In vivo observation of age-related
structural changes of dermal collagen inhuman facial skin using collagensensitive second harmonic generation
microscope equipped with 1250-nm
mode-locked Cr:Forsterite laser
Takeshi Yasui
Makoto Yonetsu
Ryosuke Tanaka
Yuji Tanaka
Shu-ichiro Fukushima
Toyonobu Yamashita
Yuki Ogura
Tetsuji Hirao
Hiroyuki Murota
Tsutomu Araki

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Journal of Biomedical Optics 18(3), 031108 (March 2013)

In vivo observation of age-related structural changesof dermal collagen in human facial skin using
collagen-sensitive second harmonic generation
microscope equipped with 1250-nm mode-locked
Cr:Forsterite laser
Takeshi Yasui,a,b,c Makoto Yonetsu,a Ryosuke Tanaka,a Yuji Tanaka,a Shu-ichiro Fukushima,a,c Toyonobu Yamashita,dYuki Ogura,d Tetsuji Hirao,d Hiroyuki Murota,e and Tsutomu ArakiaaOsaka University, Graduate School of Engineering Science, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan
University of Tokushima, Institute of Technology and Science, 2-1 Minami-Josanjima, Tokushima 770-8506, Japanc
Nara Medical University, Graduate School of Medicine, 840 Shijo-cho, Kashihara, Nara 634-8521, Japand
Shiseido Research Center, 2-2-1 Hayabuchi, Tsuzuki, Yokohama, Kanagawa 224-8558, Japane
Osaka University, Graduate School of Medicine, 2-2 Yamadaoka, Suita, Osaka 565-0871, Japanb

Abstract. In vivo visualization of human skin aging is demonstrated using a Cr:Forsterite (Cr:F) laser-based, collagen-sensitive second harmonic generation (SHG) microscope. The deep penetration into human skin, as well as
the specific sensitivity to collagen molecules, achieved by this microscope enables us to clearly visualize agerelated structural changes of collagen fiber in the reticular dermis. Here we investigated intrinsic aging and/or
photoaging in the male facial skin. Young subjects show dense distributions of thin collagen fibers, whereas elderly
subjects show coarse distributions of thick collagen fibers. Furthermore, a comparison of SHG images between
young and elderly subjects with and without a recent life history of excessive sun exposure show that a combinationof photoaging with intrinsic aging significantly accelerates skin aging. We also perform image analysis based on
two-dimensional Fourier transformation of the SHG images and extracted an aging parameter for human skin. The
in vivo collagen-sensitive SHG microscope will be a powerful tool in fields such as cosmeceutical sciences andanti-aging dermatology. © 2012 Society of Photo-Optical Instrumentation Engineers (SPIE). [DOI: 10.1117/1.JBO.18.3.031108]
Keywords: nonlinear microscopy; second harmonic generation; collagen; dermatology; intrinsic aging; photoaging; tissue; dermis.
Paper 12412SSP received Jul. 1, 2012; revised manuscript received Nov. 6, 2012; accepted for publication Nov. 9, 2012; published
online Dec. 5, 2012.

1

Introduction

Skin aging is one of the most interesting topics in the field suchas cosmeceutical sciences and anti-aging dermatology, because
skin is the superficial tissue of the human body and hence isalways in public view. For middle-aged and elderly people,
skin aging is definitely important matter as aesthetic facial concerns. Skin aging falls into the following two categories: intrinsic aging and photoaging.1–3 The intrinsic aging as chronologicchanges is caused by the declining fibroblast activity with aging
as people get older and the subsequent decrease in dermal collagen fiber. The intrinsic aging is an irreversible, physiologicalphenomenon that cannot be avoided. For example, mechanicalproperties such as tension and elasticity decline gradually withage, resulting in the appearance of wrinkles and sagging. In aprevious study, reduced synthesis of collagen fibers in chronologically aged skin was demonstrated, in which cellular fibroblast aging in the aged skin contributed to decreased type I and
III collagen production.4 In contrast, photoaging results fromrepeated exposure of the skin to ultraviolet (UV) rays in sunlightbecause UV irradiation causes dermal alterations, such as

Address all correspondence to: Takeshi Yasui, University of Tokushima, Instituteof Technology and Science, 2-1 Minami-Josanjima, Tokushima 770-8506, Japan.
Tel: +81-886-656-7377; Fax: +81-886-656-7377; E-mail: yasui.takeshi@
tokushima-u.ac.jp

