Toyonobu Yamashita1, Yuki Ogura1, Yuji Tanaka2, Ryosuke Tanaka2, Takeshi Yasui2,3, Tsutomu Araki2,and Tetsuji Hirao1
1Shiseido Research Center, Yokohama, Kanagawa, Japan
2Graduate School of Engineering Science, Osaka University, Toyonaka, Osaka, Japan
3Institute of Technology and Science, Tokushima University, Tokushima, Japan

Keywords: Collagen Fibers, Imaging, Photoaging, Second Harmonic Generation

IFSCC Basic Research Award winning paper presented at the 27th IFSCC Congress, October 15-18, 2012,
Johannesburg, South Africa

INTRODUCTION
Cutaneous photoaging due to long-term
exposure to solar ultraviolet radiation
causes the degenerative structural alterations in the dermal extracellular matrix,
such as the decrease in collagen fibers
and the accumulation of abnormal elastic materials, resulting in a loss of skinelasticity and the formation of wrinkles.
These age-related alterations in extracellular matrix have been elucidated based on

histological observations of the skin specimens. However, further detailed studies
on how UV affects the dermal extracellularmatrix in healthy subjects during daily life
are hampered by the limitation of collecting skin specimens. The development of
noninvasive techniques for the assessmentof structural alterations of the extracellular matrix is, therefore, an important challenge not only in anti-aging research but

ABSTRACTSpecific visualization and assessmentof human collagen fibers were demonstrated using a developed secondharmonic-generation (SHG) microscope equipped with a Cr:forsterite
laser (1250 nm). Initially, we examined
the specific sensitivity of SHG imaging for collagen fibers using excised
human skin specimens. Age-relateddermal alterations in female subjects
were then evaluated by means of SHG
microscopy. The developed SHG microscope enabled us to observe the collagen fibers in the skin sections; their
SHG images correlated well with theIFSCC Magazine 1 | 2013

Elastica-van-Gieson-stained sections ofdermal fibers. In vivo imaging of humanskin with a wide field of view was alsoachieved. En face SHG images of facialskin of female subjects of different ages showed the structural alterations in
the collagen fiber network. Additional
image analysis of SHG images basedon two-dimensional Fourier transformation also reflected age-related alterations. In conclusion, this proposedmethodology can be a powerful toolin fields such as anti-aging dermatology and validation of the efficacy of
cosmetic products.

also in cosmetology. Recently developed
imaging systems applying non-linear optical techniques enabled us to obtain noninvasive optical images of various tissues.
Multiphoton microscopy including SHG
microscopy allows acquisition of en facesectional images targeted at a specific biological component [1, 2]. SHG microscopy
is an attractive technique for imaging of
the skin because the SHG light detectedby this microscope is specifically generated
by collagen molecules in collagen fibers
due to structural asymmetry (Fig. 1) [3].
In this study, we investigated human dermal structures and its alteration due to UV
exposure by means of a novel collagenspecific SHG microscopy system with theaim of understanding photoaging of thehuman skin.

MATERIALS AND METHODS
Optical imaging systems
We developed a reflection-mode SHG
microscope for noninvasive observation of collagen fibers in the human skin(Fig. 2, 3) with the aim of overcoming
the limitations of commercially available
in vivo multiphoton microscopes such as

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Noninvasive in situ Assessment of Structural Alteration of Human Dermis Caused by Photoaging Usinga Novel Collagen-Specific Imaging Techniqueshallow imaging depth, long image acquisition times and small scan areas. This collagen-specific SHG microscope employed
as a light source a near infrared femtosecond Cr:forsterite laser (Avesta Project,Moscow, Russia) with a long wavelength(1250 nm). Backscattered SHG light fromdermal collagen fibers with a wavelengthof 625 nm was detected by a combined
photon-counting photomultiplier tube
and pulse counter. The 3D stage (Fig. 2)
used as a mechanical human skin-holding
and fixing device was set in this system for
acquisition of a wide field of the collagenfiber images.

