JP6389721B2 - Imaging apparatus and imaging method - Google Patents

Description

  The present invention relates to an imaging device and an imaging method for imaging a sample in which a liquid is injected into a sample container.

  In medical and biological science experiments, for example, each well of a plate-shaped sample container (for example, called a microplate or a microtiter plate) provided with a large number of depressions called wells, a petri dish, a dish A liquid or gel-like fluid (for example, a culture solution, a medium, etc.) is injected into a shallow dish-type container referred to as “etc.”, and the cells cultured here are observed and measured as a sample. In recent years, it has been increasingly performed that images are captured by 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.

  In such an imaging device, for example, when illumination light is incident from above the sample and light is transmitted through the bottom of the container to capture an image, the illumination light is refracted by the meniscus action of the injected liquid surface. There is a known problem that the brightness of an image varies depending on the position. More specifically, the image becomes dark at the periphery of the sample near the container wall surface. As a technique for dealing with this problem, for example, Patent Document 1 discloses a configuration in which illumination light is incident on a sample from an oblique direction by a diaphragm provided in an illumination optical system.

JP 2012-147739 A (for example, FIG. 2)

  The size of the sample container varies, and the sample may not fit within the imaging field of view of the imaging device. In such a case, the entire sample can be imaged by changing the imaging position and performing imaging a plurality of times. However, how the influence of the meniscus appears depends on the position of the sample, and the influence of the meniscus is asymmetric in one imaging field of view, particularly in the peripheral portion of the sample. The above prior art is based on the premise that the optical axis of the optical system is coincident with the center of the container, and cannot cope with such a case.

  The present invention has been made in view of the above problems, and an object of the present invention is to provide a technique capable of obtaining an image in which unevenness in brightness due to the influence of a meniscus is suppressed even when a sample larger than an imaging field of view is imaged. To do.

  An imaging apparatus according to the present invention is an imaging apparatus for imaging a sample in which a liquid is injected into a recess having a flat bottom surface provided in a sample container, and in order to achieve the above object, the bottom surface of the recess Holding means for holding the sample container so as to be horizontal, illumination means for making illumination light enter the recess from above the sample container, and receiving light transmitted from the bottom surface of the recess An observation optical system that is provided between an imaging unit that images a sample, the sample container held by the holding unit, and the imaging unit, and that makes light emitted from the bottom surface of the recess enter the imaging unit Positioning means for positioning the imaging means with respect to the depression by integrally moving the imaging means and the observation optical system relative to the sample container in a direction parallel to the bottom surface of the depression. With the front An observation optical system includes an objective lens provided on an optical path between the holding unit and the imaging unit, an aperture stop provided on an optical path between the objective lens and the imaging unit, and the aperture stop A diaphragm position changing unit that moves the lens in the direction of the optical axis of the objective lens, and depending on the relative positions of the imaging means, the observation optical system, and the sample container, the diaphragm position changing unit Change position.

Further, in order to achieve the above object, the imaging method according to the present invention is configured so that a sample container carrying a sample in which a liquid is injected into a recess having a flat bottom surface is placed so that the bottom surface of the recess is horizontal The holding step, the imaging means for imaging the sample, and the observation optical system for allowing the light transmitted from the bottom surface of the recess to enter the imaging means, are integrated with the sample container. A step of positioning the imaging means with respect to the recess by moving it relative to a direction parallel to the bottom; a step of causing illumination light to enter the recess from above the sample container; and from the bottom of the recess And imaging the sample by causing the transmitted light to enter the imaging means using the observation optical system, and the observation optical system is an objective provided on the optical path between the sample container and the imaging means. A lens and the objective lens And an aperture stop provided on an optical path between the imaging means and the aperture position changing unit for moving the aperture stop in the optical axis direction of the objective lens, the imaging means and the observation optical system The position of the aperture stop is changed by the stop position changing unit according to the relative position between the aperture stop and the sample container.

  As will be described in detail later, by arranging the aperture stop of the observation optical system closer to the imaging means than the focal position of the objective lens, the light whose path is bent by the meniscus effect of the liquid surface is condensed and incident on the imaging means. It is possible to make it. This is because, by moving the aperture stop away from the objective lens, it is possible to increase the amount of light received by the imaging means by taking in light from a direction having a particularly large inclination with respect to the optical axis. By utilizing this fact, it is possible to compensate for a decrease in the amount of light due to the influence of the meniscus at the periphery of the sample.