Journal of Biomedical Optics

excessive secretion of matrix metalloproteinase (MMP) family,
which degrades collagen and other extracellular matrix proteins,5 and inhibition of procollagen biosynthesis resulting ina loss of collagen content.6 These alterations lead to degenerately structural changes in the skin, such as leathery texture, laxity, and deep wrinkles.7 Furthermore, repeated exposure to UV
radiation even increases the risk of skin cancer.8,9 As opposed tointrinsic aging, photoaging can be avoided by using sunshades
and/or sunscreen creams.10 Considering that skin aging appears
as a result of both intrinsic aging and photoaging, the degree ofskin aging is not always proportional to one’s age but varies
from individual to individual depending on the history of UV
exposure as well as dietary intakes such as antioxidants.11
Therefore, there is a need for an assessment technique for estimating the degree of skin aging.
The progress of skin aging is closely related to the dynamics
of dermal collagen fibers because dermal collagen fibers contribute to the morphology and mechanical properties of the skin asa structural protein. The age-related structural change in dermal
collagen has been extensively investigated based on histologicalmethods of skin biopsy specimen. For example, morphometricexamination of skin tissue section with optical microscope
showed age-dependent change of quantity and diameter of0091-3286/2012/$25.00 © 2012 SPIE

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collagen bundle in human skin.12 However, such ex vivo assessment method of dermal collagen have often limited utility due toinvasive skin biopsy. If in vivo, low-invasive assessment
methods can be achieved, the application fields of the skinassessment method will be largely increased.
Among various inspection techniques for skin, optical probe
methods are the most attractive for such in vivo assessments
because they are simple, rapid, and low invasive. Furthermore,they can be applied directly because skin is a superficial tissue.
Recent advances in ultrashort pulse lasers have opened the door
to new optical probe methods based on optical nonlinear effects
in biological tissue. When an ultrashort pulse of light is incident
on a material with a noncentrosymmetric structure, light withhalf the wavelength of the incident light, known as second
harmonic generation (SHG) light, is often produced via a nonlinear optical process.13 Although the SHG process is widelyused for wavelength conversion of laser light using a nonlinear
optical crystal, it also enables unique collagen-sensitive microscope when applied to biological tissue.14,15 This is because the
SHG light is generated specifically by collagen molecules
among various biological materials present due to the triplehelix structure of three polypeptide chains in this molecule.
Collagen-sensitive SHG microscope provides unique imaging
characteristics: high image contrast, high spatial resolution,
optical three-dimensional (3-D) sectioning, low invasiveness,
deep penetration, and no interference from background light.
Most 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.
Collagen-sensitive SHG microscope equipped with an 800nm mode-locked Ti:Sapphire laser has been employed for invivo visualizing of the dermal collagen fibers in mouse skin16
and human skin.17 Furthermore, European conformity-certified
multiphoton microscope, giving two photon fluorescence (TPF)
images of NAD(P)H, flavins, porphyrins, elastin, and melanin as
well as SHG image of collagen, is commercially available forclinical instrument.18 With respect to assessment of dermal
photoaging, combined SHG and TPF microscope gives an indicator of changes in dermal collagen and elastin content ex vivo19
and in vivo20 because degradation of collagen and excessive
deposition of abnormal elastin are observed in photoaged
skin. Very recently, the elastin-to-collagen ratio has been introduced as a new indicator for human dermal skin aging.21 On theother hand, a mode-locked Cr:F laser centered around 1250 nm
has emerged as a new source for SHG microscope, showing
decreased photodamage22–24 and enhanced penetration
depth.25 The Cr:F laser-based SHG microscope has beensuccessfully employed for imaging of dermal collagen fiberin excised mouse skin,26 excised fixed human skin,27 and invivo human skin.25,28 From the viewpoint of assessment ofskin aging, since Cr:F laser-based SHG microscope enables
us to probe down to deeper portions in the dermis-that is, thereticular dermis-it may contribute to a better understanding of
age-related structural change in the dermal collagen fibers.
For example, based on the fact that the efficiency of SHG
light is sensitive to the collagen orientation when the incident
light is linearly polarized, polarization-resolved, Cr:F laserbased SHG microscope has been applied to investigate therelation between wrinkle direction and collagen orientation inexcised UV-exposed mouse skin.29 However, few reports
have revealed that aging-related structural changes in human
Journal of Biomedical Optics

dermal collagen fibers are clearly visualized in vivo in theform of high-contrast SHG images. In this article, Cr:F laserbased SHG microscope was used for in vivo visualization of
aging-specific structural changes in human facial collagen.
Furthermore, quantitative image analysis using Fourier transformation of SHG images was performed to identify characteristic
structures in dermal collagen fibers with and without skin aging.

2

Materials and Methods

2.1

Experimental Setup

Figure 1(a) shows the experimental setup of our collagensensitive SHG microscope. Because the setup is described indetail elsewhere,25,29 only a brief description of it is given
here. A 1250-nm mode-locked Cr:Forsterite laser (Avesta
Project Ltd., CrF-65P, pulse duration ¼ 90 fs, mean power ¼
200 mW, repetition rate ¼ 73 MHz) pumped by an Ytterbium
fiber laser emitting at 1064 nm was used as a laser source.
After converting the polarization of the laser light from linear
to circular, the laser beam was scanned two-dimensionally bya pair of galvano mirrors (GM). After passing through relay
lenses (RL1 and RL2), the laser beam was focused onto thesample with an objective lens (OL; Nikon Instruments Inc.,
CFI Plan 50 × H, magnification ¼ 50, NA ¼ 0.9, working
distance ¼ 350 μm, oil-immersion type). A customized attachment ring [see an inset of Fig. 1(a)] is attached to the subject’s
skin by a double-stick tape and used to suppress the motion of its
measured position. By changing the position of the attachment
ring with a stepping motor-driven translation stage, we canselect the measured position inside the skin three-dimensionally.
A portion of the generated SHG light was backscattered in thesample and then collected via the OL. The SHG light was descanned by GM and separated from the laser light by a harmonic
separator (HS, reflected wavelength ¼ 625 nm) and an infraredcut filter (F, stop wavelength > 800 nm). The SHG light was
detected by a photon-counting photomultiplier (PMT, Hamamatsu Photonics K. K., H8259-01) connected with a pulse
counter.
Using the above GM optics, SHG images of a 600 × 600-μm
region, composed of 256 × 256 pixels, were acquired at a rate of
2 s∕image. However, this imaging region was not suitable forassessment of skin aging because the morphology and mechanical properties of the skin are influenced by the macroscopic
properties of dermal collagen fiber rather than microscopic
ones. To enlarge the imaging region, we scanned the sample
position at intervals of 600 μm using a stepping motor-driven
translation stage whenever acquiring SHG image of
600 × 600-μm regions using GM. Finally, we obtained alarge SHG image with a size of 2.4 × 2.4 mm by arranging
16 SHG images in a matrix of four rows and four lines.

2.2

Evaluation of Human Dermis

Twelve Japanese male volunteers aged in their 20s to 60s with
Fitzpatrick’s skin phototype (SPT) III to IV were recruited asshown in Table 1. The cheek skin, serving as a sun-exposedsite of healthy subjects, was assessed by using the SHG microscope as shown in Fig. 1(b) and 1(c). With respect to the question of risk of biological photodamage caused by the Cr:
Forsterite laser, previous studies have reported that little damage
was observed on zebrafish embryos23 and hamster oral mucosa24
after long-term irradiation with a tightly focused laser beam.

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Fig. 1 (a) Experimental setup. λ∕2: half waveplate; PL: polarizer; λ∕4: quarter waveplate; HS: harmonic separator; F: infrared-cut filter; L: lens; PMT:
photon counting-type photomultiplier tube; GM: galvano mirrors; RL1 and RL2: relay lenses; OL: oil-immersion objective lens. Photograph of (b) invivo measurement and (c) measured position of human cheek skin.

Table 1 List of subjects.

Subject identifier

Sex

Age

History of sun
exposure

A

Male

20s

Moderate

B

Male

20s

Moderate

C

Male

20s

Moderate

D

Male

20s

Excessive

E

Male

20s

Moderate

F

Male

30s

Moderate

G

Male

30s

Moderate

H

Male

40s

Moderate

I

Male

40s

Excessive

J

Male

50s

Excessive

K

Male

50s

Moderate

L

Male

60s

Moderate

Journal of Biomedical Optics

Furthermore, to evaluate the risk of photodamage to human skin,
we examined changes in the subjects’ skin before, immediately
after, and one month after irradiation with a laser light, based oncolor measurement with a spectrophotometer (Konica Minolta,
CM-2600d) and clinical evaluation by a board-certified dermatologist. We concluded from these examinations that there wasno laser-induced photodamage to the skin. Based on thisevidence, we set the average power of the laser light radiatedonto the skin to 35 mW with a combination of a half waveplate(λ∕2) and a polarizer (PL). Written informed consent was
obtained from all subjects before the measurement. The protocol
conformed to the Helsinki Declaration and was approved by the
ethics committees for human experiments in both Osaka University and Shiseido Co., Ltd.

