JP2009236610A - Skin wrinkle evaluation method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for non-invasively and quantitatively evaluating a state of wrinkles on a skin owing to a change in the interior of the skin such as a corium layer, etc. <P>SOLUTION: The state of wrinkles on the skin is evaluated based on the orientation of collagen in the interior of the skin. Extra-short pulse light is applied preferably to the interior of the skin to detect second harmonic generation light (SHG light) generated. Measurement of the orientation of collagen in the interior of the skin is performed based on a detection result thereof. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a method for evaluating the condition of skin wrinkles. Specifically, the present invention relates to a method for non-invasively and quantitatively evaluating the state of skin wrinkles resulting from changes in the skin such as the dermis layer.

  One of the skin aging phenomena associated with aging is an increase in wrinkles. From the viewpoint of beauty and the like, there is a great interest in preventing and improving wrinkles, particularly in women. Since the most cosmetically interested parts such as the face are exposed to sunlight, the grades of wrinkles such as the corners of the eyes where the skin expands and contracts in such sun exposure parts are evaluated by humans. Standards and evaluation models based on animal models have been established. However, the details of the mechanism of wrinkle generation have not yet been clarified, and there has been no method for quantitatively evaluating parameters closely related to wrinkle formation.

Conventionally, as a method for non-invasively quantifying the skin state, a method of measuring skin viscoelasticity, keratin moisture, transdermal moisture transpiration (TEWL), or the like has been used. However, since any measurement method is performed in principle from the skin surface (outermost layer), the state of the skin surface greatly affects the quantitative value. For this reason, for wrinkles caused by changes in the skin, such as the dermis layer, such as wrinkles that are exposed to sunlight, quantitative parameters that reflect the state of wrinkles can be determined non-invasively and directly from within the skin. It was difficult to get to. In recent years, means capable of observing the internal structure of a living body as an image such as a clinical diagnostic apparatus such as an ultrasound echo, a confocal laser scanning microscope, and a skin light coherence tomography apparatus (OCT) have been developed. It was difficult to extract only collagen information and evaluate it in detail.
On the other hand, it is considered that the dermis state is greatly involved in wrinkle formation. However, in recent years, SHG light (second harmonic generation light) has attracted attention as a method for noninvasively measuring the dermis state. This is because when a living tissue is irradiated with ultra-short pulse light (eg, femtosecond order), a portion of the irradiated laser light is wavelength-converted due to the nonlinear optical properties that the collagen molecules specifically have, and the half-wavelength of the irradiated laser This technology applies the generation of light. (Non-Patent Document 4).
Structure observation of collagen in a living body using SHG light has been proposed and is useful for dermatological diagnosis (Non-Patent Document 5). In addition, in three-dimensional culture in vitro such as collagen gel, investigation of the structure of collagen using SHG light has been studied (Non-patent Document 6). Patent Document 1 describes that the degree of growth of a cultured tissue sample is evaluated by irradiating a cultured tissue sample obtained by collagen gel culture with ultrashort pulse light as incident light and detecting the generated SHG light. ing. Furthermore, Non-Patent Document 7 reports that the orientation of collagen can be analyzed using the incident laser polarization dependency of SHG light generation efficiency.
However, there has been no report evaluating the relationship between collagen orientation and wrinkles until now, and there has been no technology for evaluating the wrinkle state based on collagen orientation measured by the polarization characteristics of SHG light. .

"Forefront of skin anti-aging", published by NTS, July 2006 "Skin Measurement / Evaluation Manual Collection", Technical Information Association, November 2003 "Skin measurement and evaluation at the field level-trouble cases and countermeasures", published by Science & Technology Co., Ltd., February 2007 Roth S. et al., Biopolymers. 1981; 20 (6): 1271-90 Lin SJ. Et al., Eur J Dermatol. 2007 Sep-Oct; 17 (5): 361-6. Review Zoumi A. et al., Proc Natl Acad Sci U S A. 2002 Aug 20; 99 (17): 11014-9 Yasui T. et al., J Biomed Opt. 2004 Mar-Apr; 9 (2): 259-64 JP 2007-49990 A

  An object of the present invention is to provide a method for non-invasively and quantitatively evaluating the state of wrinkles in the skin caused by changes in the skin such as the dermis layer.