Fig. 1 Theory of SHG emission from collagen fibers. Structurally asymmetric collagen molecules
produce SHG light due to simultaneous two-photon absorption when exposed to a femtosecond
pulse laser

Specific sensitivity to collagen fibers
Frozen sections of human skin specimensfrom the face were visualized by SHG microscopy. After SHG imaging, skin sections
were stained with the Elastica-van-Gieson
method for comparison with the SHG images. These frozen sections of facial skinwere obtained from 17- and 64-year-oldAsian women.
Evaluation of human skin
The human studies were IRB-approved,
and written informed consents were obtained in all cases. Cheek skin was assessed as the sun-exposed site of healthy
female subjects aged from the 20s to 50s
by means of the SHG microscopy system.
Additionally, quantitative image analysesof collagen fibers using Fourier-transformed SHG images were performed,
focusing on the density. First, seven regions of interest (each 64 x 64 pixels insize) in the SHG images (1024 x 1024pixels in size) were selected excluding
the area of hair follicles. Then, a Fouriertransformed spectrum of the selected ROI
was obtained by two-dimensional Fourier transformation. Finally, the spectrum
width (full width at half maximum) wasdetermined as a parameter of collagendensity using Gaussian-fitted Fouriertransformed spectrum.

RESULTS AND DISCUSSION

Fig. 2 Photograph of the SHG microscope. (a) 3D-stage

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Comparison of SHG images and Elastica-van-Gieson stained section images
SHG images of frozen sections were compared with Elastica-van-Gieson-stained

IFSCC Magazine 1 | 2013section images. Representative SHG andstained section images of normal facialskin are shown in Figure 4. As expected,
the frozen section of a 17-year-old woman
reflected a stronger SHG light than that ofa 64-year-old woman (Fig. 4, a1 versusb1). In addition, the image of collagenfibers in the Elastica-van-Gieson-stained
section (Fig. 4, a2 and b2) correlated wellwith that of the non-stained SHG images(Fig. 4, a1 and b1). As a characteristic,
most of SHG light was observed specifically in the dermis. Also, collagen fibers
appearing as a pale-pink-stained color
in Elastica-van-Gieson-stained sections
showed a strong reflection in the SHG
images. We confirmed that bluish-purplestained elastic fibers resulted in less reflection in SHG images.
In vivo visualization of human cheek skin
Noninvasive visualization of the collagenfiber network in the human reticulardermis was achieved by means of ourSHG microscopy system. Highly efficient
SHG from collagen fibers due to theCr:forsterite laser led to an extremely fastacquisition rate of the SHG imaging (600x 600 µm with 256 x 256 pixels/2 seconds). This fast acquisition rate enabled
the acquisition of consecutive en faceIFSCC Magazine 1 | 2013

Fig. 4 Frozen section images. (a1 and b1): SHG images, (a2 and b2): Elastica-van-Gieson stained
section images, (a1 and a2): 17-year-old woman, (b1 and b2): 64-year-old woman (bars: 200 µm).

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Fig. 3 Experimental setup. PL = polarizer; HS = harmonic separator; F = infrared-cut filter;
L = lens, PMT = photomultiplier tube, GM = galvano mirror, RL1 and RL2 = relay lens,
OL = oil immersion lens

SHG images of human skin. Moreover,the use of a light source with a longer
wavelength (1250 nm) had the advantage of imaging with deeper penetration
than with conventional multiphoton microscopy (710-920 nm). Typical depthresolved SHG images and a wide field
SHG image obtained from cheek skinof a healthy male subject in his 60s areshown in Figure 5 and Figure 6, respectively. The SHG images demonstrateedthat collagen fiber bundles tend to bealigned parallel to the skin surface. The
depth-resolved SHG images showed thatthe papillary dermis is composed of very
tightly packed collagen fiber bundles(Fig. 5b) and reticular dermis is composed of bundles that have a largerdiameter (Fig. 5c and d) than those in
the papillary dermis. In addition, a wide
field SHG image showed that collagen fibers surrounding many hair follicles wereclearly visible as a mesh-like structure in
the reticular dermis (Fig. 6).

