value and (b) PA value within region of 0–10 mm, and (c)␪ maj value and (d) PA value within region of 0–1 mm. The ␪ maj and PA values inform direction angle and organization degree of collagenorientation, respectively. Results of the reticular dermis were compared with those of the Achilles tendon.
Journal of Biomedical Optics

䊉March/April 2004䊉Vol. 9 No. 2

263

Yasui, Tohno, and Araki
Table 1 Comparison of ␪ maj and PA values in reticular dermis and Achilles tendon.

␪ maj (deg, mean⫾SD)

Parameter
Region0–1 mm0–10 mm

0–1 mm0–10 mm

Reticular dermis

92.1⫾46.0

180.5⫾90.2

0.48⫾0.17

0.49⫾0.18

Achilles tendon

275.3⫾3.8

277.4⫾4.3

0.79⫾0.07

0.75⫾0.10

scopic region. The reticular dermis produced an interestingswell and drift of the ␪ maj value in addition to its variation ineach range. For the PA value, we observed significant differences between the two samples. Mechanical property of the
Achilles tendon is supported by the spatial distribution of ahigh PA value. We confirmed a large variance of deviations
around PA⫽0.5 in the reticular dermis. From such a characteristic swell of the ␪ maj value and a large variance of the PA
value, we can imagine that the collagen fibers in the reticulardermis become entangled successively within these spatial
scales, which is in good agreement with the anatomical findings of the dermis.

5

PA (mean⫾SD)

Conclusion

We have proposed 1-D and 2-D SHG polarimetry to evaluate
spatial distribution of the collagen orientation in the humandermis. On the basis of the imperfect figure-eight profile obtained in SHG polarimetry, collagen fibers in the reticulardermis were characterized as having approximately uniaxial
orientation. We also demonstrated the probing of the collagenorientation in the papillary dermis across the epidermis, which
provides a potential of in vivo monitoring of the dermis collagen in clinical medicine.
Distribution measurement of the collagen orientation using
macroscopic 2-D and microscopic 1-D SHG polarimetry indicated that the reticular dermis exhibits almost a uniaxial collagen orientation within a microscopic region and such thecollagen orientation becomes gradually disordered with theincrease of the probing area due to the mixing of the various
orientations. From these results, we conclude that the proposed measurement system is a powerful tool for the monitoring of the structural orientation of the collagen fibers in thehuman dermis.

3.
4.

5.

6.

7.

8.

9.

10.

11.
12.
13.

14.

15.
16.

Acknowledgments
Portions of this work were supported by a Grant-in-Aid for
Scientific Research Nos. 15650094 and 15300161, from the
Ministry of Education, Culture, Sports, Science and Technology of Japan. The authors would like to thank K. Shimabayashi and H. Kawaguchi for their technical help in experiments.

17.

18.

19.

References
1. B. Eyden and M. Tzaphlidou, ‘‘Structural variations of collagen in
normal and pathological tissues: role of electron microscopy,’’ Micron 32, 287–300 共2001兲.
2. V. J. James, L. Delbridge, S. V. McLennan, and D. K. Yue, ‘‘Use of

264

Journal of Biomedical Optics

䊉March/April 2004䊉Vol. 9 No. 2

20.

x-ray diffraction in study of human diabetic and aging collagen,’’
Diabetes 40, 391–394 共1991兲.
S. Osaki, ‘‘Distribution map of collagen fiber orientation in a wholecalf leather,’’ Anat. Rec. 254, 147–152 共1999兲.
J. M. Schmitt, A. H. Gandjbakhche, and R. F. Bonner, ‘‘Use of polarized light to discriminate short-path photons in a multiply scattering medium,’’ Appl. Opt. 31, 6535– 6546 共1992兲.
S. P. Morgan and M. E. Ridgway, ‘‘Polarization properties of lightbackscattered from a two layer scattering medium,’’ Opt. Express 7,395– 402 共2000兲.
D. Huang, E. A. Swanson, C. P. Lin, J. S. Schuman, W. G. Stinson,
W. Chang, M. R. Hee, T. Flotte, K. Gregory, C. A. Puliafito, and J. G.
Fujimoto, ‘‘Optical coherence tomography,’’ Science 254, 1178 –1181
共1991兲.
J. M. Schmitt, M. Yadlowsky, and R. F. Bonner, ‘‘Subsurface imagingof living skin with optical coherence microscopy,’’ Dermatology(Basel, Switz.) 191, 93 共1995兲.
A. M. Rollins, R. Ung-Arunyawee, A. Chak, R. C. K. Wong, K.
Kobayashi, M. V. Sivak, and J. A. Izatt, ‘‘Real-time in vivo imagingof human gastrointestinal ultrastructure by use of endoscopic opticalcoherence tomography with a novel efficient interferometer design,’’
Opt. Lett. 24, 1358 –1360 共1999兲.
J. F. de Boer, T. E. Milner, M. J. C. van Gemert, and J. S. Nelson,‘‘Two-dimensional birefringence imaging in biological tissue using
polarization sensitive optical coherence tomography,’’ Opt. Lett. 22,
934 –936 共1997兲.
J. F. de Boer, S. M. Srinivas, A. Malekafzali, Z. Chen, and J. S.
Nelson, ‘‘Imaging thermally damaged tissue by polarization sensitive
optical coherence tomography,’’ Opt. Express 6, 212–218 共1997兲.
W. Denk, J. H. Strickler, and W. W. Webb, ‘‘Two-photon laser scanning fluorescence microscopy,’’ Science 248, 73–76 共1990兲.
S. Fine and W. P. Hansen, ‘‘Optical second harmonic generation in
biological tissues,’’ Appl. Opt. 10, 2350–2353 共1971兲.
A. Zumbusch, G. R. Holtom, and X. S. Xie, ‘‘Three-dimensional
vibrational imaging by coherent anti-Stokes Raman scattering,’’ Phys.
Rev. Lett. 82, 4142– 4145 共1999兲.
B. M. Kim, J. Eichler, and L. B. Da Silva, ‘‘Frequency doubling of
ultrashort laser pulses in biological tissues,’’ Appl. Opt. 38, 7145–
7150 共1999兲.
S. Roth and I. Freund, ‘‘Second harmonic generation in collagen,’’ J.
Chem. Phys. 70, 1637–1643 共1979兲.
P. Stroller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik,‘‘Polarization-modulated second harmonic generation in collagen,’’
Biophys. J. 82, 3330–3342 共2002兲.
P. Stroller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da
Silva, ‘‘Polarization-dependent optical second-harmonic imaging of a
rat-tail tendon,’’ J. Biomed. Opt. 7, 205–214 共2002兲.
T. Yasui, K. Shimabayashi, H. Kawaguchi, Y. Tohno, T. Iwata and T.
Araki, ‘‘Polarimetry of second-harmonic-generation light sensitive toorientation of tissue collagen,’’ Abst. International Conference on
Optics Within Life Science VII, Switzerland, 2002, p. 19.
T. Yasui, Y. Tohno, and T. Araki, ‘‘Determination of collagen fiber
orientation in human tissue by polarization measurement of molecular second-harmonic-generation light,’’ 共unpublished兲.
P. J. Campagnola, A. C. Millard, M. Terasaki, P. E. Hoppe, C. J.
Malone, and W. A. Mohler, ‘‘Three-dimensional high-resolution
second-harmonic generation imaging of endogenous structural proteins in biological tissues,’’ Biophys. J. 82, 493–508 共2002兲.