JP2016067280A - Image processing method, control program, recording medium, and image processing device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a technique capable of discriminating between a spheroid and a foreign object efficiently and with excellent reproducibility by conducting image processing to an image formed by imaging the spheroid.SOLUTION: An image processing method comprises: a process of detecting, regarding an original image formed by imaging a spheroid, a spheroid-like object corresponding to the spheroid out of objects in the original image; a process of executing, regarding the spheroid-like object, an erosion process using a circle, which has a diameter obtained based on the size of the spheroid-like object and a smaller area than the spheroid-like object, as a structural element; and a process of executing dilation process using the structural element regarding the spheroid-like object after the erosion process.SELECTED DRAWING: Figure 4

Description

  The present invention relates to image processing performed on an image obtained by capturing spheroids, and more particularly to a technique for distinguishing spheroids included in an image from other objects.

  In medical and biological science experiments, for example, a cell to be observed is imaged with a CCD camera or the like and converted into data, and various image processing techniques are applied to the image data for observation and analysis. It is becoming. That is, the cells are imaged by a camera or the like and used for observation in a state where the cells to be observed are held in an appropriate container called a well plate or dish.

  In this case, an image other than the observation object may appear in the image. For example, an image of streaky foreign matter caused by fine dust or dirt mixed in the atmosphere, scratches or dirt on the container may appear in the image. Such an image of a foreign substance hinders observation. As a technique considering this problem, there is one described in Patent Document 1, for example. In this technology, an image of a container (well) is captured in advance without carrying cells, and a difference from the image in the state where cells are present is taken to eliminate the image of foreign matter such as scratches and dust. doing.

JP 2012-159794 A

  In the above prior art, it is necessary to image the container before and after loading the cells, and the procedure is complicated. In addition, it is not possible to deal with foreign matter that enters the container or leaves the container after the pre-imaging. For this reason, the technique which can distinguish the image of a cell and the image of a foreign material directly from the image imaged in the state containing a cell and a foreign material is desired. In particular, when the object to be imaged is a cell agglomeration (spheroid) made up of a large number of cells, it is expected to be distinguished from the image of a foreign object by utilizing the property that the cells aggregate in a spherical shape. Has not been established so far.

  This invention is made in view of the said subject, and provides the technique which can perform the distinction with a spheroid and a foreign material efficiently and with the outstanding reproducibility by performing image processing to the image which imaged the spheroid. The purpose is to do.

  In order to achieve the above object, an image processing method according to the present invention detects a spheroid-like object corresponding to the spheroid in an object region in the original image for an original image in which a spheroid is imaged, and the spheroid Performing a erosion process using a circle having a diameter obtained based on the size of the spheroid-like object and having a smaller area than the spheroid-like object as a structural element, and the spheroid-like object after the erosion process And a step of executing a duration process using the structural element for the object.

  Here, the “erosion process” is one of image morphological processes and means a process of contracting an object area in an image. Further, “dilation processing” is one of morphological processing and means processing for expanding an object region in an image.

  In the invention configured as described above, the spheroid region and the foreign material region are distinguished by morphological image processing including erosion processing and delation processing. Since the spheroid forms a substantially circular image in the original image, it can be distinguished from the image of the streak-like foreign material based on its shape characteristics. If the spheroid image and the foreign object image are separated from each other in the original image, the distinction between them is relatively easy. In reality, however, there are many cases in which the two overlap each other.

  Therefore, in the present invention, by executing the erosion process and the duration process, it is possible to obtain an image in which the streak image is erased while maintaining the shape of the spheroid in the original image. Here, in these morphological processes, the size setting of the structural element greatly affects the processing result. In the conventional cell image processing, the size of the structural element is set by subjective judgment and trial of the operator. However, skill is required to set the optimum size, and the processing result may vary. On the other hand, in the present invention, a circle having a diameter corresponding to the size of the detected spheroid-like object is used as the structural element. According to the new knowledge obtained by the inventor of the present application, the size of the structural element can be easily and automatically set in this way, and the spheroid and foreign matter can be efficiently distinguished by morphological processing. It becomes possible.

  In the present invention, when there are a plurality of spheroid-like objects having different sizes, it is preferable that a structural element having a different size is set for each. The original image can contain spheroids of various sizes, but by applying structural elements according to the individual sizes, the effect of removing foreign matter images in morphological processing and the shape reproducibility of the spheroid images are improved. be able to.

  The size of the spheroid-like object can be represented by the size of an approximate ellipse that approximates the spheroid-like object, for example. Although the spheroid image is generally circular, there is a disorder in the shape of the periphery, so it is not always easy to specify the size exactly, and a simpler alternative method is required. For example, the size of the spheroid-like object may be the size of an ellipse that approximately represents the spheroid-like object. Thereby, the size of the structural element can be determined by a simple calculation.

  Specifically, for example, an ellipse having the largest area among the ellipses included in the outline of the spheroid-like object can be set as the approximate ellipse. Alternatively, an ellipse having the same area as the spheroid-like object can be used as an approximate ellipse. Image processing algorithms corresponding to these methods for obtaining approximate ellipses have been put into practical use, and the processing can be simplified by applying them.

  In these cases, the diameter of the circle as the structural element is not less than (1/6) and not more than (1/2), more preferably not less than (1/4) and not more than (1/3) of the minor axis of the approximate ellipse. It may be configured as follows. According to the experiment by the present inventor, it has been found that when the size of the structural element is within the above range, a favorable processing result can be obtained. That is, it is possible to effectively remove the streak foreign object image while maintaining the shape reproducibility of the spheroid image.

  For example, the approximate ellipse may be an approximate circle that approximates a spheroid-like object. Since the spheroid is substantially circular, the size can be expressed with necessary and sufficient accuracy by circular approximation. In addition, the processing becomes simpler than when an approximate ellipse is used.

  In addition, the present invention may include a step of detecting a non-spheroid region different from a spheroid among objects in the original image based on, for example, a difference image between the image after the dilation processing and the original image. By performing the erosion process and the delation process, the image of the streak-like foreign matter is erased and the spheroid image remains, but by taking the difference between the processed image and the original image, the spheroid image is erased and the foreign matter is removed. It is also possible to extract an image.

  In addition, for example, the method includes a step of identifying a minimum inclusion circle of the spheroid-like object detected in the original image, and the ratio of the area of the minimum inclusion circle of the spheroid-like object to the area of the spheroid-like object among the spheroid-like objects is A configuration in which the erosion process and the duration process are executed for a value larger than a predetermined value greater than 1 may be used. By doing so, spheroid-like objects that originally do not contain foreign objects and do not require processing can be excluded from processing, and the original image information can be stored.

  These image processing methods can be executed using a general-purpose computer. Therefore, the present invention may be provided to a user as a control program for causing a computer to execute the above processing.

  In order to achieve the above object, an image processing apparatus according to the present invention includes an image acquisition unit that acquires an original image obtained by capturing a spheroid, and an image processing unit that performs image processing on the original image. The processing means executes the image processing described above. In the invention configured as described above, by performing the above-described image processing, it is possible to easily and efficiently distinguish the spheroid image and the foreign object image included in the original image.

  In the present invention, the image acquisition means may include a holding unit that holds a carrier that carries spheroids, and an imaging unit that images the carrier and creates an original image. Thereby, an original image can be acquired about various spheroids.

  According to the present invention, the structural element according to the size of the spheroid-like object detected in the original image is applied to the morphological processing, so that the spheroid and the foreign object can be efficiently distinguished.

1 is a side view showing a schematic configuration of an image processing apparatus according to the present invention. It is a figure which shows the principle of the area | region separation process in this embodiment. It is a flowchart which shows the image processing in this embodiment. It is a flowchart which shows an area | region separation process. It is a figure which shows typically the process of the image in a region separation process.

  FIG. 1 is a side view showing a schematic configuration of an image processing apparatus according to the present invention. The image processing apparatus includes an imaging unit 1 that images a sample in a liquid injected into a recess called a well W formed on an upper surface of a well plate WP, and an image that performs image processing on the captured image. And a processing unit 2.

  The well plate WP is generally used in the fields of drug discovery and biological science. The well plate WP is formed in a cylindrical shape with a substantially circular cross section on the top surface of a flat plate, and the bottom surface is transparent and flat. Are provided. 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. Note that the size of the well plate and the number of wells targeted by the imaging unit 1 are not limited to these and may be arbitrary, for example, 384 holes.