  In the invention configured as described above, it is possible to image different positions of the sample by integrally moving the imaging means and the observation optical system with respect to the sample container. In this case, the appearance of the influence of the meniscus differs depending on the imaging position, but the influence can be reduced by adjusting the position of the aperture stop. That is, a brighter image can be obtained for the peripheral portion of the sample that becomes dark due to the influence of the meniscus by increasing the amount of incident light by adjusting the position of the aperture stop.

  In this way, by combining the configuration for changing the imaging position and the configuration for changing the position of the aperture stop in the observation optical system, it is possible to suppress a light amount decrease due to the influence of the meniscus at each position of the sample, Therefore, according to the present invention, an image with little brightness unevenness can be taken.

  In the present invention, for example, the movable range of the aperture stop may be configured to include a focal position of the objective lens and a position closer to the imaging means than the focal position. If the influence of the meniscus does not appear in the imaging field, it is preferable to arrange the aperture stop at the focal position of the objective lens. When there is an influence of meniscus, it is preferable to arrange an aperture stop closer to the imaging means than the focal position. If the focal position of the objective lens and the position closer to the image pickup means are within the movable range of the aperture stop, it is possible to deal with any situation.

  Further, for example, the imaging unit may be positioned at different positions with respect to the sample container to change the imaging position, and the imaging may be performed a plurality of times. In this way, even if the sample is wider than the imaging field, each part can be included in the image. For example, in one imaging, a part of the sample is captured in the imaging visual field of the imaging means, and the imaging is performed a plurality of times by changing the position, so that each part of the sample can be imaged without omission.

  In this case, for example, in two different imaging operations, it is more preferable to partially overlap the imaging field of the imaging means on the sample. The principle of the present invention is to compensate for the decrease in the amount of light due to the meniscus effect by the increase in the amount of light by adjusting the aperture position. However, the effect of compensating for such variation in the amount of light is not always obtained in the same manner in the entire imaging field of view. That is, how the amount of light increases due to aperture position adjustment depends on the size of the observation optical system. Therefore, in consideration of the size of the sample, even if the light amount fluctuation is offset in one area of one image, the effect may not appear in another area or it may be overcompensated. . If the same region of the sample is included in multiple images and taken, the effect of adjusting the aperture position for that region varies from image to image, so it is possible to take measures such as adopting an image that exhibits the most favorable effect for that region. It becomes.

  For example, you may provide the structure for synthesize | combining the several image imaged in each imaging position. By doing so, an image corresponding to the entire sample can be created from a plurality of images obtained by individually capturing each part.

  Further, for example, a configuration in which the position of the aperture stop is changed based on information in which the relative positions of the imaging unit, the observation optical system, and the sample container are associated in advance with the position of the aperture stop. The appearance of the meniscus can be predicted to some extent from the shape of the container. If the imaging position and the aperture stop position are associated in advance based on the prediction, when positioning the imaging means and the observation optical system, the aperture stop can be immediately placed at an appropriate position.

  Further, for example, the observation optical system may be configured to include an imaging lens that converges the light that has passed through the aperture stop on the optical path between the aperture stop and the imaging unit. According to such a configuration, the image quality can be improved by adjusting the incident direction of the light focused on the imaging means.

  For example, the illumination means may be configured to allow diffused light to enter the concave surface. According to the knowledge of the inventors of the present application, particularly when a sample to be imaged includes cells having a three-dimensional structure, a favorable imaging result is obtained when diffused light is used as illumination light in this way.

  Alternatively, for example, the illumination unit may include a light source and a reduction unit that reduces the degree of diffusion of light incident on the recess from the light source. In particular, when the sample to be imaged has, for example, cells distributed in a plane along the bottom surface of the depression, a good imaging result can be obtained by using illumination light with a suppressed degree of diffusion.

  According to the present invention, by combining the configuration for changing the relative position between the imaging means and the sample container and the configuration for changing the position of the aperture stop in the observation optical system, the meniscus is changed at each position of the sample. It is possible to suppress a decrease in the amount of light due to the influence and to capture an image with little unevenness in brightness.

It is a figure which shows schematic structure of one Embodiment of the imaging device concerning this invention. It is a figure which shows typically the light which permeate | transmits a dish. It is a figure which shows the effect at the time of making the position of an aperture stop variable. It is a figure which shows the relationship between an imaging position and an effective area | region. It is a figure explaining the imaging principle of an original image. It is a flowchart which shows the image creation process in this embodiment. It is a figure which shows the other structural example of an illumination part.

