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1> REPLACE THIS LINE WITH YOUR PAPER IDENTIFICATION NUMBER (DOUBLE-CLICK HERE TO EDIT) <Photonic-crystal-fiber-coupled, hand-held,polarization-resolved second-harmonicgeneration microscope for in vivo visualizationof dermal collagen fibers in human skin
Yuki Ogura, Kosuke Atsuta, Eiji Hase, Takeo Minamikawa, and Takeshi Yasui
Abstract—Second-harmonic-generation (SHG) microscopy is a
powerful tool for in vivo monitoring of collagen fibers in humanskin. Furthermore, polarization-resolved SHG microscopy canprovide additional insights regarding the direction of collagenfibers, i.e., collagen fiber orientation. However, their practical use
in the dermatological field is still limited due to the bulky andcomplicated setup. In this paper, we constructed a photoniccrystal-fiber-coupled, hand-held polarization-resolved SHG
microscope for in vivo monitoring of collagen fibers in human skin.
Fiber delivery of ultrashort pulse light was achieved without
significant change of the linear polarization by a large-mode-area
photonic-crystal-fiber whereas the SHG microscopy setup was
enclosed into a hand-held probe head. The combination of PCF
with the hand-held probe head largely enhances the flexibility ofmeasurement sites in the human skin.
Index Terms—Optical microscopy, Optical harmonic
generation, Biophotonics, Biomedical optical imaging.

I. INTRODUCTIONSECOND-harmonic-generation microscopy (SHG-M) is
an interesting new tool for observing collagen fiber inbiological tissues [1]. SHG-M provides unique imaging
characteristics: high image contrast, high spatial resolution,
optical three-dimensional (3D) sectioning, low invasiveness,
deep penetration, and no interference from background light.
More importantly, by using the naturally endogenous SHG
process as a contrast mechanism, the structure of collagen fibers
in tissue can be clearly visualized in vivo without additional
staining. For example, SHG-M equipped with a 800-nm modelocked Ti:Sapphire laser has been used for selective
visualization of collagen fibers in skin [2, 3], cornea [4], bone[5], cartilage [6], tendon [7], and cultured cell model [8].
Furthermore, combination of SHG-M and polarimetry, namely
polarization-resolved SHG-M or PR-SHG-M, can provide
additional insights regarding the direction of collagen fibers, i.e.,

Manuscript received April 1, 2018. (Corresponding author: Takeshi Yasui.)
Yuki Ogura is the Graduate School of Advanced Technology and Science,
Tokushima University, Tokushima, Tokushima 770-8506, Japan.
Kosuke Atsuta and Eiji Hase were the Graduate School of Advanced
Technology and Science, Tokushima University, Tokushima, Tokushima 7708506, Japan.

collagen fiber orientation [9-12]. This is because the efficiencyof SHG light is sensitive to collagen fiber orientation when theincident light is linearly polarized: strong SHG light for parallel
configuration and weak SHG light for perpendicularconfiguration. In spite of such high functionality, the clinicaluse of SHG-M or PR-SHG-M is still restricted.
One reason for this restriction is in the bulky, complicated,
fixed SHG-M setup. From the viewpoint of patent’s burden
reduction in clinical applications, characteristics ofcompactness, convenience, robustness, maintenance-free, andflexibility are required in SHG-M. To meet these requirements,(a) miniaturization of SHG-M probe, (b) miniaturization oflaser source, and (c) transmission of laser light via optical fiber
are important technical elements. Since commercializedcompact optics and mechanical components can be used for
packing optical components into a hand-held probe, a hand-held
SHG-M probe was constructed by use of custom design lensand 3D microscanner [13]. However, use of the mode-locked
Ti:Sapphire laser conflicts with a compact and simple SHG-M.
Furthermore, although the ultrashort pulse laser light could bedelivered to the hand-held SHG-M probe by a 2.5-m double
clad fiber (DCF), use of such long DCF needs the bulky external
dispersion compensator (grating pair) to avoid the pulse
broadening caused by the DCF. Recently, a 1030-nm modelocked Yb-fiber laser, in place of the mode-locked Ti:Sapphirelaser, has been used for the hand-held SHG-M probe [14]. Such
all-fiber SHG-M benefits from cost effectiveness, fiber optics
flexibility, and compactness; howver, the external dispersion
compensator was still needed. Although very low risk ofthermal or photochemical damage of the tissue is anotheradvantage of SHG-M at this wavelength band, use of thelonger-wavelength laser light is favorable to further suppress
the damage.
The wavelength within the range from 1200 to 1300 nm is
the best for optical window in biological tissues because of thedecreased scattering and absorption in tissues. A promising
Takeo Minamikawa and Takeshi Yasui are with the Graduate School ofTechnology, Industrial and Social Sciences, Tokushima University, Tokushima,
Tokushima 770-8506, Japan (e-mail: yasui.takeshi@tokushima-u.ac.jp).

