Optical probe methods for in vivo assessments of cutaneous photoaging are necessary in fields such as anti-aging dermatology and skin cosmetic development. We investigated the relation between wrinkle direction and collagen orientation in ultraviolet-B-exposed (UVB-exposed) skin using polarization-resolved second-harmonic-generation (SHG) microscopy. A polarization anisotropic image of the SHG light indicated that wrinkle direction in UVB-exposed skin is predominantly parallel to the orientation of dermal collagen fibers. Furthermore, collagen orientation in post-UVB-exposed skin with few wrinkles changed from that of UVB-exposed wrinkled skin to that of no-UVB-exposed skin. The method proposed has the potential to become a powerful non-invasive tool for assessment of cutaneous photoaging.
©2009 Optical Society of America
Aging is an irreversible, physiological phenomenon that cannot be avoided. In skin, mechanical properties such as tension and elasticity decline gradually with age, resulting in the appearance of wrinkles and sagging. Furthermore, repeated exposure of ultraviolet B (UVB) rays in sunlight to the skin often accelerates skin aging and causes changes in the skin, such as mottled pigmentation, leathery texture, laxity, sallowness, and deep wrinkle. This is called cutaneous photoaging . Therefore, the need exists for an in vivo assessment technique to estimate the degree of cutaneous photoaging for studies in fields such as skin cosmetics and anti-aging dermatology.
Deep wrinkle characteristic of photoaged skin is closely related to the quantity and structure of collagen fibers in the superficial dermis because dermal collagen fibers contribute to the morphology and mechanical properties of the skin. For example, the biochemical, histochemical, and immunohistochemical experiments indicated that the dermal collagen fibers in the photoaged neck skin remarkably decrease in comparison with the intrinsically aged gluteal skin . Therefore, in vivo monitoring of dermal collagen fibers is a key step in assessment of cutaneous photoaging. Optical probe methods are attractive for such assessments because they are simple, rapid, and non-invasive. Furthermore, optical probes can be applied directly because skin is a superficial tissue. Among the optical probe methods available for skin studies [3-5], second-harmonic-generation (SHG) light is attractive for in vivo monitoring of dermal collagen fiber because SHG light is generated specifically by collagen fibers in the dermis [6, 7]. This SHG light provides unique imaging characteristics: high contrast, high spatial resolution, optical three-dimensional (3D) sectioning, non-invasiveness, and deep penetration. By using the naturally endogenous SHG process as a contrast mechanism, the structures of collagen fiber in the tissues can be clearly visualized without additional staining, and so are free from photodamage, phototoxicity, or photobleaching. For the evaluation of cutaneous photoaging, the combination of SHG imaging with two-photon autofluorescence imaging has been applied effectively as an indicator of changes in dermal collagen and elastin content because degradation of collagen and excessive deposition of abnormal elastin are observed in photoaged skin [8, 9]. In contrast, combining SHG imaging with polarization methods can provide additional insights regarding the direction of collagen fibers, i.e., collagen orientation. The efficiency of SHG light is sensitive to collagen orientation when the incident light is linearly polarized, and hence polarization measurements of the SHG light are effective for probing collagen orientation in the dermis [10, 11] and other tissues [11, 12]. However, few reports have examined changes in collagen orientation caused by wrinkling in photoaged skin despite the fact that the wrinkle formation is closely related to directional property of collagen fiber and repeated mechanical tension to the skin.
Here, the relation between wrinkle direction and collagen orientation in experimentally UVB-exposed skin was investigated as a model of cutaneous photoaging, based on 3D imaging of collagen orientation using polarization-resolved SHG microscopy.
2. Experimental setup
Figure 1 shows the experimental setup of a polarization-resolved SHG microscope. Conventionally, a mode-locked Ti:Sapphire laser with a center wavelength near 800 nm is used as the light source for SHG microscopes, and the resulting SHG light near 400 nm is detected [6-12]. However, multiple scattering and absorption by the skin at these wavelengths often results in insufficient probing depth when monitoring dermal collagen across the epidermis with a reflection setup. For polarization-resolved SHG microscopy, multiple scattering events depolarize the detected SHG light. Although it is demonstrated that polarization of the SHG light is maintained during a depth of about 100 μm , one have to consider an effect of depolarized SHG light in the deeper region of tissues and therefore decreased sensitivity to collagen fiber orientation . Another problem is the risk of biological photodamage in the vicinity of the focal spot caused by nonlinear absorption and/or ionization of the incident laser light [14, 15]. Those problems can be improved through the use of an ultrashort pulse laser with a longer wavelength to decrease multiple scattering and absorption by tissues. A promising laser source for SHG microscopy that substitutes for the Ti:Sapphire laser is a mode-locked Cr:Forsterite laser centered near 1250 nm because absorption and scattering by tissues reaches a minimum near wavelengths of 1300 nm [16, 17]. By substituting a 800-nm Ti:Sapphire laser for a 1250-nm Cr:Forsterite laser, reduced scattering coefficient of skin are decreased from 15 cm-1 to 7 cm-1 for the fundamental light and from 100 cm-1 to 30 cm-1 for the SHG light . Direct comparisons between Cr:Forsterite-laser-based and Ti:Sapphire-laser-based nonlinear microscopes indicated that the Cr:Forsterite-laser-based microscope resulted in decreased photodamage , greater penetration depth , and more rapid imaging rate . Therefore, a 1250-nm mode-locked Cr:Forsterite laser pumped by a 7.5 W Ytterbium fiber laser running at 1064 nm (Avesta Project Ltd., CrF-65P) was used for this study. The laser pulse has a duration of 90 fs and an average power of 200 mW at a repetition rate of 73 MHz. For rapid acquisition of SHG images, the laser beam is scanned two-dimensionally by a pair of galvano mirrors (GM). After passing through relay lenses (RL1 and RL2), the laser beam is focused onto the sample with an objective lens (OL; Nikon Instruments Inc. CFI Plan 50×H, magnification = 50, NA = 0.9, WD = 350 μm, oil-immersion). The oil-immersion OL is effective for suppressing reflection loss and spherical aberrations caused by mismatching of refractive index at the sample surface. The OL can be moved along the optical axis by a piezoelectric transducer (PZT, stroke length = 350 μm). Combination of the GM and the PZT enables optical 3D sectioning images of SHG light. Average power of the laser light on the sample was set to be 42 mW by a neutral density filter (ND). Setting the level of laser power to a wavelength of 1250 nm does not cause laser damage even to living cells [21-23]. A portion of the generated SHG light is backscattered into the sample and then collected via the OL. The SHG light is de-scanned by the GM and separated from the laser light by a harmonic separator (HS, reflected wavelength = 625 nm) and an infrared-cut filter (F, stop wavelength > 800 nm). The SHG light is detected by the combination of a photon-counting-type photomultiplier (PMT, Hamamatsu Photonics K. K., H8259-01) and a pulse counter. For the polarization measurement of the SHG light, a half waveplate for 1250 nm (λ/2) is inserted in the optical path and used for polarization control of the laser light. Since this λ/2 acts as a full waveplate to the 625-nm SHG light, the polarization angle of the SHG light does not change by passing through the λ/2 in the return SHG path.
To visualize overall tissue structure of the skin and determine the depth from the skin surface, a confocal imaging setup of the reflectance fundamental light (RC light) also was added to the microscope. The reflectance fundamental light from the sample passes through the HS and is partly reflected by a beam splitter (BS). A confocal section is composed of a pair of lens with a focal length of 50 mm (CL1 and CL2) and a 50-μm diameter pinhole (PH). The fundamental light passing through the confocal setup then is detected by an infrared photoreceiver (New Focus 2011).