  FIG. 1 is a diagram showing a schematic configuration of an embodiment of an imaging apparatus according to the present invention. The imaging apparatus 1 is an apparatus that images a sample in which a liquid is injected into a recess formed on the upper surface of a dish D that is a sample container. Hereinafter, in order to uniformly indicate the directions in the drawings, XYZ orthogonal coordinate axes are set as shown in FIG. Here, the XY plane represents a horizontal plane and the Z axis represents a vertical axis. More specifically, the (+ Z) direction represents a vertically upward direction.

  The dish D is a shallow dish-shaped container having a flat bottom and a transparent bottom and opening upward, and having a substantially circular shape in plan view, and has a diameter of, for example, about several tens of millimeters. A predetermined amount of a liquid as a medium M is injected into the dish D, and cells C, bacteria, or the like cultured under predetermined culture conditions are cultured in the liquid, and a sample to be imaged by the imaging apparatus 1 is previously stored. Have been made. The medium M may be added with an appropriate reagent, or may be gelled after being injected into the dish D in a liquid state.

  The imaging apparatus 1 includes a holder 11 that holds the dish D in a substantially horizontal posture by contacting the peripheral edge of the lower surface of the dish D that holds the sample, an illumination unit 12 that is disposed above the holder 11, and a lower part of the holder 11. The imaging unit 13 is provided, and a control unit 14 having a CPU 141 that controls the operation of each unit.

  The illumination unit 12 emits appropriate diffused light (for example, white light) toward the dish D held by the holder 11. More specifically, for example, a combination of a white LED (Light Emitting Diode) light source 121 as a light source and a diffusion plate 122 can be used as the illumination unit 12. The sample carried on the dish D is illuminated from above by the illumination unit 12.

  An imaging unit 13 is provided below the dish D held by the holder 11. In the imaging unit 13, an objective lens 131 is disposed immediately below the dish D. The optical axis of the objective lens 131 is directed in the vertical direction (Z direction), and the aperture stop 132, the imaging lens 133, and the imaging device 134 are arranged in order from top to bottom along the optical axis OA of the objective lens 131. Furthermore, it is provided. 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 (Z direction), and these constitute an observation 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. However, as long as the distance (optical path length) between the parts satisfies a predetermined relationship, the optical path is folded by a reflecting mirror or the like. May be.

  The imaging unit 13 can be moved in the XYZ directions by a mechanical drive unit 146 provided in the control unit 14. Specifically, based on a control command from the CPU 141, the mechanical drive unit 146 integrally connects the objective lens 131, the aperture stop 132, the imaging lens 133, and the imaging device 134 that constitute the imaging unit 13 in the X direction and the Y direction. The image pickup unit 13 moves in the horizontal direction with respect to the dish D. With the imaging unit 13 positioned at a predetermined position in the horizontal direction with respect to the dish D, the sample in the dish D is imaged by the imaging unit 13.

  Further, the mechanical drive unit 146 moves the imaging unit 13 in the Z direction, thereby focusing the imaging unit with respect to the imaging target. Specifically, the mechanical drive unit 146 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 dish D where the sample that is the imaging target exists. 134 is integrally moved up and down (moved in the Z direction).

  Furthermore, the mechanical control unit 146 can move the aperture stop 132 up and down independently of the objective lens 131. That is, in this embodiment, the Z-direction distance between the objective lens 131 and the aperture stop 132 is variable. The variable range Rz includes the Z-direction position Zf of the focal point FP of the objective lens 131 and extends in the (−Z) direction, that is, closer to the imaging device 134. Whether or not the variable range Rz includes the (+ Z) direction from the focal point FP, that is, the side closer to the objective lens 131 is arbitrary. In the following, the Z-direction position (aperture position) of the aperture stop 131 is represented by the symbol Zs.

  When the aperture stop 132 moves up and down with respect to the objective lens 131, the imaging lens 133 and the imaging device 134 also move up and down integrally with the aperture stop 131. That is, the distance between the aperture stop 132 and the imaging lens 133 and the distance between the imaging lens 133 and the imaging device 134 are both fixed to the focal length f2 of the imaging lens 133. Accordingly, the telecentric optical system including the aperture stop 132, the imaging lens 133, and the imaging device 134 moves up and down with respect to the objective lens 131. The reason why the aperture stop 132 is moved with respect to the objective lens 131 will be described later.

  The biological sample in the dish D is imaged by the imaging unit 13. Specifically, light emitted from the illumination unit 12 and incident on the medium M from above the dish D illuminates the object to be imaged, and light transmitted downward from the bottom surface of the dish D is condensed 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 two-dimensional image sensor, and converts a two-dimensional image of the imaging object formed on the surface thereof into an electrical signal. As the light receiving element 1341, for example, a CCD sensor or a CMOS sensor can be used.