  A predetermined amount of a liquid as a medium is injected into each well W of the well plate WP, and cells, bacteria, and the like cultured in the liquid under predetermined culture conditions become imaging targets of the imaging unit 1. The medium may be one to which an appropriate reagent has been added, or it may be in a liquid state and gelled after being placed in the well W.

  The imaging unit 1 is in contact with 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, and illumination disposed above the holder 11. The unit 12, the imaging unit 13 disposed below the holder 11, and a control unit 14 that controls the operation of each unit.

  The illumination unit 12 emits illumination light Li toward the well plate WP held by the holder 11. For example, white light can be used as the illumination light Li. The illumination unit 12 illuminates the sample in the well W provided on the well plate WP from above.

  An imaging unit 13 is provided below the well plate WP held by the holder 11. In the imaging unit 13, an objective lens 131 is disposed immediately below the well plate WP. The optical axis OA of the objective lens 131 is oriented in the vertical direction, and an aperture stop 132, an imaging lens 133, and an imaging device 134 are further provided in order from top to bottom along the optical axis OA of the objective lens 131. ing. The objective lens 131, the aperture stop 132, and the imaging lens 133 are arranged so that their centers are aligned in a line along the vertical direction, and these constitute the imaging optical system 130 as a unit. In this example, the respective parts constituting the imaging unit 13 are arranged in a line in the vertical direction, but the optical path may be folded by a reflecting mirror or the like.

  The imaging unit 13 can be moved by a mechanical drive unit 141 provided in the control unit 14. Specifically, the mechanical drive unit 141 moves the objective lens 131, the aperture stop 132, the imaging lens 133, and the imaging device 134 that form the imaging unit 13 integrally in the horizontal direction, so that the imaging unit 13 becomes the well. Move horizontally with respect to W. When the imaging target in one well W is imaged, the mechanical drive unit 141 positions the imaging unit 13 in the horizontal direction so that the optical axis of the imaging optical system 130 coincides with the center of the well W.

  Further, the mechanical drive unit 141 performs focusing of the imaging unit with respect to the imaging target by moving the imaging unit 13 in the vertical direction. Specifically, the mechanical drive unit 141 includes the objective lens 131, the aperture stop 132, the imaging lens 133, and the imaging device so that the objective lens 131 is focused on the inner bottom surface of the well W where the sample that is the imaging target is present. 134 is moved up and down integrally.

  Further, the mechanical drive unit 141 moves the illumination unit 12 in the horizontal direction integrally with the imaging unit 13 when moving the imaging unit 13 in the horizontal direction. That is, the illuminating unit 12 is arranged so that the optical center thereof substantially coincides with the optical axis OA of the imaging optical system 130, and interlocks with the imaging unit 13 including the objective lens 131 when it moves in the horizontal direction. To move horizontally. As a result, regardless of which well W is imaged, the illumination condition for the well W can be kept constant and the imaging condition can be maintained well.

  A sample in the well W is imaged by the imaging unit 13. Specifically, light emitted from the illumination unit 12 and incident on the liquid from above the well W illuminates the object to be imaged, and light transmitted downward from the bottom surface of the well W is collected by the objective lens 131 and the aperture stop 132. The image of the imaging object is finally formed on the light receiving surface of the imaging device 134 via the imaging lens 133, and this is received by the light receiving element 1341 of the imaging device 134. The light receiving element 1341 is a one-dimensional image sensor, and converts a one-dimensional image of the imaging target imaged on the surface thereof into an electrical signal. As the light receiving element 1341, for example, a CCD sensor can be used. The light receiving element 1341 scans and moves relative to the well plate WP, whereby a two-dimensional image of the well W is obtained.

  The image signal output from the light receiving element 1341 is sent to the control unit 14. That is, the image signal is input to an AD converter (A / D) 142 provided in the control unit 14 and converted into digital image data. The digital image data obtained in this way is output to the outside via an interface (I / F) 143.

  The image processing unit 2 includes a control unit 20 having a CPU 201 that controls the operation of each unit of the apparatus. The control unit 20 also stores and saves a graphic processor (GP) 202 responsible for image processing, an image memory 203 for storing and saving image data, programs to be executed by the CPU 201 and GP 202, and data generated by these. And a memory 204. Note that the CPU 201 may also function as the graphic processor 202. Further, the image memory 203 and the memory 204 may be integrated.

  In addition, the control unit 20 is provided with an interface (I / F) 205. The interface 205 is responsible for exchanging information with the user and an external device. Specifically, the interface 205 is connected to the interface 143 of the imaging unit 1 via a communication line, and the CPU 201 transmits a control command for controlling the imaging unit 1 to the imaging unit 1. In addition, image data output from the AD converter 142 of the imaging unit 1 is received.

  The interface 205 is connected to an input device 21 such as an operation button, an input device such as a mouse, a keyboard, or a tablet, or a combination thereof. An operation input from the user received by the input unit 21 is transmitted to the CPU 201 via the interface 205. Further, a display unit 22 having a display device such as a liquid crystal display is connected to the interface 205. The display unit 22 presents information such as processing results to the user by displaying an image corresponding to an image signal given from the CPU 201 via the interface 205.

  The image processing unit 2 having the above-described configuration is substantially the same as the configuration of a general personal computer. That is, a general-purpose computer device can be used as the image processing unit 2 of the image processing device.

  Next, the operation of the image processing apparatus configured as described above will be described. In this image processing apparatus, it is possible to image cells, bacteria, cell clumps (spheroids), etc. cultured in each well W of the well plate WP, and the captured images can be used for various observations and analysis. Can be provided. Here, a case is considered in which this image processing apparatus is applied to the purpose of imaging spheroids cultured in the culture solution injected into the well W.

  The spheroids in the culture solution have a substantially spherical shape, and a two-dimensional image obtained by imaging the spheroid forms a substantially circular image. When a plurality of spheroids exist in the well W, each has a substantially circular image, but the shape and size are not necessarily the same. On the other hand, in the captured image, in addition to the above-described spheroid image, streaks caused by foreign matters such as fibrous dust and dirt mixed in the culture medium or attached to the surface, and scratches and dirt on the bottom surface of the well W May be reflected.

  Since the shape characteristics of the spheroid image and the foreign object image are greatly different, it is considered that the discrimination is relatively easy. However, it is not easy to distinguish between the two areas when the images are captured in a state where the two overlap each other. Hereinafter, the image processing method of the present embodiment that enables this will be described.

  FIG. 2 is a diagram showing the principle of region separation processing in the present embodiment. This area separation process is a process for separating an object in an image in which a spheroid image and a foreign object image are integrated into a spheroid area and a foreign object area. FIG. 2A is a schematic diagram illustrating an example of an image in which a spheroid and a foreign object are overlapped. An image object OB captured by overlapping a spheroid and a foreign object has a spheroid area Ra having a shape close to a circle corresponding to the spheroid and a foreign object area Rb corresponding to a streak-like foreign object connected to the spheroid. In addition, as shown to the same figure, the some foreign material area | region Rb may contact | connect one spheroid area | region Ra. In the schematic diagram shown here, the difference between the spheroid region Ra and the foreign matter region Rb is obvious, but in the actual image, the boundary between the spheroid region Ra and the foreign matter region Rb in the object OB is determined from the image content. It ’s hard.

  As a method of separating the spheroid area Ra and the foreign substance area Rb from such an image object OB, it is conceivable to apply image morphological processing. That is, as shown in FIG. 2B, an erosion process using a circle C1 having a predetermined diameter De as a structural element is performed to contract the image object. Thereby, the foreign substance region Rb having a relatively long and narrow shape is erased from the image, and the region Rc formed by erasing the peripheral portion of the spheroid region Ra remains. Next, as shown in FIG. 2C, a deletion process is performed in which a circle C2 having the same diameter De as that in the case of the erosion process is used as a structural element, thereby obtaining a region Rd in which the region Rc is expanded. This region Rd can be regarded as the foreign object region Rb removed from the original image object OB, and generally has the same shape as the spheroid region Ra.

  Since the morphological processing of the image is known, a detailed description is omitted. However, in order for such morphological processing to function effectively, it is necessary to appropriately set the sizes (specifically, the diameter De) of the circles C1 and C2 that are the structural elements. That is, if the diameter De of the structural element is too small, the foreign substance region Rb may remain without being sufficiently erased. In addition, when the diameter De of the structural element is too large, the shape reproducibility of the spheroid region Ra is deteriorated, and the small spheroid region Ra may be erased in the erosion process.

  In the conventional technology, in order to solve the above problems and to make the morphological processing function effectively, the size of the structural element is determined by subjective judgment of an expert, or the size is changed in multiple stages. To find the optimal size. However, such work requires a great deal of labor, and variations in processing results are unavoidable. In particular, when there are a plurality of image objects to be processed, such a manual response is not realistic.

  In view of this problem, the present inventor conducted experiments using various cells forming spheroids and obtained the following knowledge. That is, for an image object having a spheroid region Ra and a foreign object region Rb associated therewith, by applying a morphological process using a structural element having a size calculated based on a predetermined rule from the size of the spheroid region Ra, the spheroid region Ra and the foreign substance region Rb can be distinguished well.

  More specifically, for example, when the spheroid region Ra is regarded as a circle, an erosion process and a delation are performed using a circle having a diameter not less than (1/4) and not more than (1/3) of the diameter of the circle as a structural element. Process in order. By doing so, the shape reproducibility of the spheroid region Ra is good, and it is possible to effectively erase the spheroid region Ra while suppressing the remaining of the foreign material region Rb. Hereinafter, the image processing of the present embodiment for distinguishing the spheroid region and the foreign material region based on such knowledge will be specifically described with reference to FIGS. 3 to 5.

  FIG. 3 is a flowchart showing image processing in this embodiment. FIG. 4 is a flowchart showing the region separation process. Further, FIG. 5 is a diagram schematically showing an image processing process in this processing. This process is realized by the CPU 201 executing a stored control program stored in advance in the memory 204 to control each part of the apparatus. Among these, various processes for the image are executed by the graphic processor 202, but at least a part thereof may be executed by the CPU 201.

  First, the well plate WP carrying the spheroid as the imaging object in the well W together with the culture solution is carried into the imaging unit 1 and set in the holder 11 (step S101). Then, the imaging optical system 13 is positioned with respect to the well W to be imaged, and imaging by the imaging device 134 is performed (step S102). Thereby, the original image containing a spheroid is acquired.

  The graphic processor 202 performs predetermined image processing on the original image thus obtained, and detects an area of the image object included in the original image (step S103). A known technique can be applied to the extraction of the object in the original image. For example, it is possible to apply a method of binarizing an original image with an appropriate threshold value and dividing it into a background area and an object area.

  The graphic processor 202 further extracts a spheroid-like object including a region corresponding to the spheroid from the detected image object (step S104). The image objects detected in step S103 include those consisting only of spheroids, those consisting only of foreign matters, and those where spheroids and foreign matters overlap. Among these, an object including a spheroid, that is, an object composed only of a spheroid, and an object in which a spheroid and a foreign object overlap are extracted as a spheroid-like object.

  As a method for extracting a spheroid-like object including the spheroid region Ra, various methods capable of detecting an image object including a circular region peculiar to the spheroid are conceivable. For example, a spheroid-like object having a circularity greater than or equal to a predetermined value can be used. An object having an area larger than a predetermined circle of a predetermined size can be a spheroid-like object. Further, since the foreign substance region is generally elongated and its area is relatively small, an object having an area equal to or larger than a predetermined value can be a spheroid-like object. On the other hand, a condition for considering an object consisting only of a foreign object may be set, and an object that does not match the condition may be regarded as a spheroid-like object.

  One of the spheroid-like objects extracted in this way is selected (step S105), and the minimum inclusion circle of the object is specified (step S106). An object whose ratio of the area of the minimum inclusion circle to the area of the object is equal to or greater than a predetermined threshold value greater than 1 is set as a target of the region separation process in step S108. If the area ratio is smaller than the threshold value, step S108 is skipped. This is because an object that does not include a foreign substance area is excluded from the target of the area separation process, and the image information is stored. It also helps shorten processing time.

  FIG. 5A shows the relationship between the object and the minimum inclusion circle. The minimum inclusion circle of the object OB is a circle C3 having the smallest diameter among circles including the entire object OB. As shown in the figure, when the foreign substance region Rb extends long from the spheroid region Ra, the diameter D3 of the minimum inclusion circle C3 is much larger than the diameter of the spheroid region Ra, and the minimum inclusion circle C3 is a background region other than the object OB. Will contain a lot. Therefore, the area of the minimum inclusion circle C3 is larger than the area of the object OB.

  On the other hand, when the object OB does not include the foreign substance region Rb and consists only of the spheroid region Ra, the size of the minimum inclusion circle C3 is not significantly different from the size of the spheroid region Ra, and the area ratio approaches 1. In principle, the area ratio cannot be smaller than 1. Thus, if the area ratio between the object OB and the minimum inclusion circle C3 is close to 1, the object OB does not include the foreign substance region Rb. If the area of the minimum inclusion circle C3 is sufficiently larger than the area of the object OB, the object OB is a foreign substance. It can be determined that the region Rb is included. Therefore, an appropriate threshold value (for example, 1.1) larger than 1 is set for the area ratio between the two, and an object OB having an area ratio equal to or greater than the threshold value is set as a region separation process target. Here, the object is also processed when the area ratio is equal to the threshold value, but whether such an object is processed is arbitrary.

  When the area ratio of the minimum inclusion circle C3 to the object OB is equal to or greater than the threshold value, the region separation process shown in FIG. 4 is executed for the object. First, an approximate circle that approximates the spheroid region is specified for the object (step S201). This approximate circle is a circle that approximates the spheroid region Ra included in the object OB by a circle, and is considered to have a size equivalent to the spheroid region Ra.

  FIG. 5B shows an example of an approximate circle. Of the object OB, the spheroid Ra has a shape close to a circle, and as a method of approximating it to a circle of the same size, for example, a circle C4 indicated by a broken line in the figure does not protrude from the object OB. There is a method of using a circle having the maximum diameter among the circles that fits (herein referred to as a “maximum inclusion circle”). Further, for example, a circle having the same area as the area of the object OB including the spheroid region Ra and the foreign material region Rb (herein referred to as an “area equivalent circle”), such as a circle C5 indicated by a dotted line in the figure, is an approximate circle. You can also.

  Based on the thus obtained approximate circle (maximum inclusion circle or area equivalent circle), the diameter of the circle, which is a structural element in the morphological process, is determined (step S202). As described above, the morphological treatment in this case has a diameter of (1/6) to (1/2), more preferably (1/4) to (1/3) of the diameter of the spheroid region Ra. It has been found that good results are obtained when a circle is the structural element. However, since the spheroid region Ra and the foreign matter region Rb are undivided at this time, the size of the spheroid region Ra cannot be clearly specified. Therefore, the diameter of the approximate circle described above is regarded as the diameter of the spheroid region Ra. That is, the diameter of the circle as the structural element is determined in the range of (1/6) to (1/2) of the diameter of the approximate circle. Here, it is set to a value not less than (1/4) and not more than (1/3) of the diameter of the approximate circle.

  Increasing the diameter of the circle as the structural element increases the effect of removing foreign matter. On the other hand, if the diameter of the circle as the structural element is reduced, the shape reproducibility of the spheroid region is enhanced. The size of the structural element can be appropriately set according to the size of the spheroid and foreign matter, the purpose of processing, and the like. In any case, the size of the structural element is set according to the size of the spheroid region, thereby preventing the small spheroid from being erased by the erosion process. In order to avoid the disappearance of the spheroid region due to the erosion process even when the approximate circle is larger than the spheroid region Ra, the diameter of the circle as a structural element is less than (1/2) of the diameter of the approximate circle. Is desirable.

  As the erosion process and the duration process are sequentially performed using the structural elements thus determined (steps S203 and S204), the region Rd corresponding to the spheroid region Ra is included in the image as shown in FIG. While being reproduced, the foreign substance region Rb is erased from the image. The purpose of erasing the foreign object region Rb from the image is achieved at this point. The purpose of extracting only the spheroid region Ra from the object OB is also achieved at this time.

  Furthermore, the region separation processing of this embodiment aims to clearly distinguish these regions while leaving the image information of both the spheroid region Ra and the foreign matter region Rb. Therefore, the spheroid region Ra identified by the processing up to step S204 is expanded by an appropriate size, for example, one pixel (step S205), and the result is subtracted from the original image (step S206). Note that the number of pixels to be expanded may be two pixels.

  By subtracting the spheroid area Ra from the object OB of the original image, as shown in FIG. 5C, an area other than the spheroid in the object, that is, the foreign substance area Rb is extracted. At this time, when subtraction is performed after the spheroid region Ra is expanded, as indicated by an arrow in FIG. 5C, the spheroid region Ra is expanded between the spheroid region Ra and the foreign material region Rb by the amount of expansion. A gap is formed and both are separated on the image. If the foreign matter region Rb is simply extracted, it is not always necessary to expand the spheroid region Ra.

  Furthermore, by adding and synthesizing the image of the spheroid region Ra extracted by the duration process (step S204) to the image from which the foreign substance region Rb is extracted, as shown in FIG. 5D, the spheroid region Ra and An image in which the image information of the foreign substance region Rb is stored together and the both regions are clearly separated is obtained (step S207). This image is an image to be obtained in this processing.

  The composite image is stored in the image memory 203 (step S208). Instead of or in addition to this, the image of the spheroid region Ra obtained by the process of step S204 (FIG. 2B) and the step The image (FIG. 5C) of the foreign substance region Rb obtained by the process of S206 may be stored in the image memory 203.

  Returning to FIG. 3, the description of the flowchart will be continued. The above processing (steps S105 to S108) is executed for all the extracted spheroid-like objects (step S109). Since the diameter of the circle used as a structuring element is set individually for each object, a circle with a large diameter is applied to an object that includes a large spheroid area and a small diameter circle is applied to an object that includes a small spheroid area. The Therefore, a small spheroid is not erased by the erosion process, and each object can be appropriately processed to efficiently distinguish the spheroid area and the foreign substance area.

  The processed image is stored in the image memory 203 and is output in an appropriate form via the interface 205 (step S110). For example, the processed image is displayed on the display unit 22 and presented to the user. Alternatively, the data is output to an external device or an external storage medium via the interface 205.

  Note that although there is a concern that the image information in the object OB is lost by executing the morphological processing, it is possible to avoid this as follows, for example. The first method performs the above-described morphological processing on the binarized original image to create a mask indicating the range of the spheroid region Ra and the foreign material region Rb, and applies this mask to the original multi-valued original image. By doing so, the image information of the spheroid region Ra and the foreign material region Rb is restored. The second method is to restore the pixels in the region that is expanded when performing the duration process with the pixel values of the original image.

  As described above, in the above-described embodiment, the imaging unit 1 and the image processing unit 2 function as an “image processing apparatus” of the present invention. The imaging unit 1 functions as an “image acquisition unit” of the present invention, and the holder 11 and the imaging unit 13 function as a “holding unit” and an “imaging unit” of the present invention, respectively. Further, the image processing unit 2, particularly the graphic processor 202 functions as the “image processing means” of the present invention.

  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, in the region separation process of the above embodiment, the size of the structural element is determined by approximating the spheroid region Ra to a circle, but more generally, it is preferable to approximate the spheroid region by an ellipse. This is because the spheroid region may have a shape deviating from a circle due to distortion of the spheroids or overlapping of a plurality of spheroids on the image. By approximating the spheroid region with an ellipse, accurate approximation is possible even in such a case, and the setting of the size of the structural element can be made more preferable.

  In this case, the diameter of the circle as the structural element is preferably set based on the minor axis of the approximate ellipse. In the departure from the circular shape of the spheroid region due to the above causes, it is unlikely that the minor axis of the ellipse is significantly different from the true size of the spheroid, while the major axis may greatly exceed the true size of the spheroid. It is possible. By determining the diameter of the circle based on the minor axis, which is a smaller dimension, it is possible to avoid erasing the spheroid from the object.

  Further, for example, the image processing apparatus of the above embodiment includes the imaging unit 1 as “image acquisition means”, but may be an apparatus that does not include an imaging function. That is, in the image processing unit 2 of the above embodiment, the interface 205 acquires an original image by receiving image data from the outside. In this sense, the interface 205 has a function as an “image acquisition unit”. Yes. As described above, the present invention functions effectively even in an aspect in which an image processing apparatus having no imaging unit receives original image data from an external apparatus or an external storage medium.

  In the above embodiment, the present invention is implemented by the CPU 201 executing a control program stored in advance in the memory 204. As described above, the image processing unit 2 in this embodiment is a general-purpose computer device. Can be used. Therefore, the present invention is provided to the user as a control program for causing the computer apparatus to execute the above-described image processing on the assumption that the image apparatus is read by such a computer apparatus, and in a mode in which the program is recorded on an appropriate recording medium. Is also possible. Accordingly, for example, it is possible to add a new function to an imaging apparatus that is already in operation.

  The present invention is suitable for use in imaging spheroids for the purpose of observation, analysis, etc., and can be used for various experiments in the fields of medicine, drug discovery, and biological science, for example.

1 Imaging unit (image acquisition means)
2 Image processing unit (image processing means)
11 Holder (holding part)
13 Imaging unit 201 CPU
202 Graphic processor (image processing means)
C4, C5 Approximate circle OB Object Ra Spheroid area Rb Foreign object area

Claims (15)

For a spheroid imaged original image, a step of detecting a spheroid-like object corresponding to the spheroid among objects in the original image;
For the spheroid-like object, performing a erosion process using a circle having a diameter determined based on the size of the spheroid-like object and having a smaller area than the spheroid-like object as a structural element;
And a step of executing a duration process using the structural element for the spheroid-like object after the erosion process.   The image processing method according to claim 1, wherein the structural elements having different sizes are set for the plurality of spheroid-like objects having different sizes.   The image processing method according to claim 1, wherein the size of the spheroid-like object is represented by a size of an approximate ellipse that approximates the spheroid-like object.   The image processing method according to claim 3, wherein an ellipse having the largest area among ellipses included in an outline of the spheroid-like object is set as the approximate ellipse.   The image processing method according to claim 3, wherein an ellipse having the same area as the spheroid-like object is used as the approximate ellipse.   6. The image processing method according to claim 3, wherein a diameter of a circle as the structural element is not less than (1/6) and not more than (1/2) of a minor axis of the approximate ellipse.   The image processing method according to claim 6, wherein a diameter of a circle as the structural element is not less than (1/4) and not more than (1/3) of a minor axis of the approximate ellipse.   The image processing method according to claim 3, wherein the approximate ellipse is an approximate circle that approximates the spheroid-like object.   9. The method according to claim 1, further comprising a step of detecting a non-spheroid region different from the spheroid among objects in the original image based on a difference image between the image after the dilation processing and the original image. Image processing method. Identifying a minimum inclusion circle of the spheroid-like object detected in the original image,
Among the spheroid-like objects, the erosion process and the dilation process are executed for a spheroid-like object whose ratio of the area of the minimum inclusion circle of the spheroid-like object and the area of the spheroid-like object is larger than a predetermined value larger than 1. The image processing method according to claim 1.   A control program for causing a computer to execute the image processing method according to claim 1.   The computer-readable recording medium which recorded the control program of Claim 11. Image acquisition means for acquiring an original image in which a spheroid is imaged;
Image processing means for performing image processing on the original image,
The image processing means includes a process of detecting a spheroid-like object corresponding to the spheroid in the original image, and the spheroid-like object has a diameter obtained based on a size of the spheroid-like object, and the spheroid-like object An image processing apparatus that executes a process of executing an erosion process using a circle having a small area as a structural element, and a process of executing a delation process using the structural element for the spheroid-like object after the erosion process. Image acquisition means for acquiring an original image in which a spheroid is imaged;
Image processing means for performing image processing on the original image,
The image processing device that executes the same image processing as the image processing method according to any one of claims 1 to 10.   The image processing apparatus according to claim 13, wherein the image acquisition unit includes a holding unit that holds a carrier that carries the spheroid, and an imaging unit that images the carrier and creates the original image.

Images