3

Results and Discussions

3.1

Structural Differences in Facial Collagen Fiber
Among Subjects with Different AgesFirst we investigated differences in facial collagen fiber among
male subjects with different ages. We set the probing depth
to 200 μm from the skin surface to investigate the collagenfiber in the reticular dermis by performing depth-resolved
SHG imaging before conducting the following experiments.
Figure 2(a) to 2(e), respectively, show photographs of thecheek skin (image size ¼ 58 × 43 mm) and large-area SHG
images (image size ¼ 2.4 × 2.4 mm) of a subject in his 20s(subject E), a subject in his 30s (subject G), a subject in his
40s (subject I), a subject in his 50s (subject K), and a subject
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Fig. 2 Photographs of the cheek skin (image size ¼ 58 × 43 mm) and large-area SHG images (image size ¼ 2.4 × 2.4 mm) of (a) subject in his 20s(subject E), (b) subject in his 30s (subject G), (c) subject in his 40s (subject I), (d) subject in his 50s (subject K), and (e) subject in his 60s (subject L).

in his 60s (subject L). Imperfect matching of adjacent SHG
images at the boundary is mainly due to a little discrepancy
between the image region acquired by the galvano mirror
and the movement of the stepping motor stage. In all SHG
images, the detailed structure of dermal collagen fibers wasclearly visualized. Although pores in the skin should appear
as black circles due to the absence of collagen fiber, faint signals
were observed in the centers of the pores, implying contamination of SHG image form other signals. We investigated thereason for this contamination by replacing the infrared-cut filter(pass wavelength <800 nm) with another optical filter with asharp pass-band (pass wavelength ¼ 590 to 630 nm) as
Journal of Biomedical Optics

shown in Fig. 3(a) and 3(b). The faint signals in the centersof the pores completely disappeared under insertion of the
sharp pass-band filter. Although one possible reason for thefalse signals may be multiphoton autofluorescence fromkeratin30 rich in the hair, it is difficult to specify the sourceof this faint signal at the present time.
Here we focus on structural differences in the dermal
collagen fiber among subjects with different ages. For example,
the skin of subject E indicated that thin collagen fibers weredensely distributed [see Fig. 2(a)]. Conversely, in the case of
subject L, thin collagen fibers were greatly reduced, and a coarse
distribution of thickly grown collagen fibers was enhanced

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Fig. 3 Comparison of SHG image obtained with (a) the infrared-cut filter (pass wavelength <800 nm) and (b) sharp band-pass filter(pass wavelength ¼ 590 to 630 nm).

[see Fig. 2(e)]. These age-related structural changes in dermal
collagen fibers were confirmed in the previous studies. On one
hand, ex vivo morphometric analysis of the reticular dermis indicated that fiber diameter in young subjects was thinner than thatin elderly subjects.12 On the other hand, confocal microscopy
revealed in vivo that collagen bundles were abundant and composed of fine fiber in young subjects while the dermis showedcoarse collagen and curled fibers in elderly subjects.31 The
thickness of a collagen fiber is closely related to the level ofstructural maturity in the fiber; immature fibers are thinnerand mature fibers are thicker. Procollagen, which is collagenprecursor molecule, is synthesized by dermal fibroblasts andsecreted into extracellular spaces, where it is enzymatically processed to mature collagen. Mature collagen spontaneously
forms fibrils, and then collagen fibrils build up their higher
order structures, collagen fibers. Since collagen fibrils havean estimated half-life of 17 years, fragmented collagen fibrils
accumulate with the passage of time.5 This may lead to difference of SHG image between Fig. 2(a) and 2(e). Age-related
decrease in collagen in the skin was also observed in the previous studies.12,31 This good agreement with the previous studies
indicates the high potential of SHG microscope for in vivo
assessment of age-related change in dermal collagen fiber.
Regarding the skin of the subjects in their 30s, 40s, and 50s,
we were expecting that the above structural changes in dermal
collagen fiber would advance gradually in proportion to age.
However, a series of SHG images in Fig. 2, from subjects intheir 20s to 60s, did not always indicate such a gradual change.
For example, the skin of the subject I in his 40s [see Fig. 2(c)]indicated a larger decrease of thin collagen fibers than in subject
K in his 50s [see Fig. 2(d)]. We consider that photoaging influences these results because the effect of photoaging is notalways in proportion to age, and individual subjects have different histories of UV exposure. Since males usually do not use
sunshades or sunscreen creams carefully to avoid UV exposure,their skin will be susceptible to the effects of photoaging.
Furthermore, subject I had a rich history of UV exposure byplaying rugby football throughout the years, from elementary
school to university. Although sun-protected part in the skinshould be selected as a measurement position to investigate
the intrinsic aging without the influence of photoaging, it isstill interesting to visualize actual age-related structural change
of facial collagen fiber in vivo from the viewpoint of aesthetic
facial concerns and anti-aging dermatology. Work is in progress
Journal of Biomedical Optics