  The present inventors have observed that wrinkles caused by changes in the skin, such as skin expansion and contraction, photoaging, or complex factors (skin expansion and contraction in exposed areas such as UV) observed on the surface of the skin, It was revealed for the first time that the orientation of collagen in the skin, particularly in the dermis layer, is closely related.

  For the observation of collagen in the dermis layer, an SHG (second harmonic generation light) microscope capable of noninvasively and selectively measuring the collagen fiber structure is used. This is because when a living tissue is irradiated with ultra-short pulse light (for example, femtosecond order), a portion of the irradiated laser light is wavelength-converted due to the nonlinear optical characteristics that the collagen molecules specifically have, and the half wavelength of the irradiated laser This technology applies the generation of light.

  Using the dependence of the SHG light generation efficiency on the incident laser polarization, the relationship between the presence of wrinkles on the skin surface and the orientation of collagen inside the skin was found to be correlated. . The present inventors have applied this technique to newly establish a method for evaluating skin wrinkles based on the degree of collagen orientation in the skin, thereby completing the present invention.

That is, the gist of the present invention resides in a method for evaluating skin wrinkles, comprising a step of evaluating the state of wrinkles in the skin based on the orientation of collagen in the skin (claim 1).
Here, the orientation of the collagen inside the skin is preferably the orientation of the collagen in the dermis layer.
In addition, the wrinkle state in the skin is preferably wrinkle formation caused by stretching and contracting of the skin (Claim 3), and it is also preferably wrinkle formation caused by photoaging (Claim 4).
In addition, it is also preferable that the evaluation is a prediction of the possibility and / or direction of wrinkle formation on the skin based on the orientation state of collagen in the skin (Claim 5).
Further, the evaluation method of the present invention irradiates the inside of the skin with ultrashort pulse light, detects the generated second harmonic generation light (SHG light), and measures the orientation of collagen inside the skin based on the detection result. It is preferable that the method further comprises a step of (Claim 6).
Here, it is preferable to measure the orientation of collagen in the skin based on the polarization anisotropy of the detected SHG light.
The ultrashort pulse light is preferably femtosecond mode-locked laser light.
Further, it is preferable that the ultrashort pulse light is a chrome forsterite laser light, a titanium / sapphire laser light, a fiber laser light, or a neodymium glass laser light.

  According to the evaluation method of the present invention, by using the orientation of collagen in the skin such as the dermis layer as a parameter for evaluating the wrinkle of the skin, it is possible to expand / contract the skin, photoaging, or a complex factor thereof (such as UV). It is possible to non-invasively and quantitatively evaluate the state of wrinkles caused by changes in the skin, such as the expansion and contraction of the skin in the exposed area.

  Further, by using the detection result of SHG light generated by irradiating the inside of the skin with ultrashort pulse light, it is possible to noninvasively and directly evaluate the orientation of collagen inside the skin.

  Next, the present invention will be described in detail with specific embodiments. However, the present invention is not limited to these embodiments, and any changes can be made without departing from the gist thereof. In addition, it can be implemented.

The method for evaluating skin wrinkles according to the present invention (referred to as “the evaluation method of the present invention” as appropriate) includes a step of evaluating the state of wrinkles in the skin based on the orientation of collagen in the skin.
Here, “the orientation of collagen in the skin” is preferably the orientation of collagen in the dermis layer. Collagen fibers (dermis collagen fibers) occupying about 70% of the dermis layer play an important role in determining skin morphology and mechanical properties (such as tension and elasticity), and the formation of skin wrinkles caused by aging It is thought to be deeply involved in the state.
As shown in the examples described later, the present inventors have skin with wrinkles caused by changes in the skin such as skin stretching, photoaging, or complex factors thereof (skin stretching in exposed areas such as UV). Then, it discovered that the collagen inside the skin was orientating along the wrinkle of skin. Based on this knowledge, in the evaluation method of the present invention, the presence / absence and / or direction of collagen in the skin is measured, and various states of wrinkles in the skin are evaluated based on the measurement result.
Specifically, according to a preferred embodiment, wrinkle formation in the skin is caused by changes in the skin such as skin expansion and contraction, photoaging, or complex factors thereof (skin expansion and contraction in exposed areas such as UV). Can be evaluated. That is, when the collagen inside the skin is oriented along the skin wrinkles, the wrinkles are changes in the skin, such as skin expansion and contraction, photoaging, or complex factors (skin expansion and contraction in exposed areas such as UV). It can be determined that the wrinkle is caused by the wrinkles.
Moreover, according to another suitable aspect, possibility and / or direction that a skin is wrinkled can also be estimated based on the orientation state of collagen inside the skin. That is, even when there is little (or no) wrinkle in the skin, it can be determined that there is a high possibility that wrinkles will be formed in the skin if the collagen is oriented in a certain direction inside the skin. . Moreover, the direction of the wrinkle can be predicted to be parallel to the orientation direction of collagen.