  When the horizontal extent (XY direction) of the sample in the dish D is smaller than the imaging field of view of the imaging unit 13, the imaging is performed with the optical axis OA of the observation optical system 130 aligned with the central axis of the dish D. Thus, the entire sample can be imaged. On the other hand, when the sample is larger than the imaging field of the imaging unit 13, the mechanical driving unit 146 changes the horizontal position (imaging position) of the observation optical system 130 with respect to the dish D in multiple stages, and the imaging unit 13 each time. By taking an image, the entire sample can be divided and recorded in a plurality of images.

  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) 143 provided in the control unit 14 and converted into digital image data. The CPU 141 executes image processing as appropriate based on the received image data. For example, as described above, the original image captured by dividing into a plurality of images is synthesized to execute a process of creating a synthesized image corresponding to the entire sample.

  The control unit 14 further includes an image memory 144 for storing and storing image data, and a memory 145 for storing and storing programs to be executed by the CPU 141 and data generated by the CPU 141. It may be integral. In addition, the control unit 14 is provided with an interface (I / F) 142. The interface 142 receives an operation input from the user, presents information such as a processing result to the user, and exchanges data with an external apparatus connected via a communication line.

  FIG. 2 is a diagram schematically showing light transmitted through the dish. A medium M is injected into the dish D, and a generally downward meniscus is formed on the liquid surface. In the central part of the dish D where the surface S of the medium M can be regarded as almost flat, the incident light L travels straight into the medium M, so there is no need to consider the influence of the meniscus. In this case, as shown in FIG. 2A, the distance between the objective lens 131 and the aperture stop 132 is the focal length f1 of the objective lens 131, and the aperture stop 132 is at a position corresponding to the focal point FP of the objective lens 131 (that is, The sample can be imaged satisfactorily by a general observation optical system arranged at Zs = Zf).

  On the other hand, as shown in FIG. 2B, when the imaging field includes a region close to the peripheral portion having the liquid level variation due to the meniscus, the incident light L is refracted on the medium surface S, and the optical path in the sample is change. In a general downward meniscus, the optical path is bent in the direction from the center of the dish D toward the peripheral edge. For this reason, the inclination of the principal ray when the light bent in this way enters the aperture stop 132 disposed below increases, and as a result, the amount of light that can pass through the aperture stop 132 decreases. That is, the image is darker at the peripheral edge of the dish D than at the center.

  In this embodiment, by changing the position of the aperture stop 132 in the Z direction, such a decrease in the amount of light is suppressed, and a bright image can be obtained even at the peripheral portion of the dish D. The concept will be described below.

  FIG. 3 is a diagram showing the effect when the position of the aperture stop is variable. When imaging the sample region where the influence of the meniscus appears as described above, the aperture stop 132 is closer to the imaging device 134 than the focal point FP of the objective lens 131, as shown in FIG. Consider the case where it is arranged on the (−Z) side. In this case, the light incident on the central portion of the objective lens 131 is not greatly affected. However, in the peripheral portion of the objective lens 131, the inclination of the principal ray incident on the aperture stop 132 from the objective lens 131 is small, so that the aperture is reduced. The diaphragm 132 can pass more light. That is, light refracted by the meniscus can be collected and incident on the imaging device 134, and a brighter image can be obtained.

  FIG. 3B is a diagram illustrating the relationship between the position taken in the radial direction from the dish center and the amount of light incident on the imaging device 134 from the position. A curve indicated by a solid line in FIG. 3B represents the light amount when the aperture stop 132 is disposed at the focal position of the objective lens 131 (that is, Zs = Zf). In this case, in the region Rm where the meniscus at the peripheral edge of the dish D is generated, the amount of light is reduced with respect to the central portion. As it approaches the wall surface of the dish D, the refraction of the incident light L increases, and the decrease in the amount of light becomes significant.

  On the other hand, when the aperture stop 132 is located farther than the focal position of the objective lens 131, that is, on the imaging device 134 side, as shown by a dotted line in FIG. 3B, a region facing the peripheral portion of the objective lens 131. Increases the light intensity. For this reason, in the meniscus region Rm, a decrease in the amount of light is suppressed, and a region in which the amount of light can be regarded as uniform spreads. That is, in this region, the light amount decrease due to the influence of the meniscus is canceled and corrected by the light amount increase due to the aperture position adjustment.

  However, due to the rotational symmetry about the optical axis of the observation optical system 130, the light quantