JP2020099591A - Photography and analysis method of skin capillary blood vessel - Google Patents

Abstract

To provide an analysis method of a skin capillary blood vessel which enables blood flow velocity of each capillary to be calculated simultaneously and automatically from many moving images of a skin capillary blood vessel.SOLUTION: The analysis method of a skin capillary blood vessel image is provided that comprises a step of photographing a moving image of a skin capillary blood vessel, using a microscope system comprising: an imaging optical system of wide-angle view and high definition in which a visual field length is 5-12 mm and a value obtained by dividing the horizontal resolution by the visual field length is 0.8[μm/mm] or less; and an illumination system making irradiation light from a light source oblique incident on the surface of an observation target at an incidence angle of 40-75° with respect to an optical axis of the lens, while a contact agent having a refraction index of 1.3-1.55 and the viscosity of 5.0×10-4 to 1.5×102 Pa s is interposed at the gap between the microscope system and the skin of a subject's body region. The analysis method further comprises: a step of calculating the blood flow velocity in the capillary blood vessel by performing processing of aligning between frames of the moving image using at least template matching, processing of extracting a blood flow component using the robust principal component analysis, processing of extracting a blood vessel region using an extracted blood flow component, and processing of calculating the displacement of the blood flow component between the frames using an optical flow in the blood vessel region.SELECTED DRAWING: None

Description

The present invention relates to a method for analyzing a moving image of skin capillaries.

Human skin is composed of epidermis, dermis, and its adjunct organs (sweat glands, etc.). The epidermis is a tissue located at the outermost layer and having a thickness of about 100 to 200 μm, and is composed of four layers of a basal layer, a spinous layer, a granular layer, and a stratum corneum in order from the inside of the body. The uppermost layer, the stratum corneum, is the first line of defense against external stimuli and plays an important role in maintaining homeostasis of the living body. The stratum corneum is also an important site for cosmetics. That is, the state and phenomenon such as skin groove, crest, texture or rough skin, pores, wrinkles, spots, and sunburn are closely related to disturbance of the stratum corneum structure, change in stratum corneum composition, and the like. Therefore, in order to achieve healthy skin and beautiful skin, it is necessary to keep the stratum corneum in good condition.

The keratinocytes formed in the basal layer are keratinized, and the stratum corneum is formed by differentiation into flattened stratum corneum cells via the spinous layer and granular layer. Since there are no capillaries in the epidermis (basal layer, spinous layer, granular layer, stratum corneum), oxygen and nutrients required for keratinocyte metabolism and differentiation are supplied from the dermis vascular system under the epidermis. Specifically, the capillaries in the papilla structure at the epidermis/dermis boundary and the capillaries near the epidermis outside the papilla play a final stage of supply.

Blood does not always flow through these capillaries. It is believed that the amount of blood flowing through the capillaries is controlled depending on the skin condition. For example, it is considered that the volume of blood flowing through the capillaries is large in a region where the epidermal metabolism is active.

Thus, the blood flow behavior in capillaries near the epidermis is 1) a factor that directly affects the metabolism and differentiation of keratinocytes in the epidermis, and 2) a factor that indirectly contributes to the state of the stratum corneum. 3) Ultimately, it is considered to be a factor contributing to the realization of healthy and beautiful skin. Therefore, grasping the blood flow state in the skin capillaries is useful for grasping the health condition of the skin, investigating the cause of skin troubles such as rough skin, and evaluating substances and physical actions for improving the skin condition. is there.

Conventionally, optical microscope observation is known as a method for observing the blood flow state in skin capillaries. However, in order to obtain a resolution capable of observing blood flow in capillaries (diameter of 5 to 20 μm), the observation visual field can usually secure only a few mm ² . Generally, the shape and distribution of capillaries are not uniform, and the blood flow state of the capillaries of the skin cannot be accurately grasped from the result of one observation of such a narrow region. Therefore, a large number of observations are required to obtain a significant result, which imposes a great burden on the measurer and the subject.
For example, Non-Patent Document 1 discloses that a blood flow velocity is calculated by performing image analysis on a moving image obtained by photographing a capillary blood vessel of the oral mucosa with a microscope. However, the field of view is narrow (1 mm×0.7 mm) because the image is captured by a microscope, and only a small number of capillaries can be analyzed.