A technique for quantitatively measuring the orientation of collagen in the skin is not limited, but is based on detection of second harmonic generation light (SHG (Second Harmonic Generation) light) as described below. A method is mentioned.
That is, according to a preferred aspect, the evaluation method of the present invention irradiates the inside of the skin with ultrashort pulse light, detects the generated SHG light, and measures the orientation of collagen inside the skin based on the detection result. And further comprising a step.
SHG light is a second-order nonlinear optical response generated by irradiating a non-center target substance with ultrashort pulse light having a high peak power. In a linear optical response such as normal reflection or scattering, the frequency ( It is known that the frequency of SHG light is converted to twice that of incident light (2ω) while ω) does not change.
Collagen fiber molecules present in the skin are known as biological constituents that specifically generate SHG light because they have a non-centrosymmetrical triple helical structure. The intensity of the generated SHG light depends on the concentration and orientation of collagen. By detecting this SHG light, information such as the density, distribution, orientation, etc. of collagen in the skin can be obtained. Since the collagen inside the skin is mainly localized in the dermis layer, as shown in FIG. 1, by irradiating the skin with ultra-short pulse light and detecting the generated living SHG light, collagen in the dermis layer is particularly obtained. It is possible to selectively measure information such as density, distribution, and orientation.
That is, according to the technique based on the detection of the SHG light, since the non-linear optical characteristic inherent to the collagen fiber molecule in the skin is used, tissue staining or the like is unnecessary, and in vivo (in vivo) in the state of living tissue. ) Measurement is possible. Moreover, since the intensity | strength of SHG light obtained in this way is dependent on the density | concentration of the collagen inside skin, collagen density | concentration distribution can be visualized from the intensity | strength information. Since the detection of SHG light is a technique using a nonlinear optical effect, three-dimensional imaging with extremely high spatial resolution is possible.

Examples of the ultrashort pulse light to be irradiated to obtain SHG light include femtosecond mode-locked laser light. The femtosecond mode-locked laser beam is an ultrashort pulse laser beam on the order of femtosecond ( ^10-15 seconds), and has a very high instantaneous peak power. The irradiation means of such femtosecond mode-locked laser light is not particularly limited. Examples include laser light sources such as titanium / sapphire laser, chromium / forsterite laser, fiber laser light, and neodymium / glass laser. The specific pulse length of the ultrashort pulse light is usually 300 fs (femtosecond) or less, preferably 100 fs or less. Although a minimum in particular is not restrict | limited, Usually, it is 100 fs or more. The shorter the pulse width, the higher the generation efficiency of SHG light.
Further, as the ultrashort pulse light, it is preferable to use ultrashort pulse light having a wavelength in the near infrared region. The near-infrared region is a wavelength band that absorbs and scatters less in living tissue and has good biological permeability, so if you use near-infrared ultrashort pulse light, laser light is incident on the skin through the epidermis. It is possible to induce living body SHG light and detect the backscattered SHG light through the epidermis. In addition, if near-infrared ultrashort pulse light is used, it is easy to separate from background light (diffuse reflected light, fluorescence), minimally invasive and deeply penetrating, and low thermal damage. Benefits are gained.
Specifically, it is preferable that the center wavelength of the ultrashort pulse light is usually in the range of about 700 nm to 1550 nm, in particular, about 800 nm to 1300 nm, and more preferably in the range of 1200 nm to 1300 nm. From this point of view, a chrome forsterite laser is preferable as the light source of the ultrashort pulse light. If a chrome forsterite laser is used, for example, it is possible to obtain ultrashort pulse light having a center wavelength of around 1250 nm.