> REPLACE THIS LINE WITH YOUR PAPER IDENTIFICATION NUMBER (DOUBLE-CLICK HERE TO EDIT) <laser source for this wavelength light is a mode-locked
Cr:Forsterite laser centered around 1250 nm; it has emerged asa new source for SHG-M, showing decreased photodamage[15-17] and enhanced penetration depth [18]. Thanks to thesebenefits, the 1250nm-band SHG-M or PR-SHG-M has beenused in dermatological applications in vivo or ex vivo, such as
skin aging [19], skin burn [20], skin burn healing [21], skinwound healing [22], and skin wrinkling [23, 24].
Since the state-of-art large-mode-area photonic crystal fiber(LMA-PCF) technology [25] enables us to deliver the
femtosecond pulse light without the need for external
dispersion compensators, it was used for delivering of the
Cr:Forsterite laser light to the hand-held SHG-M probe [26, 27].
The 80-fs pulse light could be delivered with little pulse
broadening by a 75-cm LMA-PCF even though no external
dispersion compensator was used [27]. However, there are no
attempts to expand it to PR-SHG-M.
In this paper, we modified our previous fiber-coupled, handheld SHG-M [27] for PR-SHG-M and evaluated its
performance. We demonstrated in vivo visualization of collagenfiber in human skin.
II. MATERIALS AND METHODS
A. Experimental Setup
Figure 1(a) shows the experimental setup of the hand-held
PR-SHG-M. A mode-locked Cr:Forsterite laser (Cr:F-L;
Avesta Project Ltd., CrF-65P, center wavelength = 1250 nm,
pulse duration = 80 fs, mean power = 250 mW, repetition rate
= 73 MHz) pumped by a 1064-nm Ytterbium fiber laser wasused as a laser source. We used a LMA-PCF (Thorlabs, Inc.,
LMA-25, core diameter = 25µm, NA = 0.05, length = 75cm)
for fiber-delivery of the laser light. In this LMA-PCF, mode
dispersion and wavelength dispersion are largely reduced by thedesigned PCF structure. Furthermore, the large mode-areadiameter suppresses unnecessary optical nonlinear effects in thefiber and hence the pulse distortion. After passing through apolarizer (P) and a half-waveplate (/2) for linear polarizationand its rotation, the laser light was incident into the LMA-PCF
with a coupling efficiency of 33 % by use of an objective lens
(Edmund Optics, magnification = 4, NA = 0.10). We did notobserve any long-term thermal or other damages to the LMAPCF input end. The matching of NA between the LMA-PCF
and the objective lens will further improve the coupling
efficiency. The laser light propagated in the LMA-PCF and thenwere fed into a hand-held SHG-M probe. The SHG-M wascomposed of a fiber collimator, a pair of galvano-mirrors (GM),relay lenses (RL1 and RL2), a harmonic separator (HS;reflected wavelength = 625 nm), an objective lens (OL; Nikon
Instruments Inc., CFI Plan 50×H, magnification = 50, NA = 0.9,
working distance = 350 µm, oil-immersion type), an opticalband-pass filter (BPF; transmitted wavelength = 625 nm), anda photon-counting photomultiplier (PMT; Hamamatsu
Photonics K. K., H7155-01). After passing through the fiber
collimator, GM, RL1, RL2, and HS, the laser beam was focused
onto the sample with the OL. The SHG light back-scatteredfrom the sample was reflected by HS, is filtered by the BPF,

2

and then was detected by PMT. While the focus of the laser
light was two-dimensionally scanned over the sample by GM,
it was scanned along the depth direction by a translation stage.
These optical systems were enclosed in the lens tube system(Thorlabs, Inc.) [see Fig. 1(b)]. For comparison, Fig. 1(c) shows
an optical photograph of our previous SHG-M [19]. Since
volume of this hand-held SHG-M (310mm wide, 150mm height,
and 50mm depth) was only 5 % of that of our previous SHG-M(700mm wide, 400mm height, and 400mm depth), one can holdit by hand as shown in Fig. 1(b).

