Image processing method, image processor, image capturing device, and image capturing method for generating omnifocal image

A plurality of captured images is first acquired by capturing images of an object while changing a focal position along an optical axis. Then, variations in magnification among the captured images are acquired. On the basis of the variations in magnification, corresponding pixels in the captured images are specified, and definition is compared among the corresponding pixels. Then, an image reference value indicating the number of a captured image that is to be referenced as the luminance value of each coordinates in an omnifocal image is determined on the basis of the result of comparison of the definition. The omnifocal image is thereafter generated by referencing the luminance value in the captured image indicated by the image reference value for each coordinates. In this way, the omnifocal image that reflects the position and size of the object accurately can be generated.

CROSS REFERENCE

This application is the U.S. National Phase under 35 U.S.C. § 371 of International Application No. PCT/JP2017/020066, filed on May 30, 2017, which claims the benefit of Japanese Application No. 2016-138163, filed on Jul. 13, 2016 and Japanese Application No. 2016-181225, filed Sep. 16, 2016, the entire contents of each are hereby incorporated by reference.

TECHNICAL FIELD

The present invention relates to an image processing method, an image processor, an image capturing device, and an image capturing method for generating an omnifocal image on the basis of a plurality of captured images.

BACKGROUND ART

Patent Literature 1 discloses a device for observing the culture conditions of biological cells by capturing images of the cells at high resolutions. The device according to Patent Literature 1 captures images of cells held along with a culture solution in a container with a camera. Such a device may in some cases has difficulty in focusing on all cells in the culture solution in one image capture. Thus, the device captures images a plurality of times while changing the focal position of the camera and combines a plurality of obtained images to generate an omnifocal image that focuses on all the cells.

Patent Literature 2 discloses a conventional technique for generating an omnifocal image. With the device according to Patent Literature 2, the magnifications of images change with a change in the focal position of a camera because the optical system from specimens to image capturing means is non-telecentric. Thus, the device according to Patent Literature 2 corrects and unifies the magnifications of images and then generates an omnifocal image (see, for example, FIG. 2 of Patent Literature 2). The magnifications are corrected on the basis of pre-saved design information about an enlarging optical system and positional information about specimens (see, for example, paragraph 0054 of Patent Literature 2).

CITATION LIST

Patent Literature

SUMMARY OF INVENTION

Technical Problem

In the case of capturing images of cells in a culture solution as in the device according to Patent Literature 1, a concave meniscus is formed in the surface of the culture solution by surface tension. Thus, light is refracted at the surface of the culture solution. As a result, the magnification of each image (the width of the field of view) varies depending the focal position of the camera. Therefore, such variations in magnification among images obtained by a plurality of image captures need to be taken into consideration when generating an omnifocal image.

For example, it is conceivable to generate an omnifocal image by correcting the magnification of each image, as in Patent Literature 2, in accordance with the amount of variations in magnification among images obtained by a plurality of image captures. However, there is the problem that the size of each cell or the intervals of a plurality of cells in each image can change if the magnification of the image is corrected.

In particular, in the case where a plurality of omnifocal images obtained with different fields of view is combined to generate one resultant image, the positions and sizes of cells need to be matched between adjacent omnifocal images. However, if the magnification of each image is corrected as described above, the positions and sizes of cells do not match with high accuracy between adjacent omnifocal images. This causes an image disturbance at the boundaries of the omnifocal images in the resultant image.

Also, the meniscus affects differently depending on various conditions such as the shape of the container, the type of the culture solution, elapsed time, and culture environments. Thus, it is not possible to correct the magnifications of images on the basis of information prepared in advance as in Patent Literature 2.

The present invention has been made in view of such circumstances, and it is an object of the present invention to provide an image processing method, an image processor, an image capturing device, and an image capturing method that can generate an omnifocal image with high accuracy even if the magnifications of images vary depending on the focal position of a camera.

Solution to Problem

In order to solve the problems described above, a first invention of the present application is an image processing method for generating an omnifocal image on the basis of a plurality of captured images. The method includes the steps of a) acquiring a plurality of captured images by capturing an image of an object while changing a focal position along an optical axis, b) acquiring variations in magnification among the plurality of captured images, c) specifying corresponding pixels in the plurality of captured images and comparing definition among the corresponding pixels on the basis of the variations in magnification, d) determining an image reference value on the basis of a comparison result obtained in the step c), the image reference value being a number of one of the captured images that is to be referenced as a luminance value of each coordinates in an omnifocal image, and e) generating an omnifocal image by referencing the luminance value in the captured image indicated by the image reference value for each coordinates.

A second invention of the present application is an image processor for generating an omnifocal image on the basis of a plurality of captured images. The image processor includes an image storage that stores a plurality of captured images by capturing an image of an object while changing a focal position along an optical axis, a magnification variation acquisition part that acquires variations in magnification among the plurality of captured images, an image reference value determination part that specifies corresponding pixels in the plurality of captured images and compares definition among the corresponding pixels on the basis of the variations in magnification to determine an image reference value that is a number of one of the captured images that is to be referenced as a luminance value of each coordinates in an omnifocal image, and an omnifocal image generator that generates an omnifocal image by referencing the luminance value in the captured image indicated by the image reference value for each coordinates.

A third invention of the present application is an image capturing device for capturing an image of an object to generate an omnifocal image. The image capturing device includes a camera that captures an image of the object, a projector that emits light toward the object, a moving mechanism that changes a focal position of the camera along an optical axis, and a controller that controls the camera, the projector, and the moving mechanism and processes an image acquired by the camera. The controller executes the steps of a) acquiring a plurality of captured images by causing the camera to capture an image while causing the moving mechanism to change the focal position, b) calculating variations in magnification among the plurality of captured images, c) performing reciprocal correction of the variations in magnification for each of the plurality of captured images, and d) generating an omnifocal image by using the plurality of captured images having undergone the reciprocal correction.