A specific method for grasping the orientation of collagen from the detection result of SHG light includes a method for grasping based on the polarization anisotropy of the detected SHG light. This method utilizes the fact that the generation efficiency of SHG light in a living body strongly depends on the relationship between the direction of polarization of incident laser light and the direction of orientation of collagen.
The biological SHG light is sensitive to the central axis of the collagen fiber molecule, that is, the orientation state, because it originates from the non-centrosymmetric property of the structure of the collagen fiber molecule. FIG. 2 shows the relationship between the polarization state of incident laser light and the orientation of collagen fibers related to the generation of biological SHG light. As shown in FIG. 2, when the laser light is incident from the cross-sectional direction of the collagen fiber, the structure of the collagen fiber is centrally symmetric with respect to any polarized light, so that no SHG light is generated. On the other hand, for incidence from other directions, living body SHG light is generated due to the non-centrosymmetrical structure of the collagen fiber. In this case, the generation efficiency of SHG light strongly depends on the relationship between the polarization of incident laser light and the orientation of collagen fibers. For example, strong biological SHG light is generated when the two are parallel to each other, while it is very weak when they are orthogonal to each other. Therefore, the orientation state of the collagen fibers can be evaluated by analyzing the polarization anisotropy of the SHG light.

Specifically, the SHG light intensity obtained when the polarization direction of the incident laser light is orthogonal to the orientation direction of the collagen fibers is I _、, and is obtained when the polarization direction of the incident laser light is parallel to the orientation direction of the collagen fibers. If the SHG optical signal intensity is I _‖ , the polarization anisotropy of the SHG light can be quantified by an α value defined by the following formula (I).
If the value of α obtained by the above formula (I) is close to 1, the collagen fibers are oriented perpendicular to the polarization direction of the incident laser light, and conversely if close to −1, the polarization direction of the incident laser light. This means that the collagen fibers are oriented in parallel. Therefore, if this α value is calculated and analyzed, the orientation of the collagen fibers can be grasped.
As an example of a specific analysis method, for example, the α value obtained for a predetermined skin region is three-dimensionally imaged, and the orientation of collagen in the skin of the skin region is obtained from the three-dimensional image of the obtained α value. State distribution information can be extracted. Furthermore, a detailed collagen orientation distribution can be obtained by statistically processing the α value distribution. For example, create a histogram of the α value, determine the average collagen orientation in the measurement area from the peak position of the histogram, and determine how well the collagen is aligned from the distribution width (full width at half maximum) of the histogram. Evaluation becomes possible. It is also possible to simply perform these evaluations from the average value of α values.
As described above, since there has been no means capable of visualizing the collagen orientation of the dermis layer, which is thought to be deeply related to the formation of wrinkles in the skin, with high sensitivity without contact, the present invention. It can be said that the technique based on the detection of SHG light used in is an innovative technique in skin measurement.

  Next, a specific mode for achieving the evaluation method of the present invention will be described. In addition, the evaluation method of this invention is not limited to the following forms.

FIG. 3 is a schematic configuration diagram showing an example of an SHG microscope system that can be used for irradiation with ultrashort pulse light and detection of SHG light in the evaluation method of the present invention. This SHG microscope system includes a light source 1, a stage 11 on which a subject 10 is arranged, a photodetector (for example, a photomultiplier tube) 13, and a computer 14. Between the light source 1 and the subject 10, an ND filter (neutral density filter) 2, a polarizer 3, a phase difference plate 4 (for example, a half-wave plate, an electro-optic modulator, etc.), a mirror 5, and a dichroic mirror 6, the galvanometer mirror 7, the first lens 121, the second lens 122, and the objective lens 8 are arranged in this order. Between the subject 10 and the photodetector 13, the objective lens 8, the second lens 122, the first lens 121, the galvano mirror 7, the dichroic mirror 6, and the SHG light transmission filter 12 are arranged in this order. The photodetector 13 is connected to the computer 14. The objective lens 8 is attached to the piezo stage 9. A solid line X indicates incident laser light (ultra short pulse light), and a dotted line Y indicates reflected SHG light.
First, the ultrashort pulse light X is emitted from the light source 1, adjusted to an appropriate laser light intensity by the ND filter 2, and then passes through the polarizer 3 and the phase difference plate 4. Polarized light obtained according to the type of the phase difference plate is reflected by the mirror 5, passes through the dichroic mirror 6, and then is reflected by the galvano mirror 7. Further, after passing through the first lens 121 and the second lens 122, the light is condensed on the subject 10 on the stage 11 by the objective lens 8. The backscattered component Y of the SHG light generated in the subject 10 is collected by the objective lens 8 and the same optical path as that of the incident laser light X is reversed. The SHG light Y passes through the second lens 122 and the first lens 121 and is reflected by the galvanometer mirror 7. The dichroic mirror 6 reflects only the SHG light Y, and only the SHG light Y is extracted by the SHG light transmission filter 12. The extracted SHG light Y is detected by the photodetector 13. Then, the detection result is input to the computer 14 and an imaging process is performed.
The incident laser light can be converted into linearly polarized light in an arbitrary direction by the phase difference plate 4 (for example, a half-wave plate) after being converted into linearly polarized light by the polarizer 3. Furthermore, the beam spot on the subject 10 can be scanned at high speed in the two-dimensional plane of the subject 10 by the galvanometer mirror 7, the first lens 121, and the second lens 122. Alternatively, the subject 10 can be scanned in a two-dimensional plane by the stage 11. Further, it is possible to scan the beam spot in the depth direction of the subject 10 by the piezo stage 9 to which the objective lens 8 is attached. In this way, it is possible to analyze a two-dimensional or three-dimensional spatial distribution of SHG polarization anisotropy.
In the system shown in FIG. 3, for example, the mirror 5 is not an essential component and can be omitted as appropriate. When in-plane two-dimensional distribution measurement is unnecessary, the galvanometer mirror 7, the first lens 121, and the first lens 122 are connected to the piezo stage 9 when distribution measurement in the depth (optical axis) direction is not required. Can be omitted as appropriate.