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Fig. 1. (a) Schematic diagram and (b) optical photograph of hand-held PRSHG-M. (c) Optical photograph of conventional SHG-M.

B. In vivo measurement of human skin
To evaluate the risk of photodamage to human skin, we
previously examined changes in the subjects’ skin before,
immediately after, and one month after irradiation with a laser
light, based on color measurement with a spectrophotometer(Konica Minolta, CM-2600d) and clinical evaluation by aboard-certified dermatologist [19]. We concluded from these
examinations that there was no laser-induced photodamage tothe skin under irradiation of 35-mW laser light, corresponding
to the damage threshold of 6.2 MW/cm2. Based on this evidence,
we set the average power of the laser light radiated onto the skinto 30 mW. Written informed consent was obtained from thesubject before the measurement. The protocol conformed to theHelsinki Declaration and was approved by the ethics committee
for human experiment in Tokushima University (No. 14003).
We measured the SHG image of collagen fiber at the cheek skinin the 20’s male subject.
III. RESULTS AND DISCUSSION
A. Fiber delivery of linearly-polarized pulse light
In PR-SHG-M, while the polarization angle of a linearlypolarized laser light is rotated, the corresponding SHG images,
namely polarization-resolved SHG images, are acquired [9-12].
When its polarization angle is rotated before propagating in the
LMA-PCF as shown in Fig. 1(a), the LMA-PCF has to maintainthe same polarization condition during the propagation. We firstevaluated the polarization characteristics of the laser light
before and after propagating in a 75-cm LMA-PCF with acurvature radius of 7.5 cm when the vertically linear-polarizedlaser light is incident into the LMA-PCF. To this end, we placed
an additional analyzer before or after the LMA-PCF [not shownin Fig. 1(a)] and analyzed the polarization characteristics byrotating the analyzer at a step of 20 degree. Figure 2 shows acomparison of polarization characteristics (a) before and (b)after propagating in the LMA-PCF, which are illustrated as aradar graph of the optical intensity with respect to thepolarization angle. In this radar graph, the major axis gives thedominant polarization direction, and a ratio of the minor axis tothe major axis is corresponding to the extinction ratio. For
example, a figure-of-eight profile indicates the linear
polarization whereas a circular profile is given by the circularpolarization or completely random polarization. Since thesimilar figure-of-eight profile was confirmed in Fig. 2(a) and2(b), the linearly polarized light was maintained during the
propagation of the LMA-PCF. Even though the LMA-PCF used
is not a polarization-maintained fiber, such polarizationmaintaining capability is an interesting profile in the LMA-PCF.

Fig. 2. Comparison of polarization characteristics (a) before propagating in the
LMA-PCF and (b) after propagating in the LMA-PCF with a curvature radiusof 7.5 cm.

We next evaluated the polarization characteristics of the laser
light after propagating in the LMA-PCF when the linearpolarization angle of the incident laser light is rotated by /2.
Figure 3(a) shows a relation of the dominant polarization
direction between the incident light and the output light fromthe LMA-PCF. A good linear relation was confirmed between
them. On the other hand, Fig. 3(b) shows the extinction ratiowith respect to the linear-polarization angle of the incident laser
light. The extinction ratio largely changed depending on thelinear-polarization angle. One possible reason for such change
is in bending of the LMA-PCF with a curvature radius of 7.5
cm; however, it is not clear at the present stage. Suchdependence has to be compensated for precise polarizationresolved SHG imaging.

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leading to the reddish  image. On the other hand, Fig. 5 shows(a) IH image, (b) IV image, and (c)  image when the collagenfiber orientation of the sample was directed at the verticaldirection. In this case, IH image was darker than IV image, andhence the  image became bluish. Such the reddish  image in
Fig. 4(c) and the bluish  image in Fig. 5(c) correctly reflected
the collagen fiber orientation of the tendon specimen. In thisway, we confirmed the effectiveness of the constructed handheld PR-SHG-M setup for evaluation of collagen fiber
orientation.

