Sample modulation method for X-ray imaging

Abstract

The present invention aims to provide a method for observing skin tissue using X-ray micro-CT. By using acetone as a pretreatment solution and a solution containing iodine as a staining solution for staining, it is possible to identify appendages such as sweat glands, sebaceous glands, and hair follicles in the skin tissue.

Description

Technical Field

[0001] The present invention relates to the technical field of X-ray imaging of skin samples. Background Art

[0002] X-rays have high transmittance, and on the other hand, transmittance varies depending on the material. Therefore, the difference in transmittance of X-rays is used in the medical field and the field of non-destructive internal inspection. The technology of processing X-rays with computers to obtain three-dimensional images is called computed tomography (CT). Regarding CT, if the influence of exposure to radiation is not considered, the resolution can be extremely high, and three-dimensional images of microscopic structures at or above the microscope level can be obtained. Such CT is called X-ray micro-CT, and although it is not preferably applied to biological bodies, it is suitable for more detailed observation of the internal three-dimensional structure of the obtained tissue.

[0003] Since the development of X-ray micro-CT, it has become possible to obtain and analyze cross-sectional images of various materials such as isolated tissues, resin molded products, and small electronic components.

[0004] Prior art literature

[0005] Patent Literature

[0006] Non-patent document 1: Journal of Cell Science (2016), vol. 129, pp. 2483-2492 Summary of the invention

[0007] Problems to be solved by the invention

[0008] The inventors of the present invention attempted to use X-ray micro-CT to analyze the three-dimensional structure of human skin tissue samples. The results showed that they were able to identify differences in large structures such as the epidermis, dermis, and subcutaneous fat layer, but were unable to identify structures such as appendages included in the skin structure.

[0009] Means for solving problems

[0010] Therefore, the present inventors conducted in-depth research on the settings of the X-ray micro-CT apparatus and the preparation of tissue samples. The present inventors pre-treated the skin tissue sample with acetone, stained it with a solution containing iodine, and provided it to the X-ray micro-CT apparatus. Surprisingly, the appendages as micro-organs contained in the skin could be identified, thereby completing the present invention.

[0011] Specifically, the present invention relates to a method for observing a skin sample using an X-ray imaging method. The observation method of the present invention is characterized by comprising the following steps:

[0012] A step of contacting a skin sample with a solution containing acetone;

[0013] a step of contacting the skin sample with a solution containing iodine; and

[0014] The process of obtaining a three-dimensional image of a skin sample using an X-ray computed tomography device.

[0015] In another embodiment, the present invention also relates to a method for preparing a skin sample for X-ray imaging. The preparation method of the present invention is characterized by comprising the following steps:

[0016] a step of contacting the skin sample with a solution containing acetone; and

[0017] A step of contacting a skin sample with a solution containing iodine.

[0018] In still another embodiment, the present invention also relates to a method for detecting skin cavitation, which uses the observation method of the present invention to measure the depth of the lowest part of the sweat gland.

[0019] Effects of the Invention

[0020] By observing the skin sample prepared by the skin sample preparation method of the present invention, the structure of the appendages as fine tissues contained in the skin can be observed. In addition, by measuring the depth of the sweat glands as one of the appendages, skin cavitation can also be detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure shows three-dimensional images obtained by X-ray micro-CT using (1) PFA-iodine-containing solution (Lugol), (2) PFA-phosphotungstic acid, (3) acetone-iodine-containing solution (Lugol), and (4) acetone-phosphotungstic acid as a combination of pretreatment solution and staining solution.

[0022] Figure 2 The three-dimensional image obtained by X-ray micro-CT using a combination of acetone and iodine-containing solution shows organs (hair, arrector pili muscles, sweat glands, and sebaceous glands) and tissues (epidermis, dermal layer).

[0023] Figure 3A represents an external photograph of normal skin and skin with severe sagging, and an internal image obtained by ultrasound. In normal skin, the boundary between the dermal layer and subcutaneous fat is flat, while in skin with severe sagging, severe dermal defects are observed. Figure 3 B is a graph showing the relationship between the size of dermal cavities (defect severity) and skin elasticity (skin firmness). The larger the dermal cavities (Large), the lower the skin elasticity, and the smaller the dermal cavities, the higher the skin elasticity (High). The correlation counts are expressed by Pearson's correlation coefficient.

[0024] Figure 4 A represents a three-dimensional image of a portion including a sweat gland obtained by X-ray micro-CT. A cavity is observed on the lower side of the sweat gland. Figure 4 B is a stained photograph of a tissue section of a sweat gland, a hair follicle, and a sebaceous gland. Cavitation exists in the sweat gland, but no cavitation exists in the hair follicle and the sebaceous gland. Figure 4 C is a graph showing the ratio of sweat glands and dermal cavities co-localized. The ratio of none co-localized is about 10%. The significant difference was determined by Student's t-test.