A fourth invention of the present application is an image capturing method for capturing an image of an object to generate an omnifocal image. The image capturing method includes the steps of a) acquiring a plurality of captured images by capturing an image of the object with a camera while changing a focal position of the camera along an optical axis, b) calculating variations in magnification among the plurality of captured images, c) performing reciprocal correction of the variations in magnification for each of the plurality of captured images, and d) generating an omnifocal image by using the plurality of captured images having undergone the reciprocal correction.

Advantageous Effects of Invention

According to the present invention, an omnifocal image can be generated with high accuracy.

DESCRIPTION OF EMBODIMENTS

1.1. Configuration of Image Capturing Device

FIG. 1is a perspective view illustrating an example of a well plate9that is set in an image capturing device1. The well plate9is a generally plate-like specimen container having a plurality of wells (depressions)91. The material for the well plate9is, for example, a transparent resin that transmits light. As illustrated inFIG. 1, the plurality of wells91is arranged regularly in the upper surface of the well plate9. Each well91holds therein a plurality of biological cells93targeted for image capture, along with a culture solution92. Note that the shape of the wells91when viewed from above may be circular as inFIG. 1or may be other shapes such as rectangles.

FIG. 2illustrates a configuration of the image capturing device1according to an embodiment of the invention. This image capturing device1is a device that captures images of the plurality of cells93in the well plate9a plurality of times while changing the focal position of a camera40and combines a plurality of resultant captured images to generate a composite image (omnifocal image) for observation that focuses on all the cells93and has less blurring.

The image capturing device1is used in, for example, a screening process of narrowing down chemical compounds serving as candidates for pharmaceuticals in the field of research and development of pharmaceuticals. In the screening process, a person in charge adds chemical compounds of different concentrations or compositions to the plurality of wells91of the well plate9. Then, the image capturing device1is used to capture images of cells93in each well91of the well plate9. The culture conditions of the cells93are thereafter compared and analyzed on the basis of the obtained images to verify the effects of the chemical compounds added to the culture solution92.

Alternatively, the image capturing device1may be used in research and development of pluripotent stem cells such as IPS cells or ES cells to observe cell differentiation, for example.

As illustrated inFIG. 2, the image capturing device1according to the present embodiment includes a stage10, a projector20, a projector moving mechanism30, the camera40, a camera moving mechanism50, and a controller60.

The stage10is a pedestal that holds the well plate9. The position of the stage10in the image capturing device1is fixed during at least image capture. The stage10has a rectangular opening11penetrating in the up-down direction in the center. The stage10also has a ring-shaped supporting surface12at the edge of the opening11. The well plate9is fitted in the opening11and supported in a horizontal position by the supporting surface12. The top and bottom of each well91are thus exposed without being blocked off by the stage10.

The projector20is disposed above the well plate9held on the stage10. The projector20has a light source such as LEDs. The light source of the projector20emits light during image capture, which will be described later. Thus, the projector20applies light downward. Note that the projector20needs only apply light from the side opposite to the camera40toward the well plate9. Therefore, the light source itself of the projector20may be disposed at a position off the top of the well plate9and configured to apply light to the well plate9via an optical system such as a mirror.

The projector moving mechanism30is a mechanism for moving the projector20horizontally along the upper surface of the well plate9held on the stage10. For example, a mechanism for converting rotational motion of a motor into rectilinear motion through a ball screw is used as the projector moving mechanism30. The image capturing device1can dispose the projector20at a position above each well91by operating the projector moving mechanism30. Although only one direction indicated by arrow A1is illustrated inFIG. 2as the direction of movement of the projector20, the projector moving mechanism30may be configured to move the projector20in two directions (left-right direction and depth direction inFIG. 2) along the upper surface of the well plate9.

The camera40is disposed below the well plate9held on the stage10. The camera40includes an optical system such as a lens and an image sensor such as a CCD or a CMOS. In the case of capturing an image, the camera40captures an image of part of the well plate9while the projector20applies light toward that part of the well plate9. Accordingly, an image of cells93in the well plate9is acquired in the form of digital data. The captured image is input from the camera40to the controller60.

The camera moving mechanism50is a mechanism for changing the height and horizontal position of the camera40while maintaining the posture of the camera40. As illustrated inFIG. 2, the camera moving mechanism50includes an up-and-down movement mechanism51and a horizontal movement mechanism52.

The up-and-down movement mechanism51is a mechanism for moving the camera40up and down. For example, a mechanism for converting rotational motion of a motor into rectilinear motion through a ball screw is used as the up-and-down movement mechanism51. The height of the camera40is changed by operating the up-and-down movement mechanism51. This changes the distance between the well plate9held on the stage10and the camera40(i.e., shooting distance between the cells93and the camera40). The camera40according to the present embodiment has a fixed focal length. Accordingly, the focal position of the camera40moves up and down along the optical axis as the position of the camera40moves up and down.

The horizontal movement mechanism52is a mechanism for moving the camera40and the up-and-down movement mechanism51horizontally as an integral unit. For example, a mechanism for converting rotational motion of a motor into rectilinear motion through a ball screw is used as the horizontal movement mechanism52. The image capturing device1can dispose the camera40at a position under each well91by operating the horizontal movement mechanism52. Although only one direction indicated by arrow A2is illustrated inFIG. 2as the direction of movement of the camera40by the horizontal movement mechanism52, the camera moving mechanism50may be configured to move the camera40in two directions (left-right direction and depth direction inFIG. 2) along the lower surface of the well plate9.

Note that the projector moving mechanism30and the horizontal movement mechanism52described above are driven in synchronization. Accordingly, the projector20and the camera40are always disposed at the same position when viewed from above. That is, the projector20and the camera40move the same distance in the same direction, and when the camera40is disposed at a position under a given well91, the projector20is always disposed at a position above that well91.