Further, in Patent Document 1, an image acquisition system using a scanner scans a skin surface of a body part of a subject to acquire a scan image, and a microscope is used based on image information such as capillaries reproduced in the scan image. A method of identifying the field of view for observation is disclosed. However, this technique does not directly capture a moving image of a capillary with a wide field of view and high resolution, and does not disclose a technique for analyzing a blood flow velocity in the capillary.

JP, 2016-214567, A

Dobbe, J.G.G. et al. Medical and Biological Engineering and Computing 2008 46(7), pp. 659-670.

The present invention relates to a method for analyzing skin capillaries, which can simultaneously and automatically calculate the blood flow velocity of each capillary from a plurality of moving images of skin capillaries.

That is, the present invention relates to the following.
The field of view is 5 to 12 mm, and the wide field of view and high resolution imaging optical system, in which the value obtained by dividing the horizontal resolution by the field of view is 0.8 [μm/mm] or less, and the irradiation light from the light source. A microscope system including an illumination system that obliquely enters the surface of the observation target at an incident angle of 40 to 75° with respect to the optical axis of the lens is used, and a refractive index of 1.3 is provided in the gap between the microscope system and the skin of the subject body part. ˜1.55 and a viscosity of 5.0×10 ^{−4 to} 1.5×10 ² Pa·s, a step of capturing a moving image of skin capillaries via a contact agent, and a step of capturing in this step For a moving image of skin capillaries, at least template matching is used to align the frames of the moving image, robust principal component analysis is used to extract the bloodstream component of the moving image, extracted bloodstream component A step of calculating a blood flow velocity in a capillary by performing a process of specifying a blood vessel region using the process of calculating a displacement of a blood flow component between frames using an optical flow in the specified blood vessel region. A method for analyzing a skin capillary image, including:

According to the method of the present invention, a moving image of skin capillaries is captured in a wide field of view with high resolution, and image analysis is performed on the captured moving image, so that blood flow velocities of a large number of capillaries can be simultaneously and automatically obtained. Can be calculated. This makes it possible to accurately analyze the blood flow velocity in the skin capillaries with a small number of times of imaging.

The whole block diagram of the observation device of a skin condition. The whole block diagram of the observation device of a skin condition. A: Appearance of oblique incidence ring illumination system, B: Appearance of oblique incidence illumination system, C: Definition of incident angle. The elements on larger scale of the skin contact part. Image example of skin capillaries. Whole field of view (7.5 mm x 5.6 mm). Image processing is applied to the area (5.6 mm x 0.8 mm) inside the broken line. FIG. 6 is a schematic diagram of an image processing procedure. FIG. 6 is an image showing the enlargement in the area surrounded by a square in FIG. 5 at one frame intervals. The broken line indicates the blood vessel region structure, and Δ indicates the leading portion of red blood cells flowing in the capillaries. An example of the analysis result in the area surrounded by a square in FIG. From the left, the extraction result of the blood flow component, the extraction result of the blood vessel region, and the analysis result of the blood flow velocity for this region (the broken line portion is the blood vessel region). An example of the analysis result in the circled area of FIG. From the left, a partial enlargement of the original image, a blood flow component extraction result for this region, a blood vessel region extraction result, and a blood flow velocity analysis result (broken line portion is a blood vessel region).

The method for analyzing a skin capillary image of the present invention is to obtain a capillary dynamic image of a body part of a subject with a wide field of view and high resolution, and analyze the blood flow state of the capillary based on the image data. ..
<Capture blood vessel image acquisition>
In the present invention, a moving image of skin capillaries has a visual field length of 5 to 12 mm, and a wide visual field and high resolution in which a value obtained by dividing the horizontal resolution by the visual field length is 0.8 [μm/mm] or less. The imaging system and the illumination system that makes the irradiation light from the light source obliquely incident on the surface of the observation target at an incident angle of 40 to 75° with respect to the optical axis of the lens are used. It is carried out by photographing in the gap of the skin of the site through a contact agent having a refractive index of 1.3 to 1.55 and a viscosity of 5.0×10 ^{−4 to} 1.5×10 ² Pa·s. ..