UVB-exposed albino hairless mice (HR-1) were selected as a model of photoaging. Wrinkles were induced by repetitive low-dose UVB irradiation to the back of the hairless mice according to the method by Kligman with slight modification [24, 25]. Four types of skin samples were prepared: UVB-exposed skin (16 weeks old), post-UVB-exposed skin (22 weeks old), and two types of age-matched control skin (16 and 22 weeks old). Figure 2 shows the sample preparation protocol and optical photographs of each sample surface. The skin was exposed intermittently to low-dose UVB light from 6 to 16 weeks of age. We uniformly exposed the whole body of the mice in the cage to broadband UVB light (wavelength = 270 ~ 400 nm). Exposure dose of UVB light is gradually increased with advance in photoaging. Briefly, we set the initial dose at 36 mJ/cm2 and subsequently increased to 54, 72, 108, 126, 144, 162, 180, and 198 mJ/cm2 at 1 week intervals and finally to 216 mJ/cm2 at the ninth and tenth weeks . Exposure time per day was set within the range from 2 to 12 minutes, and the frequency of the irradiation was set at three times per week. Total UVB exposure dose was approximately 4.6 J/cm2. This treatment resulted in the generation of deep wrinkles perpendicular to the meridian line of the body as shown in the lower-left photograph in Fig. 2 (UVB-exposed skin). The back skin around the backbone (size = 30 mm by 30 mm, thickness = 2 mm), which is most advanced area of cutaneous photoaging in the body, was excised from the mice. In contrast, the post-UVB-exposed skin was prepared by maintaining the mice with no UVB exposure for 6 weeks after completing the 10-week UVB exposure. In post-UVB-exposed skin, the wrinkles resulting from 10-week UVB exposure almost disappeared because of remodeling of the extra-cellular matrix shown in the lower-right photograph in Fig. 2. To compare UVB-exposed and post-UVB-exposed skin, 16- and 22-week-old mice that had not been exposed to UVB radiation were used as age-matched controls. The skin of the control mice was not wrinkled as shown in the upper-left and upper-right photographs in Fig. 2. We respectively examined three mice for UVB-exposed and age-matched control skins (16 weeks old) and two mice for post-UVB-exposed and age-matched control skins (22 weeks old). All the skin specimens were measured and analyzed immediately after excision from the mice.
4. Results and discussions
4.1 UVB-exposed skin
First, the depth-resolved reflectance confocal (RC) image and SHG image of the UVB-exposed skin samples (16 weeks old) were obtained to visualize distribution of dermal collagen fibers in the skin. The incident laser light was linearly polarized in the vertical direction, which is parallel to the meridian line of the body. The upper and middle rows of Fig. 3 show the typical RC and SHG images at depths of 50, 100, 150, 200, and 250 μm from the skin surface. Also, left and center panels of (Media 1) shows the change in both images as probing depth varied from 0 to 300 μm in 5-μm intervals. The area of each image is 800 μm by 800 μm, composed of 256 pixels by 256 pixels, with an image acquisition rate of 10 sec/image. Use of the mode-locked Cr:Forsterite laser provided a probing depth greater than 200 μm on SHG imaging of the dermal collagen fibers across the epidermis at the reflection setup. Since the meridian line of the sample is in the vertical direction of the images, the deep wrinkles induced by UVB exposure run along the horizontal direction of the image. The RC and SHG imaging modes provide insight into distribution of dermal collagen fibers in the overall tissue structure of the skin. In both imaging modes, the hair follicles are aligned perpendicular to the meridian line. To investigate effects of the UVB-induced wrinkling on the skin, the RC and SHG images of age-matched control skin samples (16 weeks old) were obtained as shown in the upper and middle rows of Fig. 4 (left and center panels in (Media 2)). Distribution of the hair follicles in the controls was similar to that of the UVB-exposed samples. However, no significant difference in the structure of dermal collagen fibers between SHG images of the two kinds of samples was observed.
To obtain a detailed distribution of collagen fiber orientation in the dermis, polarization-resolved SHG imaging was applied to the samples. Two polarization-resolved SHG images were acquired by adjusting polarization of the incident laser light at the vertical and horizontal directions. The vertical polarization was set parallel to the meridian line of the sample, while the horizontal polarization was perpendicular to it. Therefore, the horizontal polarization was parallel to wrinkle direction for the UVB-exposed samples. However, it was difficult to recognize differences in collagen orientation from a direct comparison between the two polarization-resolved SHG images (not shown). Therefore, polarization anisotropy of SHG light (α) to investigate the collagen orientation quantitatively was defined using the following equation: 
where IV and IH are SHG intensities when the incident light is vertically and horizontally polarized, respectively. Then the α image was calculated by substituting IV and IH values at each pixel of two polarization-resolved SHG images for Eq. (1). The α image reflects the distribution of collagen orientation because strong SHG light is observed when laser polarization is parallel to collagen orientation, and SHG light is considerably weaker if laser polarization is perpendicular to collagen orientation. Collagen orientation is uniaxial for α = ±1 and random or biaxial for α = 0. The sign of α value provides the dominant direction of the collagen orientation: positive for a vertical orientation and negative for a horizontal orientation. The bottom rows in Figs. 3 and 4 (right panel in (Media 1) and (Media 2)) show the α image of the UVB-exposed sample and control, respectively. The color scale of those images indicates the direction of collagen orientation, with the vertical direction indicated by blue, horizontal by red, and neutral by white. A significant difference in collagen orientation between UVB-exposed and control samples was confirmed (e.g., the vertical collagen orientation prevails in the control sample). In general, when mechanical tension is repeatedly applied to tissues, remodeling of the tissues occurs due to the change of elastic fibers networks and/or crosslinking of collagen fibers. Stress-relaxation tests revealed that the collagen fibers become aligned with the loaded axis when skin is stretched in uniaxial tension . In back skin, collagen fibers tend to align along direction of the muscle fiber, namely the meridian line of body, due to repeated mechanical stretch. A previous macroscopic study using microwave polarimetry supported this finding of biomechanical collagen orientation on the back skin of four-footed animals . The results of collagen orientation measurement shown in Fig. 4 are consistent with the biomechanical finding. Conversely, the UVB-exposed sample was dominated by horizontal collagen orientation although the vertical orientation was observed in regions between adjacent hair follicles. Furthermore, the horizontal collagen orientation coincides with the direction of the UVB-induced wrinkles. Similar tendency was observed in most of the skin specimens excised from three UVB-exposed mice. The UVB exposure may disturb the regular remodeling mechanism of collagen fibers on back skin.
Whether UVB exposure affects distribution of collagen orientation along the depth direction is interesting because the number of collagen fibers in the photoaged superficial dermis decrease . A 3D dataset of the α value was obtained by successively depth-resolved α imaging at 5-μm depth intervals (see right panel of (Media 1) and (Media 2)), and then successive cross-sectional α images were obtained at a 3.1-μm lateral interval by rearranging the 3D dataset of the α value with respect to the lateral direction perpendicular to wrinkle direction. The resulting cross-sectional α images and their movies of UVB-exposed and control samples are shown in Fig. 5 and (Media 3) (image size = 800-μm width × 300-μm depth). Cross-sectional α images show a significant difference in collagen orientation between both samples similar to the depth-resolved α images in Figs. 3 and 4, in which the UVB-exposed sample was dominated by horizontal collagen orientation (red color) while the vertical collagen orientation (blue color) prevails in the control sample. However, remarkable depth dependence of collagen orientation was not observed in the samples. To further investigate the depth dependence of collagen orientation, the mean value of each depth-resolved α image (α2D) was calculated and plotted with respect to depth from the skin surface. Figure 6 shows the depth dependence of the α2D values for UVB-exposed and control samples. The α2D value for the UVB-exposed sample was gradually decreased with the depth. Although penetration of the UVB radiation is confined to the epidermis and shallow part of the dermis , it has been confirmed in the previous studies of mice and human skins that the biochemical change of dermal collagen caused by the UVB radiation extended to the deep part of the dermis [29, 30]. Therefore, we consider that the negative α2D slope reflects the biochemical change in the overall dermal collagen. In contrast, the α2D curve for the control sample showed no characteristic dependence of collagen orientation on depth. Furthermore, the α2D curve in Fig. 6 shows a large deviation between the UVB-exposed and age-matched control skins (16 weeks old). To confirm the significant difference of such deviation, examination of the five different positions of UVB-exposed and the control samples were examined, followed by calculation of the mean α value of 3D volume (α3D value) for each sample. Figure 7 presents the statistics of the five α3D values for UVB-exposed and age-matched control samples (16 weeks old), showing the significant difference in α 3D values between them (n = 5, P < 0.05).
These results suggest a mechanism for formation of UVB-induced wrinkles as follows. Dermal collagen fibers of non-exposed back skin tend to align along the meridian line of the body because of the repeated mechanical tension. In contrast, UVB exposure to the skin stimulates production of collagen-degrading enzymes, such as matrix metalloproteinases (MMP) [31, 32]. The MMP decomposes the collagen fiber and its network, and then promotes wandering of fibroblasts in the dermis. The wandering fibroblasts tend to align along the direction perpendicular to the repeated mechanical stretch, and actively synthesize renewal collagen fiber along the same direction. Since the mechanical stretch of back skin is repeated along the meridian line of the body, i.e. vertical direction, the dominant orientation of the renewal collagen fibers is horizontal. UVB-damaged collagen fibers are rapidly replaced to renewal collagen fibers in the UVB-exposed skin, as a result, the collagen orientation of the UVB-exposed sample changes from the vertical to the horizontal direction. Such the change of the collagen orientation increases horizontal tensile strength of the skin, resulting in formation of deep wrinkles parallel to the horizontal collagen orientation.
4.2 Post-UVB-exposed skin
When 16-week-old UVB-exposed mice were maintained for another 6 weeks with no UVB exposure (post-UVB-exposed mouse), most of the UVB-induced wrinkles disappeared through remodeling of the extra-cellular matrix. Therefore, collagen orientation in post-UVB-exposed sample and age-matched control (22 weeks old) was determined to further investigate the relation between collagen orientation and UVB-induced wrinkles. Figures 8 and 9 show five depth-resolved images of RC light, SHG light, and α value at 50-μm intervals for both samples. In addition, consecutively depth-resolved images at 5-μm intervals are illustrated in (Media 4) and (Media 5). The α image enriched in blue for 22-week-old control skin indicates that the dominant collagen orientation is vertical and parallel to the meridian line of the body, which is similar to that in 16-week-old control skin. In contrast, post-UVB-exposed samples produced neutral α images in which blue and red colors are mixed in equal quantities. Although vertical collagen orientation (blue) of post-UVB-exposed samples was decreased when compared with the age-matched control, but the decrease was less remarkable than that of the UVB-exposed samples (see Fig. 3 and (Media 1)). Depth dependence of the α2D value for post-UVB-exposed and age-matched control samples (22 weeks old) also are shown in Fig. 6 for comparison with UVB-exposed and age-matched control samples (16 weeks old). Furthermore, the α3D values of the post-UVB-exposed samples are compared with those of the other three samples in Fig. 7. Two significant differences in α3D value were further found among those samples (n = 5, P < 0.05). One was between the post-UVB-exposed (22 weeks old) and UVB-exposed samples (16 weeks old), which indicates a relation between UVB-induced wrinkles and collagen orientation because wrinkles observed in the UVB-exposed samples nearly disappeared in post-UVB-exposed samples. Another significant difference was found between the post-UVB-exposed and age-matched control samples (22 weeks old). Although the UVB-induced wrinkles nearly disappeared in the post-UVB-exposed samples, dermal collagen orientation of the skin did not completely recover as did those of the age-matched control sample. Thus, the imperfect remodeling and/or irreversible damage of the extra-cellular matrix explain the differences in collagen orientation. In contrast, no significant difference was found between the 16-week-old control and the 22-week-old control. Interestingly, the α3D values of the post-UVB-exposed samples recovered and shifted closer to the values of the age-matched controls, suggesting that UVB-induced wrinkles are closely related to the orientation of dermal collagen fibers. Therefore, collagen orientation represented by the α3D value can be used as an indicator of UVB-induced wrinkles and, thus, the degree of cutaneous photoaging. Work is in progress to monitor changes in collagen orientation during the growth process of UVB-induced wrinkles.
Polarization-resolved SHG microscopy was applied to UVB-exposed and post-UVB-exposed skin to investigate the relation between dermal collagen orientation and UVB-induced wrinkles. The resulting α imaging of UVB-exposed samples indicated that UVB-induced wrinkles are orientated in the direction of the collagen fiber. Furthermore, collagen orientation in post-UVB-exposed skin with few wrinkles changed from that of UVB-exposed skin to that of the age-matched control samples. These results indicate a close relation between UVB-induced wrinkles and collagen orientation. Thus, the proposed method provides an in vivo indicator to evaluate cutaneous photoaging.
This work was supported by Grants-in-Aid for Scientific Research Nos. 19650117 and 20240044 from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, and the Cosmetology Research Foundation, Japan.
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