WO2016158719A1 - Image processing method, control program, and image processing device - Google Patents

Abstract

The present invention is provided with: a step for specifying a subject area including an object corresponding to a spheroid, from a captured raw image of the spheroid (step S103); a step for detecting a circular area having the maximum degree of separation within the subject area, using a circular degree-of-separation filter operation (step S105); and a step for outputting a value proportional to the degree of separation of the circular area calculated using the circular degree-of-separation filter operation, the value being outputted as an index value indicating the roundness of the spheroid specified by the circular area (step S106).

Description

Image processing method, control program, and image processing apparatus

The present invention relates to a technique for evaluating a spheroid included in an image by image processing based on an original image obtained by imaging the spheroid.
Cross-reference to related applications The disclosures in the specification, drawings, and claims of the following Japanese application are incorporated herein by reference in their entirety:
Japanese Patent Application No. 2015-071655 (filed on Mar. 31, 2015).

In medical and biological science experiments, cell clusters (spheroids) in which a large number of cells gather in a spherical shape are cultured and observed. A spheroid with higher cell activity has a shape closer to a sphere. From this, it is performed to evaluate how the spheroid has a shape similar to a circle in an image obtained by imaging the spheroid.

Examples of techniques for evaluating the roundness of an object such as a cell included in an image include the techniques described in Patent Documents 1 to 3. In these techniques, the roundness of an object included in an image is evaluated by the roundness obtained from the ratio of the area of the object to the circumference.

International Publication No. 2012/147403 Japanese Patent No. 4521572 Japanese Patent Laid-Open No. 2006-079196

When the object is a single cell, the above-described conventional evaluation method can achieve a certain result. On the other hand, when the object is a spheroid, good results are not always obtained. The reason is as follows. Spheroids are composed of a large number of cells, and irregularities due to the contours of individual cells appear on the outer periphery of the spheroids. Therefore, the circumference tends to be estimated longer than a circle of the same size. As a result, the calculated roundness does not necessarily indicate the roundness of the spheroid.

Thus, in order to evaluate a spheroid, an index that quantitatively represents its roundness is required. However, a technique for obtaining such an index value has not been established so far.

This invention is made in view of the said subject, and it aims at providing the technique which can obtain | require the index value which represents the roundness of a spheroid quantitatively from the image which imaged the spheroid.

In order to achieve the above object, the image processing method according to the present invention specifies a target region including an object corresponding to the spheroid from an original image in which a spheroid is captured, and a circular separability filter operation to determine the target. A step of detecting a circular region having a maximum degree of separation in the region, and a value proportional to the degree of separation of the circular region calculated by the circular separation degree filter calculation, the spheroid specified by the circular region And outputting as an index value indicating roundness.

In order to achieve the above object, the image processing apparatus according to the present invention includes an image acquisition unit that acquires an original image in which a spheroid is captured, and a target area that includes an object corresponding to the spheroid in the original image. A detection means for executing a circular separability filter calculation to detect a circular area having a maximum value of the separability, and a value proportional to the separability of the circular area calculated by the circular separability filter calculation Output means for outputting as an index value indicating the roundness of the spheroid specified by the region.

Further, another aspect of the present invention is a control program that causes a computer to execute each step of the above-described image processing method.

In the invention configured as described above, the circle having the maximum degree of separation among the circles drawn in the target region of the original image is specified by the circular separation degree filter calculation. This circle is considered to have a high probability of corresponding to the outline of the spheroid existing in the target region. From this, it can be said that the circular region specified by this circle approximately represents the spheroid region. If the spheroid is close to a circle in the image, the value of the degree of separation is large, and if the deviation from the circle is large, the value of the degree of separation is small. Therefore, the value of the separation degree itself is information that quantitatively indicates how close the spheroid is in the shape of a circle in the image.

Therefore, in the present invention, a value proportional to the value of the degree of separation obtained for the circular region estimated to be a spheroid is output as an index value indicating the roundness of the spheroid. The index value obtained in this way indicates the roundness of the spheroid quantitatively and objectively.

According to the present invention, an index value that quantitatively represents the roundness of a spheroid can be obtained by performing a circular separability filter operation using an image obtained by imaging the spheroid.

The above and other objects and novel features of the present invention will become more fully apparent when the following detailed description is read with reference to the accompanying drawings. However, the drawings are for explanation only and do not limit the scope of the present invention.

1 is a diagram illustrating a schematic configuration of an imaging apparatus as an embodiment of an image processing apparatus. It is a 1st figure which shows typically the external appearance of a spheroid. It is a 2nd figure which shows the external appearance of a spheroid typically. It is a 3rd figure which shows the external appearance of a spheroid typically. It is a flowchart which shows operation | movement of this imaging device. The example of the original image obtained by imaging a well is shown. The 1st example of the image of the object area | region cut out from the original image is shown. The 2nd example of the image of the object area | region cut out from the original image is shown. The 3rd example of the image of the object area | region cut out from the original image is shown. It is a flowchart which shows the processing content of circular separation degree filter calculation. It is a 1st figure which shows the relationship between the shape of a spheroid, and a corresponding circular area | region. It is a 2nd figure which shows the relationship between the shape of a spheroid, and a corresponding circular area | region. It is a 3rd figure which shows the relationship between the shape of a spheroid, and a corresponding circular area | region. It is a figure which shows an example of a display image.

FIG. 1 is a diagram showing a schematic configuration of an imaging apparatus as an embodiment of an image processing apparatus according to the present invention. The imaging apparatus 1 is an apparatus that images spheroids (cell conglomerates) that are cultured in a liquid injected into a recess called a well W formed on the upper surface of a well plate WP. Hereinafter, in order to uniformly indicate the directions in the drawings, XYZ orthogonal coordinate axes are set as shown in FIG. Here, the XY plane represents a horizontal plane and the Z axis represents a vertical axis. More specifically, the (+ Z) direction represents a vertically upward direction.

The well plate WP is generally used in the fields of drug discovery and biological science, and a plurality of wells W are provided on the upper surface of a flat plate. The cross section of the well W is formed in a substantially circular cylindrical shape, and the bottom surface is transparent and flat. Although the number of wells W in one well plate WP is arbitrary, for example, 96 (12 × 8 matrix arrangement) can be used. The diameter and depth of each well W is typically about several mm. The size of the well plate and the number of wells targeted by the imaging apparatus 1 are not limited to these and are arbitrary. For example, those having 12 to 384 holes are generally used. Further, the imaging apparatus 1 can be used not only for well plates having a plurality of wells but also for imaging spheroids cultured in, for example, a flat container called a dish.

A predetermined amount of liquid as the medium M is injected into each well W of the well plate WP, and spheroids cultured in the liquid under predetermined culture conditions are imaging targets of the imaging apparatus 1. The medium M may be added with an appropriate reagent, or may be in a liquid state and gelled after being put into the well W. Commonly used liquid volume is about 50 to 200 microliters. As will be described later, the imaging apparatus 1 can target a spheroid cultured in the medium M, for example, on the inner bottom surface of the well W, as an imaging target.

The imaging device 1 includes a holder 11, an illumination unit 12, an imaging unit 13, and a control unit 14. The holder 11 abuts on the peripheral edge of the lower surface of the well plate WP that holds the sample together with the liquid in each well W and holds the well plate WP in a substantially horizontal posture. The illumination unit 12 is disposed above the holder 11. The imaging unit 13 is disposed below the holder 11. The control unit 14 includes a CPU 141 that controls operations of these units.

The illumination unit 12 emits appropriate diffused light (for example, white light) toward the well plate WP held by the holder 11. More specifically, for example, a combination of a white LED (Light-Emitting-Diode) light source as a light source and a diffusion plate can be used as the illumination unit 12. The spheroid in the well W provided in the well plate WP is illuminated from above by the illumination unit 12.

An imaging unit 13 is provided below the well plate WP held by the holder 11. In the imaging unit 13, an imaging optical system 131 is disposed immediately below the well plate WP, and the optical axis AX of the imaging optical system 131 is oriented in the vertical direction (Z direction).

The imaging unit 13 images the spheroid in the well W. Specifically, light emitted from the illumination unit 12 and incident on the liquid from above the well W illuminates the imaging target. Light transmitted downward from the bottom surface of the well W is incident on the light receiving surface of the image sensor 132 via the imaging optical system 131. An image of the imaging target imaged on the light receiving surface of the imaging element 132 by the imaging optical system 131 is captured by the imaging element 132. As the image sensor 132, for example, a CCD sensor or a CMOS sensor can be used, and either a two-dimensional image sensor or a one-dimensional image sensor may be used.

The imaging unit 13 can be moved in the XYZ directions by a mechanical control unit 146 provided in the control unit 14. Specifically, the mechanical control unit 146 moves the imaging unit 13 in the X direction and the Y direction based on a control command from the CPU 141. As a result, the imaging unit 13 moves in the horizontal direction with respect to the well W. Further, focus adjustment is performed by moving the imaging unit 13 in the Z direction. When the imaging object in one well W is imaged, the mechanical control unit 146 positions the imaging unit 13 in the horizontal direction so that the optical axis AX coincides with the center of the well W. When the imaging device of the imaging unit 13 is a one-dimensional image sensor, a two-dimensional image can be captured by scanning the imaging unit 13 in a direction orthogonal to the longitudinal direction of the image sensor. With such an imaging method, it is possible to perform non-contact, non-destructive and non-invasive imaging on a spheroid that is an imaging target. Therefore, damage to the spheroid due to imaging can be suppressed.

Further, when moving the imaging unit 13 in the XY direction, the mechanical control unit 146 moves the illumination unit 12 in the XY direction integrally with the imaging unit 13 as indicated by a dotted arrow in the drawing. That is, the illumination unit 12 is arranged so that the optical center thereof substantially coincides with the optical axis AX of the imaging unit 13, and moves in the XY direction in conjunction with the imaging unit 13 moving in the XY direction. Thereby, no matter which well W is imaged, the center of the well W and the light center of the illumination unit 12 are always located on the optical axis of the imaging unit 13. Therefore, it is possible to maintain the imaging condition favorably while keeping the illumination condition for each well W constant.

The image signal output from the image sensor of the imaging unit 13 is sent to the control unit 14. That is, the image signal is input to an AD converter (A / D) 143 provided in the control unit 14 and converted into digital image data. The CPU 141 executes various image processing based on the received image data. The control unit 14 further includes an image memory 144 for storing and storing image data, and a memory 145 for storing and storing programs to be executed by the CPU 141 and data generated by the CPU 141. These may be an integral memory. The CPU 141 executes various control processes described later by executing a control program stored in the memory 145.

Besides, the control unit 14 is provided with an interface (IF) unit 142. The interface unit 142 receives an operation input from the user and presents information such as a processing result to the user. The interface unit 142 exchanges data with an external device connected via a communication line. Note that the control unit 14 may be a dedicated device including the hardware described above. Further, a control unit 14 that incorporates a control program for realizing a processing function described later in a general-purpose processing device such as a personal computer or a workstation may be used. That is, a general-purpose computer device can be used as the control unit 14 of the imaging device 1. In the case of using a general-purpose processing device, it is sufficient that the imaging device 1 has a minimum control function for operating each unit such as the imaging unit 13.

The imaging device 1 configured as described above has a function of imaging a spheroid cultured in the well W and a function of calculating the roundness of the spheroid based on the captured image. The “roundness” here is an index value that quantitatively indicates how close the spheroid has a circular shape in the captured image.

2A to 2C are diagrams schematically showing the appearance of the spheroid. The spheroid Sp1 shown in FIG. 2A is an example composed of cells with relatively high viability (viability, cell activity). As shown in the figure, in the spheroid Sp1 with high viability, a shape close to a sphere is formed by a large number of cells C. Therefore, in the two-dimensional image obtained by imaging the spheroid Sp1, the outline of the spheroid Sp1 is substantially circular.

On the other hand, when a part of the cells constituting the spheroid is weakened or killed, the binding force between the cells is weakened and the contour becomes indefinite, like the spheroid Sp2 shown in FIG. 2B. Thus, how close the imaged spheroids are to a circle or how far apart is important information for evaluating the activity of cells constituting the spheroids. Therefore, an index value that quantitatively represents how close the spheroid in the image is to a perfect circle is useful.

In the conventional technology, “roundness” or “circularity” is generally used as an index value indicating how close the contour of the imaged object is to a circle, that is, the “roundness” of the spheroid. In these methods, the object has a shape that is close to a circle, depending on the ratio of the radius of the circle that circumscribes the contour of the object or the ratio of the area of the object and the circumference of the contour. Is expressed.

However, spheroids are a collection of many cells. Therefore, as shown in FIG. 2A, even if the spheroid Sp1 appears to be approximately circular, microscopically, irregularities due to the surfaces of the individual cells C appear in the outline. Therefore, there are cases where the “roundness” of the spheroid cannot be appropriately represented by an index value using roundness or circularity. In addition, since an actual spheroid has a three-dimensional shape, as shown in FIG. 2C, a shadow caused by illumination light may cause the spheroid Sp1 image to be shaded. In this case, the contour of the spheroid cannot be appropriately extracted, and as a result, the roundness or circularity obtained for the spheroid may have an inappropriate value.

In this embodiment, in order to improve the above-described problems of the prior art, the “roundness” of the spheroid is introduced as a new index value. Hereinafter, the operation of the imaging apparatus 1 from imaging of the spheroid to calculation of the roundness will be described.

FIG. 3 is a flowchart showing the operation of this imaging apparatus. This operation is realized by causing the CPU 141 of the control unit 14 to execute a control program prepared in advance in accordance with a user instruction and causing each unit of the imaging apparatus 1 to perform a predetermined operation defined by the program. When the well plate WP on which the sample containing spheroids is cultured is set on the holder 11 by the user or the transport robot (step S101), the imaging unit 13 images the well W (step S102). By performing imaging a plurality of times as necessary, each well W is imaged and an original image is acquired.

The object region having characteristics different from the background region is extracted from the captured original image by image processing (step S103). Various methods for extracting an object region from an image by image processing are known, and an appropriate method can be applied in this embodiment as well. For example, an area in which pixel values of pixels constituting the original image are in a predetermined range can be set as an object area. Further, for example, an area surrounded by edges extracted by edge extraction can be set as an object area. As will be described later, step S103 may be omitted.

Next, a setting input for a target region to be subjected to the following processing is received from the captured original image (step S104). The setting input is given to the control unit 14 when the user operates a keyboard, a mouse, a tablet, or the like as an input device provided in the interface unit 142, for example.

4A to 4D are diagrams schematically showing examples of images. More specifically, FIG. 4A shows an example of an original image obtained by imaging the well W. 4B to 4D show examples of images of target regions cut out from the original image. As shown in FIG. 4A, spheroids are distributed at various locations in the well W in the original image. Here, seven spheroids Sp3 to Sp9 are included, but the number of spheroids varies, and of course, one may be sufficient.

The original image can include spheroids having various characteristics. For example, spheroids having a shape close to a circle (Sp4 to Sp8), spheroids having a more irregular shape (Sp3, Sp9), spheroids (Sp4 to Sp6) that are close to each other, and the like may be included.

The user designates a partial region including a spheroid for which the degree of roundness is to be obtained from such an original image as a target region. Then, a partial image in the target region is cut out from the original image, and a circular separation degree filter calculation is executed for the partial image (step S105).

The target area is preferably selected so that a partial image obtained by cutting out the target area from the original image includes one spheroid for which the roundness is to be obtained. For example, spheroids Sp8 and Sp9 have a relatively large distance from other spheroids and are isolated. In such a case, as shown in FIGS. 4B and 4C, it is preferable that the target region is set so that the partial image P includes the entire spheroid and the surrounding background region. On the other hand, in the spheroid Sp5, there are other spheroids in the vicinity. In such a case, as shown in FIG. 4D, it is preferable that the target region is selected so that the partial image P includes the entire spheroid and other spheroids are excluded as much as possible.

Note that although the target area is designated by the user here, the target area may be automatically set by the imaging apparatus 1 instead of or in addition to this. That is, when the object area is extracted from the original image in step S103, the position and size of the object area in the original image can be known. Therefore, it is possible to automatically set the target area so as to include the extracted object area.

全 て Not all extracted object areas are spheroids, and spheroids that do not need to be evaluated may be included. By making it possible to set the target area by the user's instruction input, unnecessary calculation processing can be omitted and the processing time can be shortened. For the same reason, the circular separation degree filter calculation is not performed on the entire original image, but the calculation is performed on the partial image P including the spheroid and its surroundings.

FIG. 5 is a flowchart showing the processing contents of the circular separation degree filter calculation in this embodiment. First, the average value of the pixel values included in the partial image corresponding to the designated target area, that is, the average pixel value is calculated (step S201). Then, a difference image is created in which the absolute value of the difference between the pixel value of each pixel of the original partial image and the average pixel value is the pixel value of the pixel (step S202).

Note that the difference image is a virtual intermediate image used for subsequent processing. It is only necessary to obtain the pixel value of each pixel in the difference image, and it is not necessary to actually create or output an image. In the difference image, the contrast between the object region and the other background region may be smaller than that of the original image. However, for the purpose of performing the circular separation degree filter calculation, there is an advantage that the influence of density unevenness in each of the background area and the object area can be reduced by using the difference image with respect to the average pixel value.