In the present invention, the body part includes the outside of the human body in which the state of capillaries needs to be observed, and specifically includes hands, feet, arms, legs, torso, face and the like. Such a body part may be a body part after applying cosmetics or drugs.

Hereinafter, an example of an apparatus (FIGS. 1 and 2) for carrying out the method of the present invention will be described, but the method of the present invention is not limited thereto.
FIG. 1 shows a mode of an apparatus including a photographing table 2 having a subject placement unit 1 having an opening and a microscope system 3 placed on the opposite side of the subject via the subject placement unit.
The subject placement unit 1 is a part of a photographing table on which a body part that is a subject is placed. If the subject can be observed by the microscope system 3 installed on the side opposite to the subject, the opening is filled with a transparent member. However, the member is not particularly limited, but it is usually preferable to use a glass plate or a transparent film.

The imaging table 2 includes the subject placement unit 1, and the shape and configuration thereof are not particularly limited as long as the microscope system 3 can be placed on the side opposite to the subject through the subject placement unit. For example, it may be a structure in which a flat plate including a subject placement portion is supported by the columns 6 (FIG. 1), the subject placement portion is placed on one of the surfaces, and the microscope system is placed inside the structure or the microscope system. It may be a casing (lens barrel 2a) in the shape of a rectangular parallelepiped, a cube, a cylinder or the like that can be connected and arranged (FIG. 2).
When a structure in which a flat plate including a subject placement portion is supported by a pillar is used as the imaging stand, the position of the microscope system 3 may be fixed, but the distance between the microscope system 3 and the subject and the observation field of view can be adjusted. It is preferable that the position is adjustable by being connected to a position adjusting mechanism 7 such as a movable stage (XYZ axis stage) or a jack.

The microscope system 3 includes an imaging optical system (lens 3a and camera 3c) for imaging an observation target with a wide field of view and high resolution, and an illumination system (light source) 3b for irradiating the surface of the observation target with oblique incident light of a directional light source. The type is not limited as long as it is equipped with a stereomicroscope, a polarization microscope, a microscope, and the like.

As the lens used in the present invention, a high-resolution lens capable of observing capillaries inside the skin is used. From this point of view, the horizontal resolution of the lens is 20 μm or less, preferably 10 μm or less, and more preferably 5 μm or less. The magnification of this lens is preferably 0.25 times or more, more preferably 0.5 times or more, and more preferably in order to avoid the difficulty of observation due to the large size of the apparatus and to secure a sufficient observation range. Is 0.75 times or more, and preferably 15 times or less.

The camera is not limited as long as it is capable of taking a moving image of a microscope observation image with a wide field of view and high resolution, and examples thereof include a digital camera adopting an image pickup device such as CCD or CMOS. The number of pixels of the image pickup device is preferably 4 million pixels or more, and more preferably 8 million pixels or more in order to obtain sufficient resolution.
The size of the image pickup device is preferably ¼ type (3.6×2.7 mm) or more, and preferably in order to avoid a difficulty in observation due to the large size of the device and to obtain sufficient resolution. The size is 4/3 type (17.3×13 mm) or less, more preferably 1 type (13.2×8.8 mm) or less, and more preferably 2/3 type (8.8×6.6 mm) or less.

In the imaging optical system of the present invention, the visual field length is 5 mm or more and 12 mm or less, more preferably 11 mm or less, and further preferably 10 mm or less. Further, it is 5 to 12 mm, more preferably 5 to 11 mm, and further preferably 5 to 10 mm.
The value [μm/mm] obtained by dividing the horizontal resolution by the field length is 0.8 or less, preferably 0.65 or less, and more preferably 0.5 or less.

Here, the “visual field length” is a diagonal line of a quadrangle forming the visual field. If it is a polygon, the length of the longest line segment of the diagonal line and sides is set. If it is circular/oval, it is the diameter/major axis. If the shape is indefinite, the distance between the longest two points in the closed curve is used. The “horizontal resolution [μm]” refers to the resolving power within the imaging plane and the shortest distance that can distinguish two points.

In the microscope system of the present invention, the light emitted from the light source is obliquely incident on the surface of the observation target and the image is captured, whereby the capillaries inside the skin can be imaged.
Visible light is usually used as the type of light source used. As the visible light, any light including a wavelength of 400 nm or more and less than 800 nm may be used, and blue light, red light, green light, and the like can be used in addition to white light, but visible light with different wavelengths is mixed. It is preferable to use white light. For example, a white LED light source, a halogen lamp, etc. can be used.

The incident angle of oblique incidence on the surface of the observation target is 40 degrees or more, preferably 42.5 degrees or more, more preferably 45 degrees or more, and 75 degrees or less, with respect to the optical axis of the lens. It is 65 degrees or less, and more preferably 60 degrees or less. Further, it is 40 to 75 degrees, preferably 42.5 to 65 degrees, and more preferably 45 to 60 degrees.

In a preferred embodiment, the light irradiation is performed by setting the distance between the light source and the observation object to 10 to 60 mm, preferably 10 to 40 mm, more preferably 10 to 30 mm, and then setting the light to 40 with respect to the optical axis of the lens. ˜75 degrees, preferably 42.5 to 65 degrees, more preferably 45 to 60 degrees, and preferably obliquely incident on the surface of the observation target, and the distance between the light source and the observation target is 10 to 30 mm, More preferably, the light is obliquely incident on the surface of the observation target at an incident angle of 45 to 60 with respect to the optical axis of the lens.
One aspect of an irradiation system (A: oblique-incidence oblique-ring illumination system, B: oblique-incidence illumination system) for obliquely making the light emitted from such a light source incident on the surface of the observation target, and the definition (C) of the incident angle are described. As shown in FIG.

At the time of photographing, the body part of the subject to be measured is placed in the opening of the subject placement portion. FIG. 4 shows a schematic cross-sectional view around the opening of the subject placement portion. In the configuration in which the transparent member is not used in the subject placement portion (FIG. 4A), the contact agent described below is applied to the surface of the skin and maintained in the attached state. When the fluidity of the contact agent is high, the configuration of FIG. 4B or 4C can be used. In the configuration of FIG. 4B, the contact agent is retained in the space surrounded by the transparent member, the spacer and the skin. In this case, when the body part of the subject is pressed against the upper part of the spacer, the skin surface is held without coming into contact with the transparent member of the opening. As a result, it is possible to avoid a decrease in blood flow due to compression of the skin at the imaging site by the transparent member. The spacer is a member having an opening and having a constant thickness so that the surface of the skin to be imaged is imaged when the body part of the subject to be measured is set. In the configuration of FIG. C, a flexible film is used as the transparent member, and the contact agent is held in the gap between the transparent member and the skin. Since the film is flexible, it is possible to avoid a decrease in blood flow due to pressure on the skin without holding the skin surface in contact with the transparent member. The film may be flexible and transparent, and a food wrap film or a packaging film made of polyvinyl chloride, polyvinylidene chloride, polyethylene, polypropylene or the like can be used.

The thickness of the spacer (height from the transparent member when installed on a transparent member) is such that when the body part of the subject is pressed, the skin surface protrudes inside the opening of the spacer and contacts the transparent member. It is necessary not to. Considering this point, the thickness of the spacer is preferably 0.1 mm or more, more preferably 0.5 mm or more, and preferably 6 mm or less, more preferably 1 mm or less. Further, it is 0.1 to 6 mm, preferably 0.5 to 1 mm.

The material of the spacer is preferably a material that has appropriate softness so as not to damage the skin and has appropriate elasticity that follows the shape so as to adhere to the skin/glass surface. Examples of such a material include silicone rubber, natural rubber, acrylic rubber, urethane rubber and the like, and it is preferable to use silicone rubber from the viewpoint of water resistance, oil resistance, odorless and nontoxic.
Further, the material can be any color, but when the material is colored red, blue, or the like, colorless, white, or black is preferable because it may affect subsequent image processing/analysis.

The contact agent can reduce the reflection of light at the interface between the stratum corneum (refractive index: about 1.5) and air (refractive index: about 1.0), and thus has a refractive index of 1.3 to 1.55. A fluid material having
As such a contact agent, one that is colorless and transparent is preferable. Further, from the viewpoint of conformability to the skin, the viscosity at room temperature and the skin surface temperature of 25° C. to 30° C. is 5.0×10 ^{−4 to} 1.5×10 ² Pa·s, and further from the spacer. The viscosity is preferably 3.0×10 ^{−2 to} 1.2×10 ² Pa·s from the viewpoint of viscosity so as not to flow out, and further, from the viewpoint of difficulty in entering bubbles and easy removal. It is more preferably 0×10 ^{−2 to} 40 Pa·s.

Here, the refractive index can be measured by an Abbe refractometer at 23° C. according to JIS K 7142, and can also be calculated and obtained based on literature values (Chemical Handbook (Chemical Society of Japan), etc.). .. Further, the viscosity can be determined by flow curve measurement using a commercially available rheometer or measurement using a B-type (single-cylinder rotary type) viscometer.

Suitable contact agents include, for example, glycerin (refractive index about 1.5, viscosity 8.3×10 ⁻¹ Pa·s), immersion oil (refractive index 1.52, viscosity 1.2×10 ² Pa·s). , Squalane (refractive index about 1.45, viscosity 3.0×10 ⁻² Pa·s), etc., commercially available ultrasonic jelly such as pro jelly (Gex Co., Ltd.) (refractive index about 1.3, viscosity about 40 Pa)・S), water (refractive index about 1.3, viscosity 8.9×10 ⁻⁴ Pa·s), rapeseed oil, etc. (refractive index about 1.5, viscosity 4.3×10 ⁻² Pa·s), edible Examples thereof include oils, and of these, glycerin, squalane, ultrasonic jelly, and rapeseed oil are more preferable.

Thus, the microscope system captures a microscopic image of the skin capillaries. The captured image data is displayed on the image display unit 5 such as a television monitor, and the image processing unit 4 performs image analysis processing described later.
In the microscopic image taken, it is observed that red blood cells flow in the capillaries. Hereinafter, pores, hair, stains, sweat glands, etc. in the moving image will be referred to as “background components”, and a component showing the flow of red blood cells, which is obtained by removing the background components from the moving image, will be referred to as “blood flow component”.

<Analysis of skin capillary images>
For the captured moving image of skin capillaries, at least the process of aligning the frames of the moving image using template matching, the process of extracting the blood flow component of the moving image using robust principal component analysis, the extracted blood The blood flow velocity in the capillaries is calculated by performing the process of identifying the blood vessel region using the flow component and the process of calculating the displacement of the blood flow component between frames using the optical flow in the blood vessel region. .. The details of each process are described below.

i) Positioning between Frames of Moving Image Using Template Matching Template matching is a method for finding an area similar to the image set as the “template” from the input image. The moving image of the acquired skin capillaries contains the blurring of the image derived from the body movements of the subject and the measurer, and the blood vessel itself moves in the moving image, and the blurring speed is calculated when calculating the blood flow velocity. There is a risk of doing it. This blurring is corrected by searching and extracting a similar region in the image by template matching.

In template matching, a part is extracted from the first frame of the acquired moving image of capillaries and set as a “template”, and the other frames are used as the input image. This makes it possible to search for similar regions in all frames in the moving image, that is, the same location of the shooting target. By cutting out the searched region and connecting them in the same order as the original moving image, a “blurred image” in which the blood vessel of interest is always at the same position in the moving image is created.
Prior to the "template" excerpt, a range to be subjected to image processing is selected from the original image. The size of this image processing range is 5 mm or more, which is larger than a general microscope image, and less than 12 mm, which is smaller than the size of a moving image to be captured.

In the search for a similar region, the “template” is compared with a region of the same size in the input image, and the degree of similarity is calculated. This similarity is calculated by comparing the brightness ("pixel value") of pixels at the same position in the two images. The calculation method for calculating the degree of similarity is not particularly limited, but for example, a method in which the smaller the sum of squares of differences in pixel values or the sum of absolute values is, the higher the degree of similarity is, or the normalized cross-correlation is close to 1 A method of calculating the degree of similarity is higher. Specifically, for example, the matchTemplate function of OpenCV 2.4.11 (Intel corporation) is used, and the normalized cross-correlation is used to evaluate the similarity.

ii) Extraction of Blood Flow Component from Moving Image Using Robust Principal Component Analysis Next, the blood flow component is extracted from the shake-corrected capillary moving image using the robust principal component analysis method.
According to the robust principal component analysis, it is possible to separate/extract minute components in a moving image having large temporal fluctuations in pixel values from components having large scales and small temporal fluctuations in pixel values. In the robust principal component analysis, the target moving image is represented in the form of a matrix and then decomposed into the form of the sum of the low rank matrix and the sparse matrix. As a method of decomposition, a term for evaluating the low rank of a low rank matrix and a term for evaluating the sparsity of a sparse matrix are provided, and the latter is multiplied by a weight λ to obtain the sum of two terms as an evaluation function. The components are separated so as to minimize As the calculation method, for example, the method described in Z. Lin et al., The Augmented Lagrange Multiplier Method for Exact Recovery of Corrupted Low-Rank Matrics, UIUC Technical Report UILU-ENG-09-2215, November 2009” can be used.

In the present invention, the weight λ is
And Capillary moving images are small and have large temporal fluctuations in moving images.The movement of red blood cells in capillaries is decomposed into sparse components, resulting in large scales and small temporal fluctuations. Background components such as are decomposed into low rank components.

iii) Identification of blood vessel region using blood flow component In the blood flow component of the moving image extracted by performing the robust principal component analysis, white particles indicating the flow of red blood cells in the blood vessel flow in a dark background. You can see the situation. In the present invention, the blood vessel region is specified by creating a mask image in order to limit the analysis target region to within the blood vessel region.
The mask image is created by the following processing.
1) The pixel values of all frames of the moving image of the blood flow component are averaged. As a result, the area where blood is flowing has a large pixel value, and thus is drawn as a continuous white area.
2) A binarized image (for example, Otsu's binarized) is used to create a binary image (white in the blood vessel region through which red blood cells pass, black in the other regions).
3) By the expansion process and the contraction process, the discrete white area derived from noise is removed.

iv) Blood flow velocity calculation using optical flow The blood flow velocity is calculated by detecting the movement of the brightness (pixel value) gradient in the moving image for each location in the image and expressing it as a vector. Is adopted.
The moving image of the blood flow component extracted by performing the robust principal component analysis is limited to the inside of the blood vessel region in the mask image showing the blood vessel region obtained by averaging and binarizing the image. The displacement of the blood flow component is detected by the optical flow.
As the optical flow, there are known algorithms such as the Lucas kanade method and the Horn Schunkk method, and the optical flow is not particularly limited. For example, the Lucas kanade method can be used, and its execution can be performed by using the calcOpticalFlowPyrLK function of OpenCV 2.4.11 (Intel corporation) and setting the size of the search window to 10 pixels square.

Example 1 (see FIG. 1 and FIG. 4-B)
(1) Acquisition of a moving image of skin capillaries A CMOS camera (BU1203, 12 million pixels, 1/1.7 type sensor, Toshiba Terry) has a working distance of 68 mm and a magnification of 1× lens (VS-TCT1-65/S, VS Optics Co., Ltd.) was placed facing upward and connected. The camera is connected to an XYZ axis stage that is a combination of an XY axis stage (TSD-602C, Sigma Koki Co., Ltd.) and a Z axis stage (B33-60KGA, Suruga Seiki Co., Ltd.), and the distance to the skin surface and the visual field can be adjusted. And A light source unit (a ring-shaped white LED light source (OPDR) capable of obliquely entering at an incident angle of 60° with respect to the lens optical axis so that the distance to the observation target is 45 mm above the optical axis (Z axis) of the lens. -LA74-48W-2, manufactured by Optex FA)). The subject's forearm inner part was placed on the subject placement part (a plate having a 15 mm aperture) of the imaging table so that the skin surface was located at the focal position of the lens, and an image was taken. Using this configuration, a moving image of skin capillaries was captured for 10 seconds at a frame rate of 30 frames per second. In the opening, a cover glass is placed as a transparent member, a spacer is a silicon rubber having a rectangular opening of 15 mm×25 mm with a thickness of 1.5 mm, and a rapeseed oil is used as a contact agent (refractive index about 1.5, viscosity 4 .3×10 ⁻² Pa·s) was used.
FIG. 5 shows the entire field of view photographed with 7.5 mm×5.6 mm, 4000 pixels×3000 pixels.