The controller60is configured by, for example, a computer. The controller60has a function of controlling the operation of each part of the image capturing device1and a function serving as an image processor for generating an omnifocal image on the basis of a plurality of captured images input from the camera40.FIG. 3is a block diagram illustrating connection between the controller60and each part of the image capturing device1. As schematically illustrated inFIG. 3, the controller60includes a processor61such as a CPU, a memory62such as a RAM, and a storage63such as a hard disk drive. The storage63stores a control program P1for controlling the operation of each part of the image capturing device1, and an image processing program P2for generating an omnifocal image on the basis of captured images input from the camera40.

As illustrated inFIG. 3, the controller60is communicably connected to each of the projector20, the projector moving mechanism30, the camera40, the up-and-down movement mechanism51, and the horizontal movement mechanism52described above. The controller60controls the operations of the above-described parts in accordance with the control program P1, thereby causing the processing for capturing images of cells93held in each well91of the well plate9to proceed. The controller60also generates an omnifocal image by processing captured images input from the camera40in accordance with the image processing program P2.

1.2. Image Capturing Process

Next, the operations of the aforementioned image capturing device1will be described.FIG. 4is a flowchart illustrating a flow of the image capturing process performed by the image capturing device1.FIG. 5illustrates the image capturing process performed for one well91.

When the well plate9has been set on the stage10of the image capturing device1and an instruction to start operation has been input to the controller60, the controller60first operates the up-and-down movement mechanism51. Thereby, the camera40is disposed at a predetermined height (step S1). According to the present embodiment, the height of the camera40can be changed in five stages (from a first height H1to a fifth height H5) as illustrated inFIG. 5. At the start of the image capturing process, the camera40is first disposed at the highest first height H1.

Next, the controller60operates the projector moving mechanism30and the horizontal movement mechanism52. Thereby, the projector20and the camera40are respectively moved to above and below a well91targeted for image capture (step S2). Then, the controller60captures an image of cells93held in that well91by operating the projector20and the camera40(step S3). That is, the camera40captures an image while the projector20applies light L downward. Accordingly, an image of the cells93held in that well91is captured from the first height H1.

Then, the controller60determines whether or not there is the next well91targeted for image capture (step S4). If there is the next well91(yes in step S4), the controller60operates the projector moving mechanism30and the horizontal movement mechanism52. Thereby, the projector20and the camera40are respectively moved to above and below the next well91(step S2). Then, the controller60captures an image of cells93held in that well91by operating the projector20and the camera40(step S3).

In this way, the controller60repeats the movement of the projector20and the camera40(step S2) and the image capture (step S3). Accordingly, images of all the wells91targeted for image capture in the well plate9are captured from the first height H1.

If there remain no wells91for which image capture has not yet been performed (no in step S4), the controller60determines whether or not to change the height of the camera40(step S5). Here, if there remains a height at which image capture has not yet been performed among the five heights H1to H5prepared in advance, the controller60determines to change the height of the camera40(yes in step S5). For example, when the image capturing process at the first height H1has ended, the controller60determines to change the height of the camera40to the next height, i.e., the second height H2.

In the case of changing the height of the camera40, the controller60operates the up-and-down movement mechanism51so as to move the camera40to a height to which the height of the camera40ought to be changed (step S1). This changes the focal position of the camera40. Then, the aforementioned processing in steps S2to S4is repeated. Accordingly, an image of cells93taken from the changed height is acquired for each well91of the well plate9.

As described above, the controller60repeats the change in the height of the camera40(step S1) and the acquisition of captured images for the plurality of wells91(steps S2to S4). Accordingly, five images taken from the five heights H1to H5are acquired for each of the plurality of wells91of the well plate9.

1.3. Generation of Omnifocal Image

Next, image processing for generating an omnifocal image on the basis of a plurality of captured images input from the camera40will be described.

When the aforementioned steps S1to S5are completed, five captured images D1to D5taken with different shooting distances are obtained for each well91of the well plate9. However, the surface of the culture solution92in each well91has a concave meniscus formed under the influence of surface tension as illustrated inFIG. 5. Thus, light L emitted from the projector20is refracted when passing through the culture solution92, and becomes diffused light. Therefore, the five captured images D1to D5have different magnifications. Also, the magnitude of diffusion of the light L differs for each well91. Accordingly, the amounts of variations in magnification among the five captured images D1to D5also differ for each well91.

FIG. 6illustrates an example of the five captured images D1to D5acquired for one well91. The captured images D1to D5are images taken by the camera40disposed at the heights H1to H5, respectively. Each of the captured images D1to D5includes one or two images out of two cells93held in the well91. The cell93on the right side in the diagram is most sharply in focus in the captured image D2taken by the camera40disposed at the height H2. The cell93on the left side in the diagram is most sharply in focus in the captured image D4taken by the camera40disposed at the height H4.

Under the influence of the aforementioned meniscus, the magnifications of the captured images D1to D5increase as the height of the camera40decreases (i.e., as the shooting distance between the cells93and the camera40increases). Thus, the captured image D1has a lowest magnification and the captured image D5has a highest magnification among the five captured images D1to D5. Accordingly, if these captured images D1to D5are simply combined, blurring around each cell93will increase as in an omnifocal image DA inFIG. 7(comparative example).FIG. 7illustrates an example (comparative example) of the omnifocal image obtained by simply combining the captured images.

FIG. 8is a flowchart illustrating a flow of image processing for generating one omnifocal image DA from the five captured images D1to D5.

When the five captured images D1to D5have been obtained, the controller60first corrects errors in each of the captured images D1to D5(step S6). Here, the controller60corrects variations in position among the captured images D1to D5, the variations being caused by machine errors in the image capturing device1. For example, if the horizontal movement mechanism52has a known positioning error, the positions of the captured images D1to D5are each corrected by an amount corresponding to the positioning error. This increases the accuracy of calculation of the amounts of variations in magnification and the amounts of parallel displacement among the five captured images D1to D5in the next step S7.

Next, the controller60calculates the amounts of variations in magnification and the amounts of parallel displacement among the five captured images D1to D5(step S7). Here, the controller60detects how much the sizes of the cells93or the positions of the cells93change among the five captured images D1to D5. In this way, the magnitude of variations in magnification caused by the meniscus of the culture solution92is calculated.

FIG. 9is a flowchart illustrating an example of processing that is performed in step S7. According to the present embodiment, the amount of variations in magnification and the amount of parallel displacement are obtained for each pair of adjacent images when the five captured images D1to D5are arranged in order of the focal position.FIG. 10schematically illustrates processing for obtaining the amount of variations in magnification and the amount of parallel displacement for the two captured images D2and D3.

In step S7, a plurality of candidate images is first created by enlarging or reducing one of the two captured images to each preset magnification (step S71). In the example inFIG. 10, the captured image D3having a higher magnification (narrower field of view), out of the two captured images D2and D3, is reduced to each preset magnification in order to create a plurality of candidate images D31, D32, D33, and so on.

Then, template matching is performed between the other of the two captured images and each of the plurality of created candidate images (step S72). In the example inFIG. 10, template matching is performed between the captured image D2and each of the plurality of candidate images D31, D32, D33, and so on as indicated by arrows T. Specifically, each of the candidate images D31, D32, D33, and so on is displaced parallel relative to the captured image D2. Then, a matching score at each position is calculated. The matching score is, for example, an evaluation value that indicates similarity of the image and that is calculated by a known method such as Sum of Squared Difference (SSD), Sum of Absolute Difference (SAD), Normalized Cross-Correlation (NCC), or Zero-mean Normalized Cross-Correlation (ZNCC).

The controller60obtains a maximum value S of the matching score and the amount of parallel displacement M at that time for each of the candidate images D31, D32, D33, and so on (step S73). Then, a candidate image having a highest value for the maximum value S of the matching score is determined as a selected image for the captured image D3(step S74). When the selected image has been determined, the controller60determines the magnification of the selected image as the amount of variations in magnification between the two captured images D2and D3. Also, the amount of parallel displacement M when the above-described matching score of the selected image becomes the maximum value S is determined as the amount of parallel displacement between the two captured images D2and D3(step S75).

The controller60executes the above-described processing in steps S71to S75for each pair of adjacent images when the five captured images D1to D5are arranged in order of the focal position. In this way, the amount of variations in magnification and the amount of parallel displacement are determined for each pair of images.

When the amount of variations in magnification and the amount of parallel displacement between each pair of images have been determined, the controller60uses one of the five captured images D1to D5(e.g., captured image D1) as a reference image and calculates the amounts of variations in magnification and the amounts of parallel displacement for the other captured images with respect to the reference image (step S76). For example, the amount of variations in magnification for the captured image D3with respect to the captured image D1is assumed to be a value obtained by multiplying the amount of variations in magnification between the two captured images D1and D2and the amount of variations in magnification between the two captured images D2and D3. Also, the amount of parallel displacement for the captured image D3with respect to the captured image D1is assumed to be a value obtained by adding the amount of parallel displacement between the two captured images D1and D2and the amount of parallel displacement between the two captured images D2and D3after correcting the amounts of variations in magnification.

Refer back toFIG. 8. When the processing in step S7is completed, next, the controller60reciprocally corrects the captured images other than the reference image among the five captured images D1to D5on the basis of the amounts of variations in magnification and the amounts of parallel displacement calculated in step S76(step S8).FIG. 11illustrates an example of the five captured images D1to D5reciprocally corrected. In the example inFIG. 11, the captured image D1having a lowest magnification is used as a reference, and each of the other four captured images D2to D5is reduced on the basis of the amount of variations in magnification and displaced in parallel on the basis of the amount of parallel displacement.

Thereafter, the controller60generates the omnifocal image DA using the reference image and the four captured images reciprocally corrected (step S9). The aforementioned reciprocal correction in step S8allows the positions of the cells93to match among the captured images D1to D5as illustrated inFIG. 11. Thus, the omnifocal image DA with less blurring as illustrated inFIG. 12can be obtained by combining these captured images D1to D5.FIG. 12illustrates an example of the omnifocal image obtained by combining the reciprocally corrected captured images.

In particular, this image capturing device1calculates the amounts of variations in magnification and the amounts of parallel displacement on the basis of the captured images D1to D5input from the camera40, instead of storing the amounts of variations in magnification and the amounts of parallel displacement in the controller60in advance. Thus, even if the amounts of variations in magnification and the amounts of parallel displacement change due to the shape of the meniscus of the culture solution92, the omnifocal image DA can be generated in consideration of the changed amounts of variations in magnification and the changed amounts of parallel displacement. Accordingly, a high-quality omnifocal image DA can be generated for each well91of the well plate9.

An image capturing device according to Embodiment 2 will be described hereinafter. Note that description of members that are similar to those in Embodiment 1 is omitted.

2.1. Configuration of Image Capturing Device

FIG. 13illustrates a configuration of an image capturing device2. As illustrated inFIG. 13, the image capturing device2according to the present embodiment includes a stage10, a projector20, a projector moving mechanism30, a camera40, a focal point moving mechanism70, a camera moving mechanism50, and a controller60.

The camera40includes an optical system41such as a lens and an image sensor42such as a CCD or a CMOS.

The focal point moving mechanism70is a mechanism for changing the focal position of the camera40. The focal point moving mechanism70according to the present embodiment moves some optics included in the optical system41of the camera40. Thereby, the focal position of the camera40is changed along the optical axis. The focal point moving mechanism70is capable of changing the focal position of the camera40minutely in the up-down direction in the vicinity of the cells93in the well plate9. For example, a compact motor is used as the focal point moving mechanism70.

The camera moving mechanism50is a mechanism for changing the horizontal position of the camera40while maintaining the posture of the camera40. The camera moving mechanism50moves the camera40and the focal point moving mechanism70horizontally as an integral unit. For example, a mechanism for converting rotational motion of a motor into rectilinear motion through a ball screw is used as the camera moving mechanism50. The image capturing device2can dispose the camera40at a specified position under a well91by operating the camera moving mechanism50. Although only one direction indicated by arrow A2is illustrated inFIG. 13as the direction of movement of the camera40by the camera moving mechanism50, the camera moving mechanism50may be configured to move the camera40in two directions (left-right direction and depth direction inFIG. 13) along the lower surface of the well plate9.

The projector moving mechanism30and the camera moving mechanism50described above are driven in synchronization. Accordingly, the projector20and the camera40are always disposed at the same position when viewed from above. That is, the projector20and the camera40move the same distance in the same direction, and when the camera40is disposed at a position under a given cell93, the projector20is always disposed at a position above that cell93.

The controller60is configured by, for example, a computer. The controller60has a function serving as a control device for controlling the operation of each part of the image capturing device2and a function serving as an image processor for generating an omnifocal image on the basis of a plurality of captured images input from the camera40.FIG. 14is a block diagram illustrating connection between the controller60and each part of the image capturing device2. As illustrated inFIG. 14, the controller60is communicably connected to each of the projector20, the projector moving mechanism30, the camera40, the focal point moving mechanism70, and the camera moving mechanism50described above.

FIG. 15is a block diagram schematically illustrating the functions implemented within the controller60. As illustrated inFIG. 15, the controller60includes an image capture controller601and an image processing part602. The image capture controller601controls the operations of the projector20, the projector moving mechanism30, the camera40, the focal point moving mechanism70, and the camera moving mechanism50in accordance with a control program P1, thereby causing the processing for capturing images of cells93held in each well91of the well plate9to proceed. The image processing part602generates an omnifocal image by processing a plurality of captured images input from the camera40in accordance with an image processing program P2.

The image processing part602includes an image storage621, an error corrector622, a magnification variation acquisition part623, an image reference value determination part624, a shadow removal processing part625, an omnifocal image generator626, and a tiling processing part627as illustrated inFIG. 15. Specific processing performed by these parts will be described later.

2.2. Image Capturing Process

Next, the operations of the aforementioned image capturing device2will be described.FIG. 16is a flowchart illustrating a flow of the image capturing process performed by the image capturing device2.FIG. 17illustrates the image capturing process performed for one well91.

When the well plate9has been set on the stage10of the image capturing device2and an instruction to start operation has been input to the controller60, the image capture controller601of the controller60first operates the focal point moving mechanism70. Thereby, the focal position of the camera40is adjusted to a predetermined height (step S11). According to the present embodiment, the focal position of camera40can be changed in five stages (from a first focal position H1to a fifth focal position H5) as illustrated inFIG. 17. At the start of the image capturing process, the focal point of the camera40is first adjusted to the highest first focal position H1.

This image capturing device2divides one well91into a plurality of regions and captures an image of each region. The controller60previously stores coordinate information about image capturing positions at which the image of each region is captured. When step S11has ended, the controller60operates the projector moving mechanism30and the camera moving mechanism50on the basis of this coordinate information. Thereby, the camera40is moved to a first image capturing position X1where a first image capture ought to be performed, and the projector20is moved to above the first image capturing position X1(step S12).

Then, the controller60operates the projector20and the camera40to capture an image from the first image capturing position X1(step S13). That is, the camera40capture an image while the projector20applies light downward. Accordingly, an image is captured from the first image capturing position X1at the first focal position H1.

Then, the controller60determines whether or not there is the next image capturing position at which image capture is to be performed (step S14). If there is the next image capturing position (yes in step S14), the controller60operates the projector moving mechanism30and the camera moving mechanism50. Thereby, the camera40is moved to the next second image capturing position X2, and the projector20is moved to above the second image capturing position X2(step S12). Then, the controller60operates the projector20and the camera40to capture an image from the second image capturing position X2(step S13).

In this way, the controller60repeats the movement of the projector20and the camera40(step S12) and the image capture (step S13). Accordingly, images are captured from all the preset image capturing positions at the first focal position H1.

If there remain no image capturing positions at which image capture has not yet been performed (no in step S14), the controller60determines whether or not to change the focal position of the camera40(step S15). Here, if there remains a focal position at which image capture has not yet been performed among the five focal positions H1to H5, the controller60determines to change the focal position of the camera40(yes in step S15). For example, when the image capturing process at the first focal position H1has ended, the controller60determines to change the focal position of the camera40to the next focal position, i.e., the second focal position H2.

In the case of changing the focal position of the camera40, the controller60operates the focal point moving mechanism70to move the focal position of the camera40to a position to which the focal position of the cameral40ought to be changed (step S11). Then, the aforementioned processing in steps S12to S14is repeated. Accordingly, images are captured from all the preset image capturing positions at the changed focal position.

As described above, the controller60repeats the change in the focal position of the camera40(step S11) and the acquisition of captured images from a plurality of image capturing positions (steps S12to S14). Accordingly, five images are captured at the five focal positions H1to H5for each of the plurality of preset image capturing positions.

2.3. Image Processing

Next, image processing for generating an omnifocal image on the basis of a plurality of captured images input from the camera40will be described.

When the aforementioned steps S11to S15are completed, five captured images D1to D5taken at different focal positions are acquired for each image capturing position. However, the surface of the culture solution92in the well91has a concave meniscus formed under the influence of surface tension as illustrated inFIG. 17. Thus, light L emitted from the projector20is refracted when passing through the surface of the culture solution92, and becomes diffused light. Therefore, the five captured images D1to D5have different magnifications. Also, the magnitude of diffusion of the light L differs for each image capturing position. Accordingly, the amounts of variations in magnification among the five captured images D1to D5also differ for each image capturing position.

FIG. 18illustrates the five captured images D1to D5taken from the second image capturing position X2inFIG. 17. The first to fifth captured images D1to D5inFIG. 18are images captured at the first to fifth focal positions H1to H5, respectively, inFIG. 17. Each of the captured images D1to D5includes one or two images out of two cells93held in the well91. The cell93on the right side in the diagram is most sharply in focus in the second captured image D2taken at the second focal position H2. The cell93on the left side in the diagram is most sharply in focus in the fourth captured image D4taken at the fourth focal position H4.

The observation of the cells93is preferably conducted at an in-focus position where the cells are in focus. However, in the case where a plurality of cells93included in one well91are at different heights (different positions in the direction of the optical axis) as illustrated inFIG. 17, it is not possible to focus on all the cells93in one captured image. Thus, the controller60of the image capturing device2combines luminance values of pixels included in the plurality of captured images D1to D5to generate an omnifocal image that focuses on all the cells93and has less blurring.

Under the influence of the aforementioned meniscus, the magnifications of the captured images D1to D5increase as the height of the focal position decreases. Thus, the first captured image D1has a lowest magnification and the fifth captured image D5has a highest magnification among the five captured images D1to D5. The positions of the cells93or the sizes of the cells93in each captured image change with the magnification of the captured image. The controller60of the image capturing device2takes such variations in magnification into consideration when generating an omnifocal image.

FIG. 19is a flowchart illustrating a flow of image processing for generating one omnifocal image from the five captured images D1to D5.

In the case of generating an omnifocal image, the controller60first stores the plurality of captured images D1to D5obtained by the aforementioned image capturing process in the image storage621(step S16). The error corrector622of the controller60corrects errors in each of the captured images D1to D5(step S17). Here, the error corrector622corrects variations in position among the captured images D1to D5, the variations being caused by machine errors in the image capturing device2. For example, if the camera moving mechanism50has a known positioning error, the positions of the captured images D1to D5are each corrected by an amount corresponding to the positioning error. This increases the accuracy of calculation of the amounts of variations in magnification and the amounts of parallel displacement among the five captured images D1to D5in the next step S18.

Next, the magnification variation acquisition part623of the controller60calculates the amounts of variations in magnification and the amounts of parallel displacement among the five captured images D1to D5(step S18). Here, the magnification variation acquisition part623detects how much the sizes of the cells93or the positions of the cells93change among the five captured images D1to D5. In this way, the magnitude of variations in magnification caused by the meniscus of the culture solution92is calculated.

The example of the processing in step S18is the same as that inFIG. 9. In step S18, the magnification variation acquisition part623executes the processing illustrated inFIG. 9.

Next, the image reference value determination part624of the controller60specifies corresponding pixels in the five captured images D1to D5(step S19). Here, on the basis of the amounts of variations in magnification and the amounts of parallel displacement obtained in step S18, pixels that are determined as being located at the same position in the well91in the captured images D1to D5are specified as the corresponding pixels.

Then, the image reference value determination part624of the controller60calculates the definition of each corresponding pixel in the five captured images D1to D5(step S20). The definition is an index indicating the sharpness of the image in the vicinity of that pixel. The definition is, for example, calculated on the basis of a change in luminance among pixels in a certain region centered on that pixel. Alternatively, other values such as the value of dispersion of luminance among peripheral pixels, a maximum luminance value, a minimum luminance value, or the luminance value of the pixel of interest itself may be used as the definition.

The image reference value determination part624of the controller60compares definition among the corresponding pixels in the five captured images D1to D5. On the basis of the comparison result, an image reference value is determined for each coordinates in the omnifocal image (step S21). The image reference value is a parameter indicating the number of a captured image that is to be referenced as the luminance value of each coordinates in the omnifocal image. For example, in the case where the luminance value of the first captured image D1is to be referenced for certain coordinates in the omnifocal image, the image reference value is set to 1.

FIG. 20is a flowchart illustrating an example of processing that is performed in step S21.FIG. 21schematically illustrates the processing performed in step S21. In step S21, definition is first compared between a pixel of interest Pa located at the same coordinates in each of the plurality of captured images and each corresponding pixel Pb in the other captured images that corresponds to the pixel of interest Pa (step S21a). InFIG. 21, the pixels of interest Pa are indicated by closed circles, and the corresponding pixels Pb are indicated by open circles. Each pixel of interest Pa and its corresponding pixels Pb in the other captured images are connected by a broken line inFIG. 21.

According to the present embodiment, the five captured images D1to D5are acquired at one image capturing position. Thus, five pixels of interest Pa and 20 corresponding pixels Pb are set for one coordinates as inFIG. 21.

Next, the image reference value determination part624calculates an evaluation value for each pixel of interest Pa (i.e., for each group consisting of one pixel of interest Pa and four corresponding pixels Pb), the evaluation value indicating the intensity of the definition of the pixel of interest Pa with respect to the definition of the corresponding pixels Pb (step S21b). The evaluation value may be calculated by, for example, dividing the definition of the pixel of interest Pa by a total value obtained from the definition of the four corresponding pixels Pb. The method of calculating the evaluation value is, however, not limited thereto.

Then, the image reference value determination part624determines the number of a captured image to which, among the pixels of interest Pa in the five captured images D1to D5, the pixel of interest Pa having a highest evaluation value belongs, as an image reference value (step S21c). For example, in the case where the pixel of interest Pa that is set in the fourth captured image D4has a highest evaluation value among the five pixels of interest Pa illustrated inFIG. 21, the image reference value of this coordinates is set to 4. In this way, the image reference value of one coordinates is determined. The image reference value determination part624executes the aforementioned processing in steps S21ato S21cfor each coordinates. As a result, the image reference value indicating a captured image that is to be referenced is determined for each coordinates in the omnifocal image DA.