Fig. 5 Depth-resolved SHG images. Serial SHG images at the different depths were acquired frommale subjects in their sixties: (a) Epidermal-dermal junction at the depth of 100 µm, (b) Papillary
dermis at 120 µm, (c) Reticular dermis at 160 µm, (d) Reticular dermis at 200 µm

resentative SHG images are shown in Fig.
7. Comparison of the SHG images at thedepth of 200 µm (reticular dermis) showeda trend in the degree of reflection to decrease with age. An SHG image of a female
subject in her 20s showed a strong reflection over the whole region, apart from hairfollicles, suggesting a high density of collagen fibers in the reticular dermis (Fig. 7,a2). On the other hand, that of a female
subject in her fifties showed a mesh-liketexture pattern (Fig. 7, d2). In this SHG image, individual collagen bundles were easily observed, suggesting a low density ofcollagen fibers due to loss of tiny collagenfibers. Additionally, the parameter of thecollagen density (spectrum width; FWHM)
calculated from Fouriour-transformed SHG
images also supported a decreasing collagen density with age because the value ofthe spectrum width was relatively narrow inthe dense distribution image pattern.
Moreover, to clarify how UV affects
dermal collagen fibers in humans, weevaluated a female subject with a »highcumulative level of UV exposure to face
(H-UV)«, and that with a »low cumulative
level (L-UV)«. Magnified collagen imagesof a subject with a high cumulative level
showed hardly observable collagen fibers
except for some thicker fibers (Fig. 8a),while an age-matched subject with a low
cumulative level showed abundant finecollagen fibers (Fig. 8b). These results indicate that SHG microscopy can be usedin the evaluation of dermal photodamage
due to cumulative UV exposure. Also, thevisualized collagen fiber network is hardly
observed in vertical-stained sections ofskin specimens with conventional histological techniques. Therefore, en face SHG
images will be useful for evaluating thecollagen architecture in detail.

CONCLUSION
Fig. 6 Wide field of the collagen fiber image acquired by SHG microscopy. Sixteen consecutive SHG
images of dermis at the depth of approximately 200 µm were obtained by computer-controlled 2D
XY-stage movement (imaging area 2.4 X 2.4 mm, major hair follicles denoted by asterisks)Age-related alteration of collagen fibers
Focusing on the dermal structural alteration as a sign of photodamage, cheeks of242

healthy female subjects of different age
were evaluated using the SHG microscope.
Photographs of individual cheek and rep-Noninvasive assessments of dermal structure in human skin were performed using
a novel collagen-specific SHG microscopy
technique. A wide field of en face collagen fiber images obtained by this newly
developed SHG microscopy enabled us toevaluate photodamaged collagen fibers inhuman skin. In conclusion, the proposed

IFSCC Magazine 1 | 2013

SCIENTIFIC PAPERFig. 7 Photographs and SHG images of female subjects of different age. Upper panels - photographs of individual cheek; lower panels - wide fieldof en face SHG images at the depth of approximately 200 µm. (a1 and a2) - subject in her 20s, (b1and b2) - subject in her 30s, (c1 and c2) - subject
in her 40s, (d1 and d2) subject in her 50s. The area of each SHG image is 2.4 X 2.4 mm and the FWHM (degree) unit 1/m[2] Tsai, T.H., Jee, S.H., Dong, C.Y., and Lin,S.J., Multiphoton microscopy in dermatological imaging, J. Dermatol. Sci., 56 (2009) 1-8.
[3] Yasui, T., Takahashi, Y., Ito, M., Fukushima, S., and Araki, T., ex vivo and in vivo
second-harmonic-generation imaging ofdermal collagen fiber in skin: comparison ofimaging characteristics between mode-locked
Cr:forsterite and Ti:sapphire lasers, Appl. Opt.,
48 (2009) D88-95.

Fig. 8 Assessment of photodamage using SHG microscopy. (a) Female subject in her fifties with ahigh level of UV exposure (H-UV) who coaches outdoor sports; (b) age-matched female subject with alow level of UV exposure who is an office worker and had used sunscreen for many years. The depthof both SHG images is approximately 200 µm (reticular dermis) and the imaging area 600 x 600 µm

optical system promises to be a powerfultool to survey photoaging and monitor
dermal alterations in UV-exposed skin.
This cutting-edge technique focusing on
extracellular matrix structure should also
be useful for validation of the efficacy ofsuncare products against UV exposure.

IFSCC Magazine 1 | 2013

References[1] Koenig, K., and Riemann, I., High-resolution multiphoton tomography of human skinwith subcellular spatial resolution and picosecond time resolution, J. Biomed Opt., 8 (2003)
432-439.

Corresponding author
Toyonobu Yamashita
Shiseido Research Center
YokohamaJapan
toyonobu.yamashita@to.shiseido.co.jp!

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