(2) Image Analysis 1) Blurring Correction Using Template Matching In the first frame of a moving image, 3000 pixels in the horizontal direction and 400 pixels in the vertical direction (5.6 mm in the horizontal direction and 0.8 mm in the vertical direction) included in all the frames in common. The area was extracted and used as a template image. Using all frames in the moving image as the input image, we searched and extracted a region similar to the template image. The excerpted image was saved as a moving image in AVI format to obtain a blur correction image. Note that the template matching was performed using the matchTemplate function of OpenCV 2.4.11, and the normalized cross-correlation was used to evaluate the similarity.

2) Robust Principal Component Analysis and Creation of Mask Image Robust principal component analysis was performed on the motion-corrected moving image obtained in 1) to acquire a moving image of blood flow component. The weight λ that controls the degree of separation of the large variation component and the small variation component is
And
In the moving image of blood flow components, white particles showing the flow of red blood cells in blood vessels were seen to flow in a dark background. From this moving image, a mask image was created by the following processes i) to iii). i) The pixel values of each frame of the moving image of the blood flow component are averaged (the area where blood is flowing is drawn as a continuous white area because the pixel value is large), ii) Otsu's binarization A binary image is created by iii) The discrete white area derived from noise is removed by expansion processing and contraction processing.

3) Calculation of blood flow velocity The mask image obtained in 2) was calculated by executing the optical flow method by Lucas-Kanade. For execution, the calcOpticalFlowPyrLK function of OpenCV 2.4.11 (Intel Corporation) was used, and the size of the search window was 10 pixels square.

(3) Results FIG. 6 shows a flow of each processing of 1) to 3) performed on the area surrounded by the broken line in FIG.
In FIG. 7, the region surrounded by a square in FIG. 5 is enlarged and shown at one frame intervals. A flow of red blood cells was observed in the capillaries inside the skin. Each of the processes 1) to 3) is performed on the entire area within the broken line in FIG. 5, and the result in the area surrounded by a square in FIG. 5 is shown in FIG. It was possible to extract the blood flow component and the blood vessel region, and to draw the arrow indicating the displacement of blood cells in the image between the two blood flow velocity frames. The blood flow velocity could be calculated by dividing the magnitude of the displacement corresponding to the length of this arrow by the time difference between frames (about 33 milliseconds).
Moreover, the result in the area enclosed with the circle in FIG. 5 is shown in FIG. As in FIG. 7, the blood flow velocity could be analyzed. Since the blood flow velocity can be analyzed simultaneously in the area surrounded by a square and a circle in the broken line region of FIG. 5, it is possible to calculate the blood flow velocity of many skin capillaries in a wide-field/high-resolution image. Was shown.

1 Subject Arrangement Section 2 Imaging Stand 2a Lens Barrel 3 Microscope System 3a Lens 3b Light Source 3c Camera 4 Image Processing Section 5 Image Display Section 6 Support 7 Position Adjustment Mechanism

Claims (1)

The field of view is 5 to 12 mm, and the wide field of view and high resolution imaging optical system, in which the value obtained by dividing the horizontal resolution by the field of view is 0.8 [μm/mm] or less, A microscope system equipped with an illumination system that obliquely enters the surface of the observation target at an incident angle of 40 to 75° with respect to the optical axis of the lens is used. ˜1.55 and a viscosity of 5.0×10 ^{−4 to} 1.5×10 ² Pa·s, a step of capturing a moving image of skin capillaries via a contact agent, and a step of capturing in this step For a moving image of skin capillaries, at least template matching is used to align the frames of the moving image, robust principal component analysis is used to extract the bloodstream component of the moving image, extracted bloodstream component And a process of calculating the displacement of the blood flow component between frames by using an optical flow in the blood vessel region. , Analysis method of skin capillary image.