[0025] Figure 5 A three-dimensional image showing the sweat glands in young skin in the 30s and old skin in the 60s. In old skin, the sweat glands shrink upward toward the epidermis. DETAILED DESCRIPTION

[0026] The present invention relates to a method for observing a skin sample using an X-ray imaging method. The method is characterized in that it comprises the following steps:

[0027] A step of contacting a skin sample with a solution containing acetone;

[0028] a step of contacting the skin sample with a solution containing iodine; and

[0029] The process of obtaining a three-dimensional image of a skin sample using an X-ray computed tomography device.

[0030] The X-ray image taking method can select any output and resolution. From the perspective of observing a three-dimensional structure, a tomogram (CT image) is preferred. In addition, from the perspective of observing tiny organs such as appendages, a method of taking images with a resolution of 0.1 μm or less, called so-called X-ray micro-CT, is preferred. From the perspective of observing smaller tissues, the resolution of X-ray micro-CT is preferably 0.1 μm or less, more preferably 0.01 μm or less, and more preferably 0.05 μm or less. The lower limit of the resolution is not particularly limited, and is affected by the capabilities of the X-ray CT device, for example, 0.0001 μm or more, or 0.001 μm or more.

[0031] The so-called skin sample refers to a sample obtained from the skin. The skin sample can be obtained in a form that includes the area to be observed. The skin sample can contain any one or more of the epidermis, dermis, and subcutaneous tissue. Skin slices can also be used, and three-dimensional cultured skin models can also be used. The animal species from which the skin sample is obtained is not limited, for example, it can be human, pig, cow, mouse, rat, rabbit, horse, etc. Human skin is particularly preferred, but pig skin, which has a structure close to that of human skin, is also preferred as observation material.

[0032] The epidermis is a skin tissue present in the outermost layer of the skin. The epidermis is mainly composed of the stratum corneum, stratum granulosum, stratum spinosum, and stratum basale. The basal cells present in the basal layer divide and move to the outer layer. In the process of this movement, denucleation occurs and flattening occurs, and differentiation occurs to the stratum corneum, and finally peels off. In addition to keratinocytes, melanocytes, Langerhans cells, Merkel cells, etc. are also present in the epidermis. There is a basement membrane at the boundary between the epidermis and the dermis, and the basement membrane bulges to form a papillary structure. The dermis is composed of a papillary layer, a subpapillary layer, and a reticular layer. From the epidermis to the dermis, secretory glands such as sweat glands and sebaceous glands, and hair organs exist sunken. As secretory glands, there are eccrine sweat glands, apocrine sweat glands, sebaceous glands, and hair glands. As hair organs, there are hair follicles, hair, and arrector pili muscles. Among the secretory glands, the apocrine sweat glands and hair glands are attached to the hair organs. It is difficult to identify these hair organs and secretory glands with conventional X-ray micro-CT, but when a sample is prepared by the sample preparation method of the present invention and observed by the observation method, these appendages can be identified.

[0033] The obtained skin sample may be stored by refrigeration or freezing after obtaining, or may be brought into contact with a solution containing acetone immediately after obtaining. The solution containing acetone may be any solution as long as it contains acetone. The content of acetone is preferably 50% or more, more preferably 80% or more, and most preferably 100% acetone.

[0034] The contact time with the solution containing acetone can be arbitrarily selected according to the size of the skin sample. For example, for a skin slice with a thickness of 5 mm, the contact time can be more than 6 hours, preferably more than 12 hours, and more preferably more than 24 hours. The upper limit of the contact time is not particularly limited, but from the perspective of experimental simplification, it is within 48 hours, and more preferably within 24 hours. The contact process with the solution containing acetone can be carried out at any temperature, for example, it can be carried out at room temperature or under refrigeration. From the perspective of processing biological samples, it is preferably carried out under refrigeration, for example, at 4°C.

[0035] In the present invention, the so-called iodine-containing solution can be any solution as long as it contains iodine or iodide. Since iodine has a high absorption rate of X-rays, it is believed that tissues can be identified by X-ray photography based on the deposition degree of iodine. Therefore, the iodine-containing solution can also be called a staining solution. Iodide can be used to help dissolve iodine. As iodides that can be contained in the iodine-containing solution of the present invention, any iodide such as hydrogen iodide, sodium iodide, potassium iodide, and carbon tetraiodide can be cited. It can be a mixed solution of iodine and potassium iodide, or it can be, for example, Lugol's reagent. The concentration of iodide and iodine in the iodine-containing solution can be used in a total concentration of 0.1 to 10%, and a concentration of 1 to 5% can be further preferably used. As a mixing ratio of iodide to iodine, it can be arbitrarily selected between 1:10 and 10:1. As a mixing ratio, it is more preferably 1:5 to 5:1, and more preferably 1:2 to 2:1. In the Lugol reagent, the total amount of potassium iodide and iodine is used at 3.75%, and the ratio thereof is 2:1.

[0036] The contact time with the solution containing iodine can be arbitrarily selected according to the size of the skin sample. For example, for a skin slice with a thickness of 5 mm, the contact time can be more than 12 hours, preferably more than 24 hours, and more preferably more than 48 hours. The upper limit of the contact time is not particularly limited. From the perspective of experimental simplification, it is within 96 hours, and more preferably within 48 hours. The contact process with the solution containing acetone can be carried out at any temperature, for example, it can be carried out at room temperature or under refrigeration. From the perspective of processing biological samples, it is preferably carried out under refrigeration, for example, at 4°C.

[0037] In the present invention, a cleaning step may be performed before or after each step in order to wash away the solution used in each step. The cleaning step may be performed by replacing the solution with any solution once or multiple times. As the solution used, from the viewpoint of processing a biological sample, phosphate buffered saline (PBS) may be used, or the solution used in the next step may be used.

[0038] After the contact process with the iodine-containing solution, in order to obtain a three-dimensional image, an X-ray computed tomography device is used for shooting. Shooting can be carried out by appropriately selecting the output in any X-ray CT device. As the output used, from the viewpoint of improving the resolution, it is preferably 10kV or more, and more preferably 25kV or more. It is preferably 100kV or less, and more preferably 50kV or less. As an example, in the μCT device (D200RSS270) of Comscan Techno, the output can be set to 50kV and the X-ray tube current 200μA for observation.

[0039] When the skin sample is visualized by X-ray micro-CT using the method of the present invention, sweat glands with a shortened length from the skin surface are found. Such sweat glands are called atrophic sweat glands. Sweat glands, especially eccrine sweat glands, atrophy. Atrophic sweat glands are mainly observed in large numbers in the skin of elderly people. It is known that the deepest part of the sweat gland is usually present at the boundary between the dermis and the subcutaneous tissue. For atrophic sweat glands, only the atrophied part moves to the skin surface side, and the moved area is replaced by the subcutaneous tissue ( Figure 3 and 4 ). The hole formed by the subcutaneous tissue bulging toward the dermis is called a dermal cavity. The size of a dermal cavity is roughly spherical with a diameter of about 10μm to 1000μm, but its shape is not fixed. Since the dermal cavity is filled with subcutaneous fat tissue, it can also be simply called a dermal cavity. It is believed that since the dermal cavity does not contain the cellular components and interstitial components contained in the dermis layer, the skin loses its elasticity and sags, leading to skin aging (PCT / JP2015 / 072140).

[0040] Therefore, by determining whether the sweat glands are atrophied, the sagging of the skin can be measured. The atrophy of the sweat glands can be determined based on the depth from the skin surface to the lowest part of the sweat glands. In addition, the distance (L) from the skin surface to the subcutaneous fat tissue in the area where the sweat glands do not exist and the subcutaneous fat tissue does not bulge can be used. 1 ), and the distance from the area where the sweat glands exist to the deepest part of the sweat glands (or subcutaneous tissue) (L 2 ) to calculate the shrinkage degree. For example, it can also be expressed by the following formula.

[0041] Sweat gland atrophy degree = L 1 -L 2 / L 1

[0042] As the depth increases, the atrophy decreases, and the skin cavities become smaller. On the other hand, as the depth decreases, the atrophy increases, and the skin cavities become larger.

[0043] Therefore, by measuring the degree of atrophy of the sweat glands using the observation method of the present invention, skin cavitation can be detected.

[0044] All documents mentioned in this specification are incorporated herein by reference in their entirety.

[0045] The embodiments of the present invention described below are for illustration purposes only and do not limit the technical scope of the present invention. The technical scope of the present invention is limited only by the description of the claims. As long as it does not exceed the purpose of the present invention, changes to the present invention, such as addition, deletion and replacement of the technical features of the present invention, can be made.

[0046] Example

[0047] Embodiment 1:

[0048] The skin samples were pre-treated with 4% PFA or acetone, immersed in Lugol or phosphotungstic acid, and washed with PBS before being imaged by CT.

[0049] The remaining skin was obtained from 17 female subjects and 20 male subjects who had undergone plastic surgery, and cut into 5 mm thickness to prepare skin samples. After washing with PBS, the skin samples were added to acetone and incubated at 4°C for 24 hours for pretreatment. For comparison, 4% PFA aqueous solution was used instead of acetone in the pretreatment solution. After incubation, the skin samples were washed with PBS. Next, the skin samples were added to an iodine-containing solution (2.5% potassium iodide and 1.25% iodine aqueous solution) obtained by diluting Lugol's reagent, and incubated at 4°C for 24 hours. For comparison, a solution containing phosphotungstic acid was used instead of the solution containing iodine. For the solution containing phosphotungstic acid, a 6.7% phosphotungstic acid aqueous solution was used as a stock solution, and 1.5 ml of water and 7 ml of 100% ethanol were added to 1.5 ml of the stock solution to prepare a staining solution, and the sample was stained. The stained skin sample was washed with PBS and measured by μCT (D200RSS270; Comscan Techno). The images obtained by X-ray microCT for the pretreatment solution-staining solution combinations: (1) PFA-solution containing iodine (Lugol), (2) PFA-phosphotungstic acid, (3) acetone-solution containing iodine (Lugol), and (4) acetone-phosphotungstic acid are shown in FIG. Figure 1 middle.

[0050] The three-dimensional image obtained by using (3) acetone-iodine-containing solution as the pretreatment-staining solution ( Figure 2 ). Figure 2The organs (hair, arrector pili muscles, sweat glands, and sebaceous glands) and tissues (epidermis, dermal layer) are shown in the figure.

[0051] Embodiment 2:

[0052] The skin appearance of two women in their 30s was photographed, and the inside of the skin was imaged using an ultrasound imaging device. One woman had normal skin appearance, but the other had sagging skin. The skin appearance and ultrasound images were shown in Figure 3 A. Even for subjects of the same age, for the skin of subjects with sagging skin, bulges of fat tissue in the dermis (hereinafter referred to as dermal cavities) were observed in the ultrasound image, while in the skin of normal subjects without sagging, no such cavities were observed. Next, the size of the dermal cavities in the skin and the relationship with the firmness of the part were measured by changing the subjects. The elasticity of the skin (skin firmness) was determined by measuring the Ur / Uf value using a skin elasticity tester (Cutometer). The results are shown in Figure 3 In B.

[0053] In order to explore the cause of dermal cavities, the inventors analyzed the three-dimensional images obtained in Example 1 and found that sweat glands ( Figure 4 A). In order to study the relationship between sweat glands and dermal cavities, sweat glands, sebaceous glands, and hair follicles were observed in the stained images of tissue sections. The results showed that dermal cavities existed specifically in sweat glands. Figure 4 B). The local coexistence rate of sweat glands and dermal cavities was calculated ( Figure 4 C).

[0054] Next, the sweat glands in the skin of people in their 30s and 60s were compared using both three-dimensional images and tissue section staining images. The results showed that the position of the sweat glands in the skin of people in their 60s became lighter and shrank with age ( Figure 5 ). It is also known that in the area where the sweat glands atrophy, the fat tissue bulges as dermal cavities.

[0055] The depth of the sweat glands in the skin of people in their 30s and 60s was found. The depth is represented by the distance from the skin surface to the deepest part of the sweat glands. In addition, the severity of dermal cavitation is represented by the distance from the skin surface to the uppermost part of the cavitation. Therefore, the correlation between the sweat gland depth and age, and the sweat gland depth and the severity of dermal cavitation was studied. The results are shown in the following table.

[0056] [Table 1]

[0057]

Claims

1. A method for observing a skin sample using an X-ray micro-CT method, comprising the following steps: A step of contacting the skin sample with a solution containing acetone for more than 6 hours; A step of contacting a skin sample with a solution containing iodine; The step of washing away the solution containing iodine; and The process of obtaining a three-dimensional image of a skin sample using an X-ray micro-CT device. The three-dimensional image is capable of identifying appendages in the skin sample, The skin sample comprises an epidermis layer, a dermis layer and a subcutaneous tissue layer.

2. The observation method according to claim 1, wherein the appendages are selected from sebaceous glands, sweat glands and hair organs.

3. The observation method according to claim 1 or 2, wherein the iodine-containing solution contains at least one of potassium iodide and iodine.

4. A method for preparing a skin sample for X-ray micro-CT, comprising the following steps: A step of contacting the skin sample with a solution containing acetone for more than 6 hours; a step of contacting the skin sample with a solution containing iodine; and The process of washing away the solution containing iodine. 5 . The method for preparing a skin sample according to claim 4 , wherein the iodine-containing solution contains at least one of potassium iodide and iodine. 6 . A method for detecting skin cavitation, comprising measuring the depth of an appendage using the observation method according to claim 1 .

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