Fig. 3. (a) Relation of the dominant polarization direction between the incident
light and the output light from the LMA-PCF. (b) Extinction ratio of the output
light with respect to the linear-polarization angle of the incident laser light.

B. Ex vivo polarization-resolved SHG imaging of sliced
tendon specimen with uniaxial collagen fiber orientation
We next performed ex vivo polarization-resolved SHG
imaging of a sliced tendon specimen by the constructed handheld PR-SHG-M setup. To obtain a spatial distribution ofcollagen fiber orientation in the dermis, orthogonallypolarization-resolved SHG imaging was applied for the samples.
Polarization anisotropy of SHG light ( ) was defined using thefollowing equation [23, 24]

where IV and IH are SHG intensities when the incident light isvertically and horizontally polarized, respectively. Then the image was calculated by substituting IV and IH values at eachpixel of two orthogonally polarization-resolved SHG images
for Eq. (1). Collagen fiber orientation is uniaxial for  = ±1 andrandom or biaxial for  = 0. The sign of  value provides thedominant direction of the collagen fiber orientation: positive fora vertical orientation (blue) and negative for a horizontal
orientation (red). Figure 4 shows (a) IH image, (b) IV image, and(c)  image (image size = 400 µm by 400 µm, pixel size = 256
pixels by 256 pixels) when the collagen fiber orientation of thesample was directed at horizontal direction. Due to thepolarization dependence of SHG light efficiency, there is asmall difference of image brightness between Fig. 4(a) and 4(b),Fig. 4. (a) IH image, (b) IV image, and (c)  image when the collagenorientation of the sample was directed at the horizontal direction.
Image size is 400 µm by 400 µm, corresponding to pixel size of 256
pixels by 256 pixels.

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Fig. 5. (a) IH image, (b) IV image, and (c)  image when the collagenorientation of the sample was directed at the vertical direction. Image
size is 400 µm by 400 µm, corresponding to pixel size of 256 pixels
by 256 pixels.

C. In vivo SHG imaging of dermal collagen fibers in humancheek skin
We performed in vivo SHG imaging of dermal collagenfibers in human cheek skin by the constructed hand-held PRSHG-M. Since the cheek skin does not have outstanding
features in the collagen fiber orientation [24], we acquired
several SHG images, in place of polarization-resolved SHG
images, at a measurement rate of 0.5 image/s to demonstrate its
in vivo SHG imaging capability of dermal collagen fibers inhuman skin. Figure 6 shows a series of SHG images at aninterval of 2 s (image size = 400 µm by 400 µm, pixel size =256 pixels by 256 pixels). The obtained image quality andcontrast were similar to those in the bulky SHG-M in theprevious study [18, 19, 24]. Although motion artefact was
confirmed in them due to the slow imaging rate, the detailed
distribution of collagen fibers was clearly confirmed. Thismeans that it is possible to perform quantitative analysis ofcollagen fiber structure based on SHG image analysis with thehand-held SHG-M [19, 28].

Fig. 6. A series of SHG images of dermal collagen fibers in humancheek skin acquired at a rate of 0.5 image/s. (a) t = 0 s, (b) t = 2 s, (c) t= 4 s, (d) t = 6 s, and (e) t = 8 s. Image size is 400 µm by 400 µm,
corresponding to pixel size of 256 pixels by 256 pixels.

Dermal collagen fibers significantly change their structure
depending on the depth while SHG-M has the intrinsic depth
selectivity. To confirm such features, we demonstrated the invivo depth-resolved SHG imaging of dermal collagen fibers inhuman cheek skin by scanning the hand-held PR-SHG-M probe
along the depth direction. Figure 7 shows a series of in vivo
depth-resolved SHG images acquired at a step of 20 µm fordermal collagen fibers in human cheek skin (image size = 1.6
mm by 1.6 mm corresponding to 256 pixels by 256 pixels,
image acquisition time ≈ 20 s). We here defined the skin surface
to be 0 µm in depth. These SHG images clearly indicated thedepth dependence of dermal collagen fiber structure: thincollagen fibers distributed at the upper dermis corresponding to
the papillary dermis whereas thick collagen fibers wereobserved in the lower dermis corresponding to the reticular

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dermis. These findings are in good agreement with our previous
researches using the bulky SHG-M [18, 19]. The maximum
probing depth of the present SHG-M is limited by the working
distance of the OL (= 350 μm) and a thickness (~ 100 µm) of athin glass plate between the OL and the sample surface, rather
than the vanished SHG light. Use of a longer working distance,high-NA OL will increase the penetration depth beyond 200μm.

IV. CONCLUSION
We constructed the fiber-coupled, hand-held PR-SHG-M by
use of the 1250-nm Cr:Forsterite laser, the 75-cm LMA-PCF,
and the hand-held probe. The LMA-PCF could deliver theultrashort pulse light without the significant change of its linear
polarization as well as its pulse duration. Also, the effectivenessof the fiber-coupled, hand-held PR-SHG-M was confirmed in
the ex-vivo polarization-resolved SHG imaging of the sliced
tendon specimen. Finally, we demonstrated in vivo SHG
imaging of dermal collagen fibers in human cheek skin. The
image quality and contrast in the hand-held PR-SHG-M arecomparable to those in the bulky SHG-M. Also, use of the long
LMA-PCF largely increases the flexibility of the hand-held
SHG-M for practical applications. Although the Cr:Forsterite
laser is still bulky for clinical application, use of a mode-locked
fiber laser [29] or mode-locked integrated external-cavity
surface emitting laser (MIXSEL) [30] is an alternative compact
laser source at this wavelength band. The fiber-coupled, handheld PR-SHG-M will be open the door for in vivo
dermatological applications in hospital as well as time-series
monitoring of collagen fiber production by cells cultured in an
incubator [8] and in vivo observation of the histological andmechanical features of tendon healing [31].

ACKNOWLEDGEMENT
The authors acknowledge Ms. Shoko Lewis of Tokushima
University, Japan, for her help in the preparation of themanuscript.

Fig. 7. Result of in vivo depth-resolved SHG imaging of dermal
collagen fibers in human cheek skin.

> REPLACE THIS LINE WITH YOUR PAPER IDENTIFICATION NUMBER (DOUBLE-CLICK HERE TO EDIT) <REFERENCES
P. J. Campagnola and C.-Y. Dong, “Second harmonic generation
microscopy: principles and applications to disease diagnosis,” Laser &Photon. Rev., vol. 5, pp. 13-26, Dec. 2011.
[2] K. Lu, J. Chen, S. Zhuo, L. Zheng, X. Jiang, X. Zhu, and J. Zhao,“Multiphoton laser scanning microscopy of localized scleroderma,” Skin
Res. Tech., vol. 15, pp. 489-495, Nov. 2009.
[3] R. Cicchi, D. Kapsokalyvas, V. De Giorgi, V. Maio, A. Van Wiechen, D.
Massi, T. Lotti, and F. S. Pavone, “Scoring of collagen organization in
healthy and diseased human dermis by multiphoton microscopy,” J.
Biophoton., vol. 3, pp. 34-43, Jan. 2010.
[4] L. Kowalczuk, G. Latour, J. L. Bourges, M. Savoldelli, J. C. Jeanny, K.
Plamann, M. C. Schanne-Klein, and F. Behar-Cohen, “Multimodal
highlighting of structural abnormalities in diabetic rat and human corneas,”
Transl. Vis. Sci. Technol., vol. 2, pp. 3, Feb. 2013.
[5] R. Ambekar, M. Chittenden, and I. Jasiuk, “Toussaint KC Jr.: Quantitative
second-harmonic generation microscopy for imaging porcine corticalbone: comparison to SEM and its potential to investigate age-relatedchanges,” Bone, vol. 50, p. 643-50, Mar. 2012.
[6] J. C. Mansfield, C. P. Winlove, J. Moger, and S. J. Matcher, “Collagenfiber arrangement in normal and dis-eased cartilage studied bypolarization sensitive nonlinear microscopy,” J. Biomed. Opt., vol. 13, art.
044020, Jul-Aug 2008.
[7] C.-Y. Park, J. K. Lee, M. Y. Kahook, J. S. Schultz, C. Zhang, and R. S.
Chuck, “Revisiting ciliary muscle tendons and their connections with thetrabecular meshwork by two photon excitation microscopic imaging,”
Invest. Ophthalmol. Vis. Sci., vol. 57, pp. 1096-105, Mar. 2016.
[8] E. Hase, O. Matsubara, T. Minamikawa, K. Sato, and T. Yasui, “In situ
time-series monitoring of collagen fibers produced by standing-cultured
osteoblasts using a second-harmonic-generation microscope,” Appl. Opt.,
vol. 55, pp. 3261-3267, Apr. 2016.
[9] P. C. Stoller, B.-M. Kim, A. M. Rubenchik, K. M. Reiser, and L. B. Da
Silva "Polarization-dependent optical second-harmonic imaging of rat-tailtendon," J. Biomed. Opt., vol. 7, pp. 205-215, Apr. 2002.
[10] P. Stoller, K. M. Reiser, P. M. Celliers, and A. M. Rubenchik"Polarization-modulated second harmonic generation in collagen,"
Biophys. J. vol. 82, pp. 3330-3342, June 2002.
[11] T. Yasui, Y. Tohno, and T. Araki, "Determination of collagen fiber
orientation in human tissue by polarization measurement of molecularsecond-harmonic-generation light," Appl. Opt., vol. 43, pp. 2861-2867,
May 2004.
[12] T. Yasui, Y. Tohno, and T. Araki, "Characterization of collagenorientation in human dermis by two-dimensional second-harmonicgeneration polarimetry," J. Biomed. Opt., vol. 9, pp. 259-264, Mar./Apr.
2004.
[13] H. Bao, A. Boussioutas, R. Jeremy, S. Russell, and M. Gu, "Second
harmonic generation imaging via nonlinear endomicroscopy," Opt.
Express, vol. 18, pp. 1255-1260, Jan. 2010.
[14] Á. Krolopp, A. Csákányi, D. Haluszka, D. Csáti, L. Vass, A. Kolonics, N.
Wikonkál, and R. Szipőcs, "Handheld nonlinear microscope systemcomprising a 2 MHz repetition rate, mode-locked Yb-fiber laser for invivo biomedical imaging," Biomed. Opt. Express, vol. 7, pp. 3531-3542,
Sep. 2016.
[15] I.-H. Chen, S.-W. Chu, C.-K. Sun, P.-C. Cheng, and B.-L. Lin,
“Wavelength dependent damage in biological multi-photon confocal
microscopy: a micro-spectroscopic comparison between femtosecond
Ti:sapphire and Cr:forsterite laser sources,” Opt. Quantum. Electron., vol.
34, pp. 1251-1266, Dec. 2002.
[16] S.-W. Chu, S.-Y. Chen, T.-H. Tsai, T.-M. Liu, C.-Y. Lin, H.-J. Tsai, andC.-K. Sun, “In vivo developmental biology study using noninvasive multiharmonic generation microscopy," Opt. Express, vol. 11, pp. 3093-3099,
Nov. 2003.
[17] S.-P. Tai, W.-J. Lee, D.-B. Shieh, P.-C. Wu, H.-Y. Huang, C.-H. Yu, andC.-K. Sun, “In vivo optical biopsy of hamster oral cavity with epi-thirdharmonic-generation microscopy,” Opt. Express, vol. 14, pp. 6178-6187,
Jun. 2006.
[18] T. Yasui, Y. Takahashi, M. Ito, S. Fukushima, and T. Araki, “Ex vivo andin vivo second-harmonic-generation imaging of dermal collagen fiber inskin: comparison of imaging characteristics between mode-locked
Cr:Forsterite and Ti:Sapphire lasers,” Appl. Opt., vol. 48, pp. D88-D95,
Apr. 2009.
[19] T. Yasui, M. Yonetsu, R. Tanaka, Y. Tanaka, S. Fukushima, T. Yamashita,
Y. Ogura, T. Hirao, H. Murota, and T. Araki, “In vivo observation of agerelated structural changes of dermal collagen in human facial skin using[1]

7

collagen-sensitive second harmonic generation microscope equipped with
1250-nm mode-locked Cr:Forsterite laser,” J. Biomed. Opt., vol. 18, art.
031108, Mar. 2013.
[20] R. Tanaka, S. Fukushima, K. Sasaki, Y. Tanaka, H. Murota, T.
Matsumoto, T. Araki, and T. Yasui, “In vivo visualization of dermal
collagen fiber in skin burn by collagen-sensitive second-harmonicgeneration microscopy,” J. Biomed. Opt., vol. 18, art. 061231, Jun. 2013.
[21] T. Yasui, R. Tanaka, E. Hase, S. Fukushima, and T. Araki, “In vivo timelapse imaging of skin burn wound healing using second-harmonic
generation microscopy,” Proc. SPIE, vol. 8948, art. 89480B, Feb. 2014.
[22] G. Deka, W.-W. Wu, and F.-J. Kao, “In vivo wound healing diagnosis
with second harmonic and fluorescence lifetime imaging,” J. Biomed.
Opt., vol. 18, art. 061222, Dec. 2012.
[23] T. Yasui, Y. Takahashi, S. Fukushima, Y. Ogura, T. Yamashita, T.
Kuwahara, T. Hirao, and T. Araki, "Observation of dermal collagen fiberin wrinkled skin using polarization-resolved second-harmonic-generation
microscopy," Opt. Express, vol. 17, pp. 912-923, Jan. 2009.
[24] Y. Tanaka, E. Hase, S. Fukushima, Y. Ogura, T. Yamashita, T. Hirao, T.
Araki, and T. Yasui, "Motion-artifact-robust, polarization-resolved
second-harmonic-generation microscopy based on rapid polarization
switching with electro-optic Pockells cell and its application to in vivo
visualization of collagen fiber orientation in human facial skin," Biomed.
Opt. Express, vol. 5, pp. 1099-1113, Apr. 2014.
[25] J. C. Knight, T. A. Birks, R. F. Cregan, P. S. J. Russell, and P. D. de
Sandro, "Large mode area photonic crystal fibre," Electron. Lett., vol. 34,
1347-1348, June 1998.
[26] S.-H. Chia, C.-H. Yu, C.-H. Lin, N.-C. Cheng, T.-M. Liu, M.-C. Chan, IH. Chen, and C. K. Sun, “Miniaturized video-rate epi-third-harmonicgeneration fiber-microscope,” Opt. Express, vol. 18, pp. 17382-17391,
Aug. 2010.
[27] K. Atsuta, Y. Ogura, E. Hase, T. Minamikawa, and T. Yasui, "In situ
monitoring of collagen fibers in human skin using a photonic-crystalfiber-coupled, hand-held, second-harmonic-generation microscope,"
Proc. SPIE, vol. 10069, art. 100692B, Feb. 2017.
[28] Y. Ogura, Y. Tanaka, E. Hase, T. Yamashita, and T. Yasui, "Texture
analysis of second-harmonic-generation images for quantitative analysisof reticular dermal collagen fiber in vivo in human facial cheek skin," Exp.
Dermatol., online publication (2018); DOI: 10.1111/exd.13560.
[29] W. Liu, S.-H. Chia, H.-Y. Chung, R. Greinert, F. X. Kärtner, and G.
Chang, “Energetic ultrafast fiber laser sources tunable in 1030–1215 nm
for deep tissue multi-photon microscopy,” Opt. Express, vol. 25, pp.
6822-6831, Mar. 2017.
[30] A. R. Bellancourt, D. J. H. C. Maas, B. Rudin, M. Golling, T. Südmeyer,and U. Keller, “Modelocked integrated external-cavity surface emitting
laser (MIXSEL),” IET Optoelectron., vol. 3, pp. 61-72, Apr. 2009.
[31] E. Hase, K. Sato, D. Yonekura, T. Minamikawa, M. Takahashi, and T.
Yasui, “Evaluation of the histological and mechanical features of tendon
healing in a rabbit model with the use of second-harmonic-generation
imaging and tensile testing,” Bone & Joint Res., vol. 5, pp. 577-585, Nov.
2016.

Yuki Ogura received her master degree in engineering fromthe Osaka University, Suita, Japan, in 2001.
Since 2001, she works in the Shiseido Research Center.,
Yokohama, Japan. She is currently a PhD student in theTokushima University, Tokushima, Japan. Here researchinterests include skin measurements and second-harmonicgeneration microscopy.
Kosuke Atsuta received his master degree in engineering fromthe Tokushima University, Tokushima, Japan, in 2016.
His area of research is second-harmonic-generation
microscopy.
Eiji Hase was born in Tokushima, Japan, in 1989. He received
the B.S., M.S. and Ph.D. degrees in engineering from
Tokushima University, Tokushima, Japan, in 2012, 2014 and2017.

> REPLACE THIS LINE WITH YOUR PAPER IDENTIFICATION NUMBER (DOUBLE-CLICK HERE TO EDIT) <From 2014 to 2017, he was a Research Associate with theJST, ERATO, MINOSHIMA Intelligent Optical Synthesizer
Project. Since 2017, he has been a Research Scientist with the
Research and Utilization Division, Japan Synchrotron
Radiation Research Institute and a Visiting Associate Professor
with the Graduate School of Technology, Industrial and Social
Sciences, Tokushima University. His research interests include
Biomedical imaging and Biomechanical analysis using
femtosecond laser and synchrotron X-ray.
Dr. Hase was a recipient of The Optical Society of Japan Best
Presentation Award in 2016, The Japanese Society for Medical
and Biological Engineering Symposium Award in 2016, andSPIE BiOS2016 in Photonics West 2016 Student Poster
Awards in 2016.
Takeo Minamikawa was born in Ibraki, Japan, in 1983. Hereceived the B.S., M.S. and Ph.D. degrees in engineering from
Osaka University, Osaka, Japan, in 2006, 2008 and 2010.
From 2010 to 2013, he was a Research Fellowship for Young
Scientists with Japan Society for the Promotion of Science.
From 2013 to 2015, he was an Assistant Professor with
Department of Pathology and Cell Regulation, Graduate Schoolof Medical Science, Kyoto Prefectural University of Medicine.
Since 2015, he has been an Associate Professor with
Department of Mechanical Science, Division of Science andTechnology, Graduate School of Technology, Industrial andSocial Sciences, Tokushima University, Tokushima, Japan. He
is the author of two book chapters, more than 30 articles, andmore than 15 inventions. His research interests include Raman
microspectroscopy and optical-frequency-comb. He holds threepatents.
Dr. Minamikawa’s awards and honors include The Japan
Society for Precision Engineering Best Presentation Award in
2016, The Japanese Society of Pathology Poster Presentation
Award in 2015, and Funai Foundation for Information
Technology Research Award in 2011.
Takeshi Yasui received the first Ph.D. degree in engineeringfrom the University of Tokushima, Tokushima, Japan, in 1997,
and the second Ph.D. degree in medical science from the Nara
Medical University, Yagi, Japan, in 2013.
From 1997 to 1999, he worked as a Post-Doctoral Research
Fellow in the National Research Laboratory of Metrology,
Japan. He was with the Graduate School of Engineering
Science, Osaka University from 1999 to 2010, and was briefly
with the University of Bordeaux I in 2007 and 2012, and with
the University of Littoral Côted'Opale in 2010 as an Invited
Professor. He is currently a Professor in the Graduate School ofTechnology, Industrial and Social Sciences, Tokushima
University, Tokushima, Japan, Vice Director of Research
Support in the same university since 2016, and an Invited
Professor in the Graduate School of Engineering Science,
Osaka University, Toyonaka, Japan. His research interests
include THz instrumentation and metrology, second-harmonicgeneration microscopy, and optical frequency comb.
Prof. Yasui is a member of the Optical Society (OSA), The
International Society for Optical Engineering (SPIE), the Japan
Society of Applied Physics, the Optical Society of Japan, theLaser Society of Japan, the Japanese Society for Medical andBiological Engineering, and the Japan Society of Mechanical8

Engineers. He received the Award for the Most Promising
Young Scientist from the Optical Society of Japan in 1998, theSakamoto Award from the Japan Society of Medical
Electronics and Biological Engineering in 2006, the Optics
Paper Award from the Japan Society of Applied Physics, andthe Funai Award from the Japan Society of MechanicalEngineers in 2009, the Original Paper Award from the Laser
Society of Japan in 2013.