The skin to be evaluated may be derived from humans or may be derived from other animals. Although skin tissue collected from a living body may be measured, in order to take advantage of the non-invasiveness that is an advantage of the measuring method of the present invention, it is preferable to use the skin of the living body as a measurement object in its original position.
The measurement target position on the skin is not particularly limited as long as it is inside the epidermis skin. However, since the collagen orientation in the dermis layer has a particularly deep relationship with wrinkles as described above, the dermis layer is measured at the measurement target position. It is preferable that The depth of the dermis layer varies greatly depending on the organism or body part, but in the case of human beings, it is usually about 0.1 mm to 4 mm deep from the skin surface.

According to the evaluation method of the present invention described above, the orientation of collagen in the skin (preferably the dermis layer) is used as a parameter for evaluating skin wrinkles, so that the skin is stretched, photoaged, or a composite thereof. It is possible to non-invasively and quantitatively evaluate the state of wrinkles caused by changes in the skin such as factors (stretching of the skin in exposed areas such as UV).
Specifically, by examining the relationship between the orientation direction of collagen in the skin (preferably the dermis layer) and the orientation direction of the wrinkle of the skin, the skin wrinkle is caused by the expansion and contraction of the skin, photoaging, or a complex factor thereof ( It can be used as an evaluation criterion for judging whether or not the skin is stretched or contracted in an exposed portion such as UV.
In addition, the possibility and / or direction of wrinkle formation on the skin can be predicted based on the orientation state of collagen inside the skin. That is, even when there is little (or no) wrinkle in the skin, it can be determined that there is a high possibility that wrinkles will be formed in the skin if the collagen is oriented in a certain direction inside the skin. . Moreover, the direction of the wrinkle can be predicted to be parallel to the orientation direction of collagen.
Further, by irradiating the inside of the skin with ultrashort pulse light (preferably chromium forsterite laser light) and using the detection result of the generated SHG light (particularly the polarization anisotropy of the SHG light), Collagen orientation can be assessed non-invasively and directly.

The evaluation method of the present invention can be used for clinical evaluation of a wrinkle inhibitor (anti-wrinkle agent). In other words, in order to suppress wrinkles due to photoaging, cosmetics (such as titanium oxide, zinc oxide, paramethoxycinnamic acid ester, paraaminobenzoic acid ester, etc.) containing various UV absorption, scattering and shielding substances as wrinkle inhibitors are used. Screens, sun protect cosmetics), and cosmetics formulated with wrinkle inhibitors with antioxidants that reduce the adverse effects caused by free radicals caused by ultraviolet rays have been proposed. It is possible to evaluate non-invasively and quantitatively how effective a wrinkle inhibitor is.
The evaluation method of the present invention can also be used for screening for a substance that becomes a wrinkle inhibitor (anti-wrinkle agent). That is, with respect to substances that are candidates for wrinkle inhibitors, if the evaluation method of the present invention is used to evaluate whether or not they actually have an effect of suppressing wrinkles, biological skin can be used as an evaluation system. Therefore, it becomes possible to select a wrinkle inhibitor more reliably and efficiently.

  EXAMPLES Hereinafter, although this invention is demonstrated further in detail using an Example, this invention is not limited to these Examples.

Six-week-old male hairless mice were irradiated with ultraviolet UV-B intermittently for 10 weeks. The UV-B intensity at the start of irradiation was 40 mJ / cm ² , the UV-B intensity was gradually increased, and the UV-B intensity at the end of irradiation was 200 mJ / cm ² . The back skin of 16-week-old male hairless mice after completion of UV-B irradiation was used as a skin sample for photoaged mice. Further, the back skin of a 16-week-old male hairless mouse that was not subjected to UV-B irradiation treatment was used as a skin sample of a control mouse.
First, each skin sample of the photoaged mouse and the control mouse was visually observed. The skin sample of the control mouse had almost no visible wrinkles, but the skin sample of the photoaged mouse had no body wrinkles. Characteristic deep wrinkles were seen in the left-right direction (direction perpendicular to the head-to-tail direction).

Next, data on SHG light was measured using the skin samples of these photo-aged mice and control mice.
As the measuring device, the measuring device shown in FIG. 3 was used. A chrome forsterite laser was used as the light source, and the wavelength of the ultrashort pulse light to be irradiated was 1250 nm, the frequency was 75 MHz, and the pulse length was 90 fs (femtosecond).
SHG light was detected by irradiating ultrashort pulse light at a depth of 100 μm, 150 μm, 200 μm, and 250 μm from the skin surface of each skin sample. In addition, the signal intensity of the SHG light is detected by changing the polarization direction of the incident ultrashort pulse laser light for each measurement site, and the polarization direction of the ultrashort pulse light is vertical and orthogonal to the wrinkle running direction of each skin sample. When the signal intensity of the SHG light obtained in the case of (when parallel to the head-to-tail direction of the mouse) is I _、, the polarization direction of the ultrashort pulse light is horizontal and parallel to the wrinkle running direction of each skin sample ( the signal intensity of the SHG light obtained when craniocaudal direction orthogonal) of the mouse as I _‖ was calculated α value using the aforementioned formula (I).

  FIG. 4 shows images of SHG light obtained from the skin surfaces of the control mouse and the photoaged mouse from the depths of 100 μm, 150 μm, 200 μm, and 250 μm from the skin surface. Also, an image of α value representing the polarization anisotropy of the SHG light and a histogram are shown in FIGS. 5 and 6, respectively.

  In both FIG. 4 and FIG. 5, the four images in the upper row represent images obtained from the skin sample of the control mouse, and the four images in the lower row are images obtained from the skin sample of the photoaged mouse. Represents. Each column represents an image obtained from the positions of depths of 100 μm, 150 μm, 200 μm, and 250 μm from the skin surface in order from the left. Each of the images in FIGS. 4 and 5 corresponds to a 400 μm × 400 μm region on the skin sample. 4 and 5, the vertical direction corresponds to the head-to-tail direction of the mouse, and the left-right direction corresponds to the direction orthogonal to the head-to-tail direction of the mouse, that is, the wrinkle direction.

  FIG. 4 is an SHG image in which collagen images of a skin sample of a control mouse and a skin sample of a photoaged mouse are detected. Furthermore, the orientation of collagen at the same position was shown by performing ellipsometry (FIG. 5).

On the other hand, in FIG. 5, as shown in the graph on the right side of each column, the portion where the α value is 0 is white, the portion where the α value takes a positive value (a value close to 1) is shown in blue, and the α value is negative. The portion taking the value of (a value close to -1) is shown in red, thereby imaging the α value.
From the α value image of FIG. 5, the skin sample of the control mouse shows a relatively isotropic collagen orientation in which red and blue are mixed, whereas the skin sample of the photoaged mouse is displayed in red overall. It can be seen that the horizontal collagen orientation is superior. The collagen orientation in the horizontal direction coincides with the running direction of the wrinkles.

  FIG. 6 is a histogram of the α value data in each image of FIG. 5 with the vertical axis representing the number of pixels and the horizontal axis representing the α value. According to the α value histogram of FIG. 6, the center of the histogram exists near zero at any depth in the skin sample of the control mouse, whereas the center of the histogram is negative in the skin sample of the photoaged mouse. It can be seen that the direction is shifted in the direction of wrinkles (the direction of wrinkles). In addition, the width of the histogram in the skin sample of the photo-aged mouse is narrower at any depth than that of the skin sample of the control mouse, and the skin sample of the photo-aged mouse has a better collagen orientation. I understand that.

From the above results, there is a correlation between the orientation of the collagen inside the skin and the direction of wrinkles generated in the exposed area of the skin due to the expansion and contraction of the skin. It can be seen that the wrinkle state can be evaluated.
It can also be seen that a technique based on detection of SHG light is effective in measuring the orientation of collagen inside the skin.
Further, the polarization anisotropy of the detected SHG light is expressed by an α value, and a histogram obtained by statistically processing the α value becomes a quantitative index indicating the orientation of collagen inside the skin, and thus wrinkles in the skin. It can be seen that it can also be used as a quantitative index representing the state.

  The most direct effect can be expected by the evaluation method of the present invention is in the field of skin cosmetics, and skin aging diagnosis is particularly interesting. Skin aging diagnosis is a field that is expected to make great progress in the future as awareness of anti-aging and skin beauty increases in recent years. In particular, if the evaluation method of the present invention can be organically integrated with cosmetic-related fields (for example, anti-wrinkle cosmetics), an extremely large market (for example, evaluation of skin wrinkles and reduced elasticity due to aging and sunburn, It is expected that cosmetics and coatings will be generated. In addition, the aging problem is a serious problem in the aging society that Japan will face in the future, and there is a great demand for skin aging diagnosis from the viewpoint of social needs.

It is a schematic diagram for demonstrating the measurement of the orientation of the collagen inside skin based on the detection of SHG light. It is a figure which shows the relationship between the polarization state of the incident laser beam regarding generation | occurrence | production of living body SHG light, and the orientation of a collagen fiber. 1 is a schematic configuration diagram illustrating an example of an SHG microphotometry system that can be used for irradiation with ultrashort pulse light and detection of SHG light in the evaluation method of the present invention. It is the image of SHG light obtained from the skin surface of a control mouse | mouth and a photo-aging mouse | mouth from the position of the depth of 100 micrometers, 150 micrometers, 200 micrometers, and 250 micrometers from the skin surface. It is an image of the α value obtained from the polarization anisotropy of SHG light obtained from the positions of depths of 100 μm, 150 μm, 200 μm, and 250 μm from the skin surface for each skin sample of the control mouse and the photoaged mouse. It is the histogram of (alpha) value calculated | required from the polarization | polarized-light anisotropy of SHG light obtained from the position of 100 micrometers, 150 micrometers, 200 micrometers, and the depth of 250 micrometers from the skin surface about each skin sample of a control mouse | mouth and a photoaging mouse | mouth.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Light source 2 ND filter 3 Polarizer 4 Phase difference plate 5 Mirror 6 Dichroic mirror 7 Galvano mirror 8 Objective lens 9 Piezo stage 10 Subject 11 Stage 12 SHG light transmission filter 13 Photo detector 14 Computer 121 First lens 122 Second lens X Incident laser beam Y SHG beam

Claims (9)

  A method for evaluating skin wrinkles, comprising a step of evaluating the state of wrinkles in the skin based on the orientation of collagen in the skin.   The evaluation method according to claim 1, wherein the orientation of collagen in the skin is the orientation of collagen in the dermis layer.   The evaluation method according to claim 1, wherein the wrinkle state in the skin is formation of wrinkles caused by expansion and contraction of the skin.   The evaluation method according to any one of claims 1 to 3, wherein the wrinkle state in the skin is wrinkle formation caused by photoaging.   The evaluation method according to claim 1 or 2, wherein the evaluation is a prediction of a possibility that the skin is wrinkled and / or a direction thereof based on an orientation state of collagen in the skin.   The method further comprises a step of irradiating the inside of the skin with ultrashort pulse light, detecting the generated second harmonic generation light (SHG light), and measuring the orientation of collagen inside the skin based on the detection result. The evaluation method according to any one of Items 1 to 5.   The evaluation method according to claim 6, wherein the orientation of collagen in the skin is measured based on the polarization anisotropy of the detected SHG light.   The evaluation method according to claim 6 or 7, wherein the ultrashort pulse light is femtosecond mode-locked laser light.   The evaluation method according to any one of claims 6 to 8, wherein the ultrashort pulsed light is chromium forsterite laser light, titanium / sapphire laser light, fiber laser light, or neodymium glass laser light.