The above-described image processing, however, involves the calculation of coordinates based on the amounts of variations in magnification. Thus, pixels in blurred portions are often selected when determining the image reference values in step S21. If such pixels are selected, for example, a shadow-like region will appear around a focused cell93in the omnifocal image DA that is to be generated in step S23described later.

In order to solve this problem, the shadow removal processing part625of the controller60performs shadow removal processing for adjusting the image reference values that have been once determined (step S22).FIG. 22is a flowchart illustrating an example of the shadow removal processing.FIG. 23schematically illustrates the shadow removal processing. The upper section ofFIG. 23shows an example of the omnifocal image DA generated without executing the shadow removal processing. In this omnifocal image DA, the image of an unfocused cell93in the first captured image D1that intrinsically should not be selected appears in the vicinity of the cell93on the right side.

As illustrated inFIG. 22, the shadow removal processing part625first compares, for each coordinates, an image reference value In and definition En of the coordinates itself with an image reference value Ip and definition Ep of each of another coordinates and peripheral coordinates thereof, the other coordinates being separated from the coordinates by a distance corresponding to variations in magnification in the direction of the variations in magnification (step S12a). The upper section ofFIG. 23shows that the image reference value In of the coordinates of interest Pn is 1, and the definition En thereof is 25. In contrast, the image reference value Ip of coordinates Pp to be compared is 2 and the definition Ep thereof is 80.

The shadow removal processing part625first determines whether or not the relationship between the image reference value In of the coordinates of interest Pn itself and the image reference value Ip of the coordinates Pp to be compared corresponds to a distance Mp between these two coordinates (step S22b). Specifically, the shadow removal processing part625determines whether or not the amount of variations in magnification between the captured images that are referenced by the image reference values In and Ip corresponds to the distance Mp between the two coordinates. If it is determined that they do not correspond, then the image reference value In of the coordinates of interest Pn is retained without alteration.

On the other hand, if it is determined in step S22bthat the relationship between the image reference values In and Ip corresponds to the distance Mp between the two coordinates, then the shadow removal processing part625determines whether or not the definition Ep of each coordinates to be compared, i.e., the coordinates Pp and the peripheral coordinates thereof, is sufficiently greater than the definition En of the coordinates of interest Pn itself (step S22c). Specifically, the shadow removal processing part625determines whether or not the definition Ep of each coordinates to be compared, i.e., the coordinates Pp and the peripheral coordinates thereof, is greater by a preset threshold value or more than the definition En of the coordinates of interest itself. If it is determined that the definition Ep is not sufficiently greater than the definition En, then the image reference value In of the coordinates of interest Pn is retained without alteration.

On the other hand, if it is determined in step S22dthat the definition Ep of each coordinates to be compared, i.e., the coordinates Pp and the peripheral coordinates thereof, is sufficiently greater than the definition En of the coordinates of interest Pn itself, then the image reference value In of the coordinates of interest Pn is replaced by the image reference value Ip of the coordinates Pp to be compared (step S22d). In the example inFIG. 23, the image reference value In of the coordinates of interest Pn is rewritten from 1 to 2. In this case, even if a blurred portion of a cell93is adopted in the omnifocal image DA as illustrated in the upper section ofFIG. 23, it is possible to replace that blurred portion by the luminance value of the captured image taken at the in-focus position (second captured image D2in the example inFIG. 23). By performing such processing for each coordinates, it is possible to remove shadows that may appear in the omnifocal image DA as illustrated in the lower section ofFIG. 23.

Note that the shadow removal processing in step S22may be executed after the generation of the omnifocal image in step S23.

Refer back toFIG. 19. When the image reference value of each coordinates has been determined, then the omnifocal image generator626of the controller60generates an omnifocal image (step S23). Here, the luminance value of each coordinates in the omnifocal image is determined by referencing the luminance values in the captured images indicated by the image reference values determined in steps S21and S22. The luminance value of each coordinates in the omnifocal image may be the luminance value of the pixel of interest Pa itself in the captured image indicated by the image reference value, or may be a different value calculated on the basis of the luminance value of the pixel of interest Pa.

FIG. 24schematically illustrates the relationship between the five captured images D1to D5and the generated omnifocal image DA. As illustrated inFIG. 24, with the technique according to the present embodiment, corresponding pixels (e.g., pixels connected by broken lines inFIG. 24) in the plurality of captured images are specified in consideration of variations in magnification. Then, the image reference value of each coordinates in the omnifocal image DA is determined by comparing definition among the corresponding pixels. Thereafter, the luminance value of the captured image indicated by the image reference value is referenced for each coordinates to generate an omnifocal image. With this technique, it is possible to generate the omnifocal image DA even if the plurality of captured images varies in magnification. It is also possible to generate the omnifocal image DA that reflects the positions and sizes of the cells93with high accuracy, because the captured images are neither enlarged nor reduced.

Refer back toFIG. 19. The tiling processing part627of the controller60arranges (tiles) a plurality of omnifocal images DA acquired with different fields of view by image captures from different image capturing positions. Accordingly, one resultant image representing the entire well91is generated (step S24). As described above, the image processing according to the present embodiment enables generating the omnifocal image DA that reflects the positions and sizes of the cells93with high accuracy. Thus, the positions and sizes of the cells93match with high accuracy between adjacent omnifocal images DA at the time of tiling. This suppresses an image disturbance at the boundaries of the omnifocal images DA in the resultant image.

While embodiments of the present invention have been described thus far, the present invention is not limited to the above-described embodiments.

If the center position of variations in magnification among the captured images is almost fixed, the reciprocal correction of the amounts of parallel displacement may be omitted from the aforementioned step S8or S18. However, if the center position of variations in magnification is liable to change in Embodiment 1, it is desirable to reciprocally correct both of the amount of variations in magnification and the amount of parallel displacement for each captured image as in the above-described embodiments. This allows the positions of the cells93in the captured images D1to D5to match with high accuracy. Also, if the center position of variations in magnification is liable to change in Embodiment 2, it is desirable to calculate both of the amounts of variations in magnification and the amounts of parallel displacement and specify corresponding pixels in the captured images on the basis of the amounts of variations in magnification and the amounts of parallel displacement in step S19as in the above-described embodiment. This increases the accuracy of specifying corresponding pixels in the captured images.

According to the above-described embodiments, the amount of variations in magnification and the amount of parallel displacement are obtained for each pair of adjacent images when the five captured images D1to D5are arranged in order of the focal position. Alternatively, the amount of variations in magnification and the amount of parallel displacement may be obtained for each pair of images that are spaced from each other. However, cells93in each captured image change only by a small amount between two adjacent images. Thus, the same cells93can more easily be associated with each other between those two captured images. Accordingly, the amount of variations in magnification and the amount of parallel displacement can be obtained with higher accuracy by the template matching in step S72.

According to the above-described embodiments, the values of the magnification and the amount of parallel displacement of the selected image are directly determined as the amount of variations in magnification and the amount of parallel displacement between two images in step S75. Alternatively, the amount of variations in magnification and the amount of parallel displacement may be calculated with higher accuracy by approximation of functions such as parabolic fitting.

According to the above-described embodiments, the captured image D1having a lowest magnification is used as a reference when reducing the other captured images D2to D5in step S8or S9. This eliminates the need for interpolation processing because the resolutions of the captured images D2to D5do not decrease. Alternatively, any one of the captured images D2to D5may be used as a reference image when enlarging or reducing the other images in step S8.

Also, the captured images D1to D5may be reduced prior to the above-described processing in step S7or S18in order to reduce computational loads on the controller60.

According to the above-described embodiments, the amounts of variations in magnification among a plurality of captured images are calculated after the captured images are acquired. However, in the case where the amount of variations in magnification does not change, such as where the captured images vary in magnification due to the characteristics of the optical system, the amounts of variations in magnification may be stored in advance in the controller60.

According to the above-described embodiments, the cells93to be observed are held in the plurality of wells91of the well plate9. Alternatively, the cells93may be held in a container other than the well plate9. For example, the cells93may be held in a petri dish. However, in the case where the well plate9is used as in the above-described embodiments, the individual wells91holding the cells93are relatively small. This makes the meniscus of the culture solution92more likely affect the captured images. Therefore, the present invention is particularly useful.

According to the above-described embodiments, the cells93are held along with the culture solution92in the well plate9. Alternatively, the cells93may be held along with a gel culture medium. The gel culture medium also has an irregular surface shape. Thus, the captured images vary in magnification depending on the shooting distance under the influence of the surface shape of the culture medium. Thus, it is not possible to obtain a fine omnifocal image by simply combining the captured images. However, if the amounts of variations in magnification and the amounts of parallel displacement are obtained and reciprocally corrected as in the above-described embodiments, it is possible to align the positions of the cells93in each captured image. Accordingly, an omnifocal image with less blurring can be generated.

According to the above-described embodiments, the cells93that are simple substances are used as objects targeted for image capture. Alternatively, the objects targeted for image capture may be cell agglomerations (spheroids) that are three-dimensional aggregates of a plurality of cells. As another alterative, the objects targeted for image capture may be substances other than cells, which are held along with a liquid or gel substance in the container.

According to the above-described embodiments, the projector20is disposed above objects targeted for image capture, and the camera40is disposed below the objects targeted for image capture. Alternatively, the projector20may be disposed under the objects targeted for image capture, and the camera40may be disposed above the objects targeted for image capture. As another alternative, a configuration is also possible in which the projector20and the camera40are disposed on the same side relative to the objects targeted for image capture, and reflected light of the light emitted from the projector20is incident on the camera40.

According to Embodiment 1 described above, the focal position of the camera40is changed along the optical axis by moving the camera40itself up and down. Alternatively, the position of the camera40may be fixed, and the optical system such as a lens may be moved to change the focal position of the camera40along the optical axis. According to Embodiment 2 described above, the focal position of the camera40is changed by moving some optics included in the optical system41of the camera40. Alternatively, the entire camera40may be moved up and down to change the focal position of the camera40within the well91along the optical axis.

Also, the focal position of the camera40relative to the container may be changed by moving the container holding the objects targeted for image capture up and down. That is, the “moving mechanism” according to the present invention may be any of the mechanism for moving some optics in the camera40, the mechanism for moving the entire camera40, and the mechanism for moving the container.

According to the above-described embodiments, the position of the container holding the objects targeted for image capture is fixed, and the projector20and the camera40are moved in the horizontal direction. Alternatively, the positions of the projector20and the camera40may be fixed, and the container may be moved in the horizontal direction. However, if the surface shape of the culture solution92changes during image capture, it is difficult to accurately calculate the amounts of variations in magnification and the amounts of parallel displacement among the captured images. Therefore, it is preferable for the positions of objects targeted for image capture to be fixed as in the above-described embodiments.

According to Embodiment 1 described above, the height of the camera40can be changed in five stages, and five captured images are acquired for each well91. Alternatively, the number of images to be captured for each well91may be in the range of two to four, or may be six or more. According to Embodiment 2 described above, the focal position of the camera40can be changed in five stages, and five captured images D1to D5are acquired for each field of view. Alternatively, the number of images to be captured for each field of view may be in the range of two to four, or may be six or more.

Each element in the above-described embodiments and variations may be combined appropriately within a range that presents no contradictions.

REFERENCE SIGNS LIST

30Projector moving mechanism

50Camera moving mechanism

51Up-and-down movement mechanism

52Horizontal movement mechanism

623Magnification variation acquisition part

624Image reference value determination part

625Shadow removal processing part

626Omnifocal image generator

627Tiling processing part

P2Image processing program

L Light

DA Omnifocal image

S Maximum value of matching score

M Amount of parallel displacement