Next, the center coordinate position and diameter of a virtual circle are provisionally set to appropriate values as parameters for calculating the degree of separation (step S203). If the object area extracted in step S103 is present in the target area, the position and size are known in advance, and parameter setting can be performed based on the information. That is, it is only necessary to set a circle whose center position and size are equal to those of the object area. The parameter setting value may be given by the user.

The degree of separation is calculated for a circle in the target area specified by the set center coordinate position and diameter (step S204). The degree of separation is calculated based on the pixel value of a pixel included in an annular outer region adjacent to the outside of the circle and the pixel value of a pixel included in an annular inner region adjacent to the inside of the circle. Is required. The width of these annular regions can be, for example, several pixels.

Steps S203 and S204 are a loop process, and the calculation of the degree of separation is repeated while variously changing the combination of the center position and the diameter of the circle. The calculation may be performed for all circles that can be drawn in the target area. However, when the position and size of the object area in the target area are known, the center position of the circle and the change range of the diameter can be limited based on the information. By doing so, the processing time can be significantly shortened compared with the case where the calculation is performed for all combinations of the center coordinate position and the diameter.

When the degree of separation is obtained for all the circles to be set, the loop process ends (step S205). The maximum value of the degree of separation thus obtained (hereinafter referred to as “maximum degree of separation”) is stored in the memory 145 together with the center position and diameter of the circle having the maximum value (step S206). As a result, a circle having the maximum degree of separation in the target region is specified.

The circle thus identified can be presumed to schematically indicate the outline of the spheroid existing in the target region. That is, there is a high probability that a circular region having the circle as an outer periphery is a spheroid region in the target region. In particular, when the spheroid has a shape close to an ideal circle, the circular region should substantially match the spheroid region.

6A to 6C are diagrams showing the relationship between the shape of the spheroid and the corresponding circular region. For example, when the spheroid Sp has a substantially circular shape like the spheroid Sp8 shown in FIG. 4A, the obtained circular region CR and the actual spheroid Sp region substantially coincide as shown in FIG. 6A. . For this reason, the obtained value of the maximum degree of separation is relatively large.

On the other hand, for example, when the shape of the spheroid Sp is distorted like the spheroid Sp9 shown in FIG. 4A, the difference between the obtained circular region CR and the actual spheroid Sp region is relatively large as shown in FIG. 6B. . The value of the maximum degree of separation obtained for a circle having a large deviation from the contour of the spheroid Sp is relatively small.

As shown in FIG. 6C, when a plurality of spheroids Sp are included in the target region, the value of the maximum separation degree is further reduced in the circular separation degree filter calculation using a single circle. The value cannot be said to appropriately index any attribute of the spheroid included in the target region. From this point, as shown in FIG. 4D, it is desirable that the target region is set so as to mainly include a single spheroid.

As described above, by performing the circular separability filter operation and detecting the circular region CR having the maximum separability, it is possible to specify the region in which the spheroid is estimated to exist in the target region. Furthermore, the value of the degree of separation in the circular region increases as the spheroid is closer to a circle. Accordingly, for example, a value proportional to the value of the maximum separation degree, which takes a larger value as the maximum separation degree increases, can be used as an index value representing the roundness of the spheroid. In the index value of the roundness based on the degree of separation, if the envelope shape of the spheroid is substantially circular, the influence of fine irregularities on the outer periphery is less likely to appear.

Referring back to FIG. 3, the operation of this device will be continued. In step S105, when a circle having the maximum degree of separation in the target area is specified, the roundness of the spheroid in the target area is calculated based on the value of the maximum degree of separation (step S106). As the roundness, a value proportional to the value of the maximum separation can be used. For example, a value obtained by normalizing the value of the maximum degree of separation as it is or multiplying by a predetermined proportionality coefficient can be set as the value of the roundness. In addition, the degree of roundness may be quantified by an appropriate calculation formula that monotonously increases with respect to the value of the maximum degree of separation.

In addition, the value of the maximum degree of separation obtained by the above calculation tends to increase as the concentration of the spheroid region increases if the concentration of the background region is the same. Therefore, when comparing the roundness of spheroids having greatly different concentrations, it is not appropriate to use the value of maximum separation as an index value as it is. In order to avoid this problem, for example, a value obtained by dividing the value of the maximum separation by the average pixel value in the spheroid region and reducing the influence of the density difference may be used as the roundness.

The roundness of the spheroid thus obtained is output in a predetermined format and presented to the user. For example, the user can be notified by displaying the result on a display (output device) provided in the interface unit 142 (step S107).

FIG. 7 is a diagram showing an example of a display image. The display mode is not limited to that shown here, but is arbitrary. Further, the roundness calculation result may be output in a manner other than being displayed on the display device in this way, for example, transmitted and output to an external recording medium or an information processing device.

The original image is displayed in the box area B1 of the display image IM, and the range of the set target area is indicated by a dotted line. In the box area B2, an enlarged image of the target area is displayed. In addition, a circular region corresponding to the maximum degree of separation specified by the circular degree of separation filter calculation is indicated by a dotted line. In the box area B3, the calculation results of the center coordinate position, the diameter, and the roundness of the spheroid of the circular area are displayed as text. Thus, the roundness is calculated for one target area, and the result is displayed.

Referring back to FIG. When the processing is completed for one target area, it is determined whether another target area is set and calculation of the roundness in that area is to be continued (step S108). If it is necessary to continue the process, the process returns to step S104 and the above process is repeated.

When the target area is specified by the user, the user can be inquired whether to continue the process. When the target area is automatically set, if there is an extracted object area whose roundness has not yet been calculated, the process for the object area is continued. Then, if the roundness calculation processing has been completed for all object regions, the processing may be terminated.

As described above, in the imaging device 1 of this embodiment, the circular separation degree filter calculation is performed on the target region designated to include the spheroids in the original image obtained by imaging. A value proportional to the value of the separation degree when the separation degree is maximized is output as an index value indicating the roundness of the spheroid in the target region. By doing so, it is possible to quantitatively and objectively express how close the spheroid included in the image has a circular shape.

Even a spherical spheroid with high cell activity has small irregularities on its surface due to individual cells. In the “roundness” obtained by the present embodiment, the influence of such unevenness is reduced. In addition, the conventional technique for expressing roundness by roundness or circularity has a problem in that an accurate value cannot be obtained unless the spheroid contour can be properly specified in advance. On the other hand, in the calculation method of the present embodiment, the roundness can be calculated without specifying the contour in advance. In this sense, the roundness can be calculated even if step S103 in FIG. 3 is omitted. In particular, when it is clear that one spheroid is included in the target region, it is possible to omit specifying the contour.

In the present embodiment, the circular separability filter calculation is not used for the purpose of searching a circular area from the original image. That is, on the premise that the position of the object corresponding to the spheroid is specified to some extent, the circular separability filter calculation is used to obtain the maximum value of the separability within the target area including this object.

The roundness of the spheroid obtained in this way becomes a large value when the envelope shape of the spheroid is close to a circle, and becomes a smaller value as it deviates from the circle. Since the deviation from the spheroid-shaped circle is mainly caused by the weakening of the spheroid-forming cells, the roundness value can be used for the evaluation of spheroid activity.

As described above, in this embodiment, the imaging device 1 functions as the “image processing device” of the present invention. The imaging unit 13 functions as the “image acquisition unit” of the present invention, the CPU 141 functions as the “detection unit” of the present invention, and the interface unit 142 functions as the “output unit” and the “accepting unit” of the present invention.

Note that the present invention is not limited to the above-described embodiment, and various modifications other than those described above can be made without departing from the spirit of the present invention. For example, the image processing apparatus of the above embodiment includes the imaging unit 13 as the “image acquisition unit” of the present invention. However, an image processing apparatus that does not include an imaging function may be used. That is, the present invention is implemented in such a manner that the interface unit 142 in the control unit 14 of the above embodiment acquires an original image by receiving image data from the outside, and the roundness is calculated using the original image. May be. In this case, the interface unit 142 functions as an “image acquisition unit”, and an imaging function is not required.

In the above embodiment, the present invention is implemented by the CPU 141 executing the control program stored in the memory 145. As described above, a general-purpose computer device can be used as the control unit 14 in this embodiment. Therefore, the present invention may be provided to the user as a control program for causing the computer apparatus to execute the above-described processing on the premise that the program is read by such a computer apparatus, and in a mode in which the program is recorded on an appropriate recording medium. Is possible. Accordingly, for example, it is possible to add a new function to an imaging apparatus that is already in operation. Further, a general-purpose computer device can function as the “image processing device” of the present invention.

As described above, the specific embodiment has been described by way of example, and in the circular separation degree filter calculation according to the present invention, at least one of the center position and the diameter in the target region is changed and set in a plurality of stages. The degree of separation may be determined. By doing so, it is possible to accurately select a circle having the maximum degree of separation from various circles that can be set in the target region.

In addition, for each pixel in the target area, a difference between the pixel value of the pixel and the average pixel value of the target area is obtained, and a circular separation filter operation is performed on the difference image using the difference as the pixel value of the pixel. Also good. By doing so, it is possible to reduce the influence of density unevenness in the object region and the background region in the target region on the calculation result of the separation degree.

Also, a value obtained by normalizing the value of the degree of separation of the circular area with the average pixel value of the pixels in the circular area may be used as an index value indicating roundness. When the concentration is different for each spheroid, the value of the degree of separation is different even if the spheroids have the same roundness. This problem can be solved by normalizing the degree of separation with the average pixel value of the circular area corresponding to the spheroid.

Further, it may be configured such that a setting input for specifying the target area from the original image is received from the user, and the target area is specified according to the setting input. By doing so, it is possible to preferentially process an object that the user desires to evaluate and present the calculation result to the user in a short time.

Further, a step of detecting an object corresponding to the spheroid from the original image by image processing may be provided, and the target region may be specified based on the detection result. By doing so, it is possible to automatically perform calculation for each object in the original image regardless of user input.

Further, in the image processing apparatus according to the present invention, the image acquisition means may have a configuration having an imaging unit that captures a sample and acquires an original image. In the image processing apparatus of the present invention, the original image may be given from the outside, but by providing the imaging unit, it is possible to consistently perform from the imaging of the original image to the calculation of the roundness of the spheroid.

Although the invention has been described with reference to specific embodiments, this description is not intended to be construed in a limiting sense. Reference to the description of the invention, as well as other embodiments of the present invention, various modifications of the disclosed embodiments will become apparent to those skilled in the art. Accordingly, the appended claims are intended to include such modifications or embodiments without departing from the true scope of the invention.

The present invention is suitable for use in evaluating spheroids for the purpose of observation, analysis, and the like, and can be used for various experiments in the medical and biological science fields, for example.

1 Imaging device (image processing device)
13 Imaging unit (image acquisition means)
14 control unit 141 CPU (detection means)
142 Interface unit (output means, accepting means)
CR circular region Sp, Sp1 to Sp9 Spheroid W well WP well plate

Claims (10)

Identifying a target region including an object corresponding to the spheroid from an original image obtained by capturing the spheroid;
A step of detecting a circular region having a maximum value of the degree of separation within the target region by a circular separation degree filter calculation;
And outputting a value proportional to the degree of separation of the circular area calculated by the circular degree of separation filter calculation as an index value indicating the roundness of the spheroid specified by the circular area. The image processing method according to claim 1, wherein, in the circular separation degree filter calculation, at least one of a center position and a diameter in the target region is changed and set in a plurality of stages, and the separation degree is obtained for each setting. For each pixel in the target region, a difference between a pixel value of the pixel and an average pixel value of the target region is obtained, and the circular separation degree filter calculation is performed on a difference image using the difference as a pixel value of the pixel. Item 3. The image processing method according to Item 1 or 2. 4. The image processing method according to claim 1, wherein the index value is a value obtained by normalizing a value of the degree of separation of the circular area with an average pixel value of pixels in the circular area. 5. The image processing method according to claim 1, wherein a setting input from a user for specifying the target area is received, and the target area is specified according to the setting input. 5. The image processing method according to claim 1, further comprising a step of detecting an object corresponding to the spheroid from the original image by image processing, and the target region is specified based on the detection result. A control program for causing a computer to execute each step of the image processing method according to any one of claims 1 to 6. Image acquisition means for acquiring an original image in which a spheroid is imaged;
In the target area including the object corresponding to the spheroid in the original image, detection means for executing a circular separability filter operation and detecting a circular area where the separability value is maximum,
An image processing apparatus comprising: an output unit configured to output a value proportional to the degree of separation of the circular area calculated by the circular degree of separation filter calculation as an index value indicating the roundness of the spheroid specified by the circular area. The image processing apparatus according to claim 8, further comprising a receiving unit that receives a setting input for specifying the target area from the original image from a user. The image processing apparatus according to claim 8 or 9, wherein the image acquisition unit includes an imaging unit that captures an image of a sample and acquires the original image.

Images