to conduct a similar experiment on female subjects. This isbecause females tend to use sunshades and/or sunscreen creams
more carefully than males, and hence their skin is less susceptible to photoaging than that of males. We expect that the age
dependence of SHG images in female subjects will reflect theeffect of intrinsic aging on dermal collagen fiber more clearly,
without a little interference by photoaging.

3.2

Influence of UV Exposure History on Facial
Collagen Fiber at Different Ages

We investigated how cumulative UV exposure influences facialcollagen fibers at different ages. Here we compared SHG imagesof facial collagen fibers between male subjects in their 20s withand without a recent life history of excessive sun exposure. Figure 4(a) and 4(b) shows photographs of the cheek skin(image size ¼ 58 × 43 mm) and large-area SHG images(image size ¼ 2.4 × 2.4 mm) of two subjects in their 20s, subject D with excessive sun exposure and subject E with moderate
sun exposure. We confirmed again that thin collagen fibers weredensely distributed in both skins, which is characteristic inyoung subjects. Furthermore, thin collagen fibers in subject
D with excessive sun exposure were somewhat less abundant
than those in subject E with moderate sun exposure, whereas
thick collagen fibers in the former were slightly more conspicuous than those in the latter. However, it was difficult to findclear differences in collagen fiber structure between them.
Next we compared two subjects in their 50s, subject J with alife history of excessive sun exposure and subject K with moderate sun exposure, as shown in Fig. 5(a) and 5(b). In contrast to
the subjects in their 20s, we confirmed a large difference infacial collagen fibers between the two subjects. Subject J
with excessive sun exposure in Fig. 5(a) showed drasticallyreduced thin collagen fibers, which had almost completely disappeared, except in areas around pores, whereas a small amountof thick collagen fibers remained. On the other hand, in subject
K with moderate sun exposure in Fig. 5(b), both thin and thickcollagen fibers still remained. Thus structural differences in collagen fiber due to UV effect were enhanced in the subjects intheir 50s compared with the subjects in their 20s.
We here discuss the reason why the structures of the facialcollagen fibers are so different between the subjects. UV irradiation to the skin inhibits the synthesis of collagen and induces its
degradation.32,33 At the same time, UV irradiation induces

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Fig. 4 Comparison of photographs of the cheek skin and large-area SHG images between subjects in their 20s with and without a recent life history ofexcessive sun exposure. (a) Subject D with excessive sun exposure and (b) subject E with moderate sun exposure.

Fig. 5 Comparison of photographs of the cheek skin and large-area SHG images between subjects in their 50s with and without a recent life history ofexcessive sun exposure. (a) Subject J with excessive sun exposure and (b) subject K with moderate sun exposure.
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elastin gene transcriptional activity, resulting in increased
deposition of elastin that replaces the degenerated collagen,referred to as solar elastosis.5 On the other hand, intrinsic
aging diminishes collagen synthesis, resulting in the decreased
density of collagen fiber in the dermis as shown in Fig. 2(e).
Therefore, UV irradiation to intrinsically aged skin makes it
easier to change the ratio of the degenerated collagen to theabnormal elastin. The SHG images sensitively reflect the factthat a combination of photoaging with intrinsic aging greatly
accelerates skin aging.
Since the elastin fibers induce third harmonic generation(THG) light under the illumination of a Cr:F laser,34 the combined microscopy of SHG and THG may give the detailed
insight regarding the collagen/elastin ratio as well as the combined microscopy of collagen-specific SHG and elastin-specific
TPF.19–21 To visualize the distribution of dermal collagen andelastin fibers in the photoaged skin, we performed simultaneous
SHG and THG imaging by incorporating an additional PMT
with a bandpass filter suitable for THG light. Figure 6(a) and6(b) shows collagen-specific SHG image (observed wavelength ¼
608 to 646 nm) and elastin-specific THG image (observed
wavelength ¼ 385 to 444 nm) of a subject in his 50s with alife history of excessive sun exposure (subject J). We were expecting that the elastin-specific THG signal was observed only in thearea where the collagen-specific SHG signal was not observed,
namely, the complementary distribution of elastin-specific THG
signal to collagen-specific SHG signal. However, actual THG signal is weak in the whole region of Fig. 6(b), whereas coarse distribution of thick collagen fibers was clearly visualized in SHG
image of Fig. 6(a). Considering that the change of the collagen/elastin ratio was confirmed in the previous studies basedon combined SHG and TPF microscopy,19–21 we can conclude
that THG signal from the abnormal elastin is too weak to detect
it in the present setup, or the abnormal elastin does not induce
THG light.
One might consider that the reason for the structural difference in the subjects in their 50s (see Fig. 5) is to make the comparison at different relative depth due to intrinsic aging-related
thinning and/or photoaging-related thickening of the epidermis
because the collagen structures changes with depth. In this case,depth-resolved SHG imaging will provide more information of
those structural changes in dermal collagen. Therefore, we performed depth-resolved SHG imaging for these subjects in their
50s. The resulting series of depth-resolved SHG images areshown in Fig. 7 (imaged region ¼ 600 × 600 μm, interval ofscanning depth ¼ 10 μm), where the surface of skin is defined
as a depth of 0 μm. In the case of subject J with excessive sun
exposure in Fig. 7(a), nonfibrous structures were observed
within a range of depths from 60 to 100 μm and then disappeared at a depth of 110 μm. We consider that these SHG images
were also contaminated by other signals due to imperfect blocking of the infrared-cut filter (pass wavelength <800 nm). Endogenous chromospheres in the epidermis, such as NAD(P)H,
FAD, retinol, tryptophan, and keratin emit autofluorescence signal via multiphoton process.16,17,30 Furthermore, local opticalinhomogeneities in the epidermis cause THG signal.26–28 At
the present time, although it is still difficult to specify the sourceof this faint signal, this faint SHG signal may give an indicatorrelated with the photoaging. Work is in progress to specify it. At
a depth of 120 μm, thick collagen fiber in the reticular dermis
appeared; however, it immediately disappeared at a depth of
140 μm. At a depth of 150 μm, even thick collagen fibers greatly
decreased. Conversely, in the case of subject K with moderate
sun exposure in Fig. 7(b), thin collagen fibers in the papillary
dermis were observed between depths of 50 and 70 μm, andthick collagen fibers were observed in the reticular dermis at
depths over 80 μm. These results show the age-related structuralchanges in dermal collagen fibers progress in the depth direction, in addition to the lateral distribution.

3.3

Image Analysis Based on Fourier Transform

The collagen-sensitive SHG microscope could visualize agerelated structural changes in dermal collagen fibers in the formof high-contrast SHG images. If a skin aging parameter can beextracted only from the SHG signal without the help of elastinspecific TPF signal, in vivo assessment of human skin aging
will be simplified. We next considered how to extract a quantitative parameter related to skin aging from those SHG images.
The intensity value in an SHG image is often influenced by
the conditions of the laser source, the scattering efficiency in theepidermis layer, and other factors, although it can give us
information about the level of structural maturity and densityof dermal collagen. One potential method to extract an aging
parameter from the present SHG images is to use image analysis, because structural changes in the dermal collagen fibers
were clearly visualized as image differences. Recently, image
analysis based on Fourier transformation has been used forFig. 6 Comparison of (a) collagen-specific SHG image and (b) elastin-specific THG image for subject J with excessive sun exposure.
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Fig. 7 Comparison of depth-resolved SHG images between subjects in their 50s with and without a recent life history of excessive sun exposure.
(a) Subject J with excessive sun exposure and (b) subject K with moderate sun exposure.

SHG images in order to assess the structure of collagenfibers.35,36 We tried to extract a quantitative parameter related
to this difference from SHG images by using Fourier transform(FT) analysis of the images.
Journal of Biomedical Optics

We analyzed SHG images of dermal collagen fibers based on
the following procedure. First, we selected region of interest(ROI; size ¼ 64 × 64 pixels) so that the ROI did not include
pores. Second, we performed 2-D FT of the selected ROI

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and obtained the FT spectrum of it. Third, we fitted a Gaussianfunction to the spectral shape to determine its spectral width [fullwidth at half maximum (FWHM)]. We consider that this FT
spectral width can be used for an indicator reflecting the difference of spatial distribution in dermal collagen fiber. Finally, after
applying the above procedure for five different ROIs in the same
subject, we calculated the mean and standard deviation of thespectral width.
Figure 8 shows a comparison of the spectral widths for all
subjects. In the case of subjects A, B, and E who indicateddense distributions of thin collagen fibers, the spectral widths
were relatively narrow. Conversely, subjects J and L, indicatingcoarse distributions of thick collagen fibers owing to intrinsic
aging and/or photoaging, had particularly wide spectral widths.
Furthermore, relatively speaking, the spectral widths increased
in proportion to age. These tendencies well reflected the characteristic structures of dermal collagen fibers in the nonaging
and aging skins, although we could not perform the statisticalanalysis of this parameter due to the limited number of subjects
in this experiment. We here consider the correlation between thequalitative and the quantitative results of SHG images, although
it is difficult to obtain the clear interpretation for the correlation
due to less spatial periodicity of collagen fibers. In the case ofyoung subjects, SHG images were obtained at low contrast due
to dense distributions of thin collagen fibers. On the other hand,elderly subjects indicated high-contrast SHG images due tocoarse distributions of thick collagen fibers. We consider thatdifference of the image contrast influences FT spectral width.
In other words, harmonic components of the fundamental spatial
frequencies were enhanced by the high image contrast, leading
to the increase of FT spectral width. At the present stage,
although this only-SHG-based parameter just shows a possibility as the aging parameter, it may enable simpler assessment of
human skin aging than conventional parameters based on combination of collagen-specific SHG signal and elastin-specific
TPF signal.19–21 Work is in progress to evaluate this parameterfrom the viewpoint of the statistical analysis by increasing thenumber of subjects. In addition to FT analysis of SHG image,combination of SHG microscopy with the polarization measurement, namely polarization-resolved SHG microscopy, mayalso give an additional means for the skin-aging parameterbased only on SHG signal because the skin wrinkling,
characteristic in the aged skin, is closely related with orientationof collagen fiber.29

Fig. 8 Comparison of FT spectral widths for all subjects.
Journal of Biomedical Optics

4

Conclusions

We evaluated the potential of a collagen-sensitive SHG microscope equipped with a Cr:Forsterite laser as an in vivo assessment tool for skin aging from the viewpoint of structuralchanges in dermal collagen fibers. Ability to probe deeper
below skin surface in this SHG microscope enables us to visualize age-related structural change of collagen fiber in the reticular dermis clearly. SHG images of human facial skin clearlyvisualized age-related structural changes in dermal collagenfibers: young subjects showed dense distributions of thin collagen fibers, whereas elderly subjects showed coarse distributions of thick collagen fibers. Furthermore, a comparison of
SHG images between subjects in their 50s with and withouta recent life history of excessive sun exposure showed that a
combination of photoaging with intrinsic aging significantly
accelerates skin aging. Such age-related structural change ofdermal collagen was also confirmed in depth-resolved SHG
imaging. Finally, to extract a quantitative parameter related toskin aging from SHG images, we defined the spectral widthof an FT spectrum of SHG image as an aging parameter. The
FT spectral width moderately reflected age-related structuralchange of dermal collagen. Therefore, the collagen-sensitive
SHG microscope will be a powerful tool of in vivo skinassessment in fields such as anti-aging dermatology and thedevelopment of skin cosmetics.

Acknowledgments
This work was supported by Grants-in-Aid for Scientific
Research Nos. 22300154, 23240069, and 23650260 from the
Ministry of Education, Culture, Sports, Science, and Technology of Japan. We are grateful to Dr. Mamoru Hashimoto of
Osaka University for fruitful discussions.

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