IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 25, NO. 1, JANUARY/FEBRUARY 2019

6801007

Photonic-Crystal-Fiber-Coupled, Hand-Held,Polarization-Resolved Second-Harmonic-Generation
Microscope for In Vivo Visualization of 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 human skin. Furthermore, polarization-resolved SHG microscopy
can provide additional insights regarding the direction of collagen fibers, i.e., collagen fiber orientation. However, their practicaluse in the dermatological field is still limited due to the bulky
and complicated setup. In this paper, we constructed a photoniccrystal-fiber (PCF) 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 PCF,
whereas the SHG microscopy setup was enclosed into a hand-heldprobe head. The combination of PCF with the hand-held probehead largely enhances the flexibility of measurement sites in thehuman 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 collagenfiber in biological 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
Manuscript received April 4, 2018; revised July 26, 2018 and August 13,
2018; accepted August 13, 2018. Date of publication August 17, 2018; date ofcurrent version September 17, 2018. (Corresponding author: Takeshi Yasui.)Y. Ogura is with the Graduate School of Advanced Technology and Science,
Tokushima University, Tokushima 770-8506, Japan (e-mail:, yuki.ogura1@to.
shiseido.co.jp).
K. Atsuta and E. Hase were with the Graduate School of Advanced Technology and Science, Tokushima University, Tokushima 770-8506, Japan (e-mail:,
truth0f.telecaster.ash@gmail.com; hase@spring8.or.jp).
T. Minamikawa and T. Yasui are with the Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima 7708506, Japan (e-mail:, minamikawa.takeo@tokushima-u.ac.jp; yasui.takeshi@
tokushima-u.ac.jp).
Color versions of one or more of the figures in this paper are available online
at http://ieeexplore.ieee.org.
Digital Object Identifier 10.1109/JSTQE.2018.2865779

mode-locked 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., collagen fiber orientation [9]–[12]. This is because theefficiency of SHG light is sensitive to collagen fiber orientation
when the incident light is linearly polarized: strong SHG lightfor 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 of compactness,convenience, robustness, maintenance-free, and flexibility are
required in SHG-M. To meet these requirements, (a) miniaturization of SHG-M probe, (b) miniaturization of laser source,and (c) transmission of laser light via optical fiber are important
technical elements. Since commercialized compact optics andmechanical components can be used for packing optical components into a hand-held probe, a hand-held SHG-M probe wasconstructed by use of custom design lens and 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 be delivered to the handheld SHG-M probe by a 2.5-m double clad fiber (DCF), use ofsuch long DCF needs the bulky external dispersion compensator(grating pair) to avoid the pulse broadening caused by the DCF.
Recently, a 1030-nm mode-locked Yb-fiber laser, in place of themode-locked Ti:Sapphire laser, has been used for the hand-held
SHG-M probe [14]. Such all-fiber SHG-M benefits from costeffectiveness, fiber optics flexibility, and compactness; howver,
the external dispersion compensator was still needed. Althoughvery low risk of thermal or photochemical damage of the tissue
is another advantage of SHG-M at this wavelength band, use of
the longer-wavelength laser light is favorable to further suppress
the damage.
The wavelength within the range from 1200 to 1300 nm is thebest for optical window in biological tissues because of the decreased scattering and absorption in tissues. A promising laser

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6801007IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 25, NO. 1, JANUARY/FEBRUARY 2019source for this wavelength light is a mode-locked Cr:Forsterite
laser centered around 1250 nm; it has emerged as a new source
for SHG-M, showing decreased photodamage [15]–[17] andenhanced penetration depth [18]. Thanks to these benefits, the1250 nm-band SHG-M or PR-SHG-M has been used in dermatological applications in vivo or ex vivo, such as skin aging [19],skin burn [20], skin burn healing [21], skin wound 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 collagen fiberin 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 was used
as a laser source. We used a LMA-PCF (Thorlabs, Inc., LMA25, core diameter = 25 µm, NA = 0.05, length = 75 cm)
for fiber-delivery of the laser light. In this LMA-PCF, mode
dispersion and wavelength dispersion are largely reduced by
the designed PCF structure. Furthermore, the large mode-areadiameter suppresses unnecessary optical nonlinear effects inthe fiber and hence the pulse distortion. After passing through apolarizer (P) and a half-waveplate (λ/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 andthe objective lens will further improve the coupling efficiency.
The laser light propagated in the LMA-PCF and then were fed
into a hand-held SHG-M probe. The SHG-M was composed ofa 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 optical band-passfilter (BPF; transmitted wavelength = 625 nm), and a photoncounting 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-scattered from the sample wasreflected by HS, is filtered by the BPF, and then was detected by

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

PMT. While the focus of the laser light was two-dimensionallyscanned over the sample by GM, it was scanned along the depth
direction by a translation stage. These optical systems wereenclosed in the lens tube system (Thorlabs, Inc.) [see Fig. 1(b)].
For comparison, Fig. 1(c) shows an optical photograph of ourprevious SHG-M [19]. Since volume of this hand-held SHG-M

OGURA et al.: PCF COUPLED, HAND-HELD, POLARIZATION-RESOLVED SHG-M

6801007probe (310 mm wide, 150 mm height, and 50 mm depth) was
only 5% of that of our previous SHG-M (700 mm wide, 400 mm
height, and 400 mm depth), one can hold it by hand as shownin Fig. 1(b).
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 a board-certified
dermatologist [19]. We concluded from these examinations that
there was no laser-induced photodamage to the 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 skin to 30 mW.
Written informed consent was obtained from the subject before
the measurement. The protocol conformed to the Helsinki Declaration and was approved by the ethics committee for humanexperiment in Tokushima University (No. 14003). We measured
the SHG image of collagen fiber at the cheek skin in the 20’s
male subject.
III. RESULTS AND DISCUSSION
A. Fiber Delivery of Linearly-Polarized Pulse LightIn 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 propagatingin the LMA-PCF as shown in Fig. 1(a), the LMA-PCF has tomaintain the same polarization condition during the propagation. We first evaluated the polarization characteristics of thelaser light before and after propagating in a 75-cm LMA-PCF
with a curvature radius of 7.5 cm when the vertically linearpolarized laser light is incident into the LMA-PCF. To this end,
we placed an additional analyzer before or after the LMA-PCF[not shown in Fig. 1(a)] and analyzed the polarization characteristics by rotating the analyzer at a step of 20 degree. Fig. 2
shows a comparison of polarization characteristics (a) beforeand (b) after propagating in the LMA-PCF, which are illustrated as a radar graph of the optical intensity with respect tothe polarization angle. In this radar graph, the major axis gives
the dominant polarization direction, and a ratio of the minor
axis to the 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 the similar figure-of-eight profile was confirmed in Fig. 2(a) and 2(b),the linearly polarized light was maintained during the propagation of the LMA-PCF. Even though the LMA-PCF used is nota polarization-maintained fiber, such polarization-maintaining
capability is an interesting profile in the LMA-PCF.
We next evaluated the polarization characteristics of thelaser light after propagating in the LMA-PCF when thelinear-polarization angle of the incident laser light is rotatedFig. 2. Comparison of polarization characteristics (a) before propagating in
the LMA-PCF and (b) after propagating in the LMA-PCF with a curvatureradius of 7.5 cm.

by λ/2. Fig. 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. Such dependence
has to be compensated for precise polarization-resolved SHG
imaging.
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 hand-held
PR-SHG-M setup. To obtain a spatial distribution of collagen fiber orientation in the dermis, orthogonally polarizationresolved SHG imaging was applied for the samples. Polarization
anisotropy of SHG light (α) was defined using the following

6801007IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 25, NO. 1, JANUARY/FEBRUARY 2019

Fig. 4. (a) IH image, (b) IV image, and (c) α 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.
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.

equation [23], [24]α=IV − IH
IV + IH(1)

where IV and IH are SHG intensities when the incident light isvertically and horizontally polarized, respectively. Then the αimage was calculated by substituting IV and IH values at eachpixel of two orthogonally polarization-resolved SHG images forEq. (1). Collagen fiber orientation is uniaxial for α = ±1 andrandom or biaxial for α = 0. The sign of α value provides thedominant direction of the collagen fiber orientation: positive fora vertical orientation (blue) and negative for a horizontal orientation (red). Fig. 4 shows (a) IH image, (b) IV image, and (c) αimage (image size = 400 µm by 400 µm, pixel size = 256 pixels
by 256 pixels) when the collagen fiber orientation of the sample
was directed at horizontal direction. Due to the polarization dependence of SHG light efficiency, there is a small difference ofimage brightness between Fig. 4(a) and 4(b), leading to the reddish α image. On the other hand, Fig. 5 shows (a) IH image, (b)IV image, and (c) α image when the collagen fiber orientationof the sample was directed at the vertical direction. In this case,
IH image was darker than IV image, and hence the α image
became bluish. Such the reddish α image in Fig. 4(c) and thebluish α image in Fig. 5(c) correctly reflected the collagen fiber
orientation of the tendon specimen. In this way, we confirmed

Fig. 5. (a) IH image, (b) IV image, and (c) α 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.

OGURA et al.: PCF COUPLED, HAND-HELD, POLARIZATION-RESOLVED SHG-M

6801007

Fig. 6. A series of SHG images of dermal collagen fibers in human cheek skinacquired 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 sizeof 256 pixels by 256 pixels.

the effectiveness of the constructed hand-held PR-SHG-M setup
for evaluation of collagen fiber orientation.
C. In Vivo SHG Imaging of Dermal Collagen Fibers in Human
Cheek Skin
We performed in vivo SHG imaging of dermal collagen fibers
in human cheek skin by the constructed hand-held PR-SHG-M.
Since the cheek skin does not have outstanding features in thecollagen 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 in human skin. Fig. 6 showsa series of SHG images at an interval of 2 s (image size = 400 µm
by 400 µm, pixel size = 256 pixels by 256 pixels). The obtained
image quality and contrast were similar to those in the bulky

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

SHG-M in the previous study [18], [19], [24]. Although motion
artefact was confirmed in them due to the slow imaging rate, thedetailed distribution of collagen fibers was clearly confirmed.
This means that it is possible to perform quantitative analysis ofcollagen fiber structure based on SHG image analysis with thehand-held SHG-M [19], [28].
Dermal collagen fibers significantly change their structure
depending on the depth while SHG-M has the intrinsic depth

6801007IEEE JOURNAL OF SELECTED TOPICS IN QUANTUM ELECTRONICS, VOL. 25, NO. 1, JANUARY/FEBRUARY 2019selectivity. 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. Fig. 7 shows a series of in vivo depthresolved SHG images acquired at a step of 20 µm for dermal
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 the depth dependence of dermal collagen fiber structure: thin collagen fibers
distributed at the upper dermis corresponding to the papillary
dermis whereas thick collagen fibers were observed in the lower
dermis corresponding to the reticular dermis. These findings arein 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 a thin glass plate between the OL
and the sample surface, rather than the vanished SHG light. Useof a longer working distance, high-NA OL will increase thepenetration depth beyond 200 µm.
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 the ultrashort pulse light without the significant change of its linear
polarization as well as its pulse duration [27]. Also, the effectiveness of the fiber-coupled, hand-held PR-SHG-M was confirmedin 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 are comparable to those in the bulky SHG-M. Also, use of the long LMAPCF largely increases the flexibility of the hand-held SHG-M
for practical applications. Although the Cr:Forsterite laser is stillbulky 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 atthis wavelength band. The fiber-coupled, hand-held PR-SHGM 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 and mechanical features of tendon
healing [31].
ACKNOWLEDGMENT
The authors would like to acknowledge Ms. Shoko Lewis of
Tokushima University, Japan, for her help in the preparation of
the manuscript.
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Takeo Minamikawa was born in Ibraki, Japan, in 1983. He received the B.S.,
M.S., and Ph.D. degrees in engineering from Osaka University, Osaka, Japan,
in 2006, 2008, and 2010, respectively.
From 2010 to 2013, he was a Research Fellowship for Young Scientists
with the Japan Society for the Promotion of Science. From 2013 to 2015, he
was an Assistant Professor with the Department of Pathology and Cell Regulation, Graduate School of Medical Science, Kyoto Prefectural University of
Medicine. Since 2015, he has been an Associate Professor with the Departmentof Mechanical Science, Division of Science and Technology, Graduate School ofTechnology, Industrial and Social Sciences, Tokushima University, Tokushima,
Japan. He is the author of two book chapters, more than 30 articles, and morethan 15 inventions. His research interests include Raman microspectroscopy
and optical frequency comb. He holds three patents.
Dr. Minamikawa was the recipient of the Japan Society for Precision Engineering Best Presentation Award in 2016, the Japanese Society of Pathology
Poster Presentation Award in 2015, and the Funai Foundation for Information
Technology Research Award in 2011.

Yuki Ogura received the master’s degree in engineering from Osaka University,
Suita, Japan, in 2001. She is currently working toward the Ph.D. degree with
Tokushima University, Tokushima, Japan.
Since 2001, she has been with the Shiseido Research Center, Yokohama,Japan. Her research interests include skin measurements and second-harmonicgeneration microscopy.

Kosuke Atsuta received the master’s degree in engineering from Tokushima
University, Tokushima, Japan, in 2016.
Since 2016, he has been with the Seiko Epson Corp., Suwa, Japan. His
current research focuses on second-harmonic-generation microscopy.

Eiji Hase was born in Tokushima, Japan, in 1989. He received the B.S., M.S., andPh.D. degrees in engineering from Tokushima University, Tokushima, Japan, in
2012, 2014, and 2017, respectively.
From 2014 to 2017, he was a Research Associate with the JST, ERATO,
Minoshima Intelligent Optical Synthesizer Project. Since 2017, he has beena Research Scientist with the Research and Utilization Division, Japan Synchrotron Radiation Research Institute, Sayo, Japan, 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 the recipient of the Optical Society of Japan Best Presentation
Award in 2016, the Japanese Society for Medical and Biological Engineering
Symposium Award in 2016, and SPIE BiOS2016 in Photonics West 2016 Student Poster Awards in 2016.

Takeshi Yasui received the first Ph.D. degree in engineering from the University of Tokushima, Tokushima, Japan, in 1997, and the second Ph.D. degree inmedical science from the Nara Medical University, Yagi, Japan, in 2013.
From 1997 to 1999, he was a Postdoctoral Research Fellow with the National
Research Laboratory of Metrology, Japan. He was with the Graduate Schoolof Engineering Science, Osaka University from 1999 to 2010, and was briefly
with the University of Bordeaux I in 2007 and 2012, and with the Universityof Littoral Côte d’Opale in 2010 as an Invited Professor. He is currently a Professor with the Graduate School of Technology, Industrial and Social Sciences,
Tokushima University, Tokushima, Japan, has been the Vice Director of Research Support with the same university since 2016, and an Invited Professor
with the Graduate School of Engineering Science, Osaka University, Toyonaka, Japan. His research interests include THz instrumentation and metrology,
second-harmonic-generation microscopy, and optical frequency comb.
Prof. Yasui is a member of the Optical Society of America, the International Society for Optical Engineering, the Japan Society of Applied Physics,
the Optical Society of Japan, the Laser Society of Japan, the Japanese Society
for Medical and Biological Engineering, and the Japan Society of MechanicalEngineers. He was the recipient of the Award for the Most Promising Young
Scientist from the Optical Society of Japan in 1998, the Sakamoto Award fromthe Japan Society of Medical Electronics and Biological Engineering in 2006,
the Optics Paper Award from the Japan Society of Applied Physics, the Funai
Award from the Japan Society of Mechanical Engineers in 2009, and the Original Paper Award from the Laser Society of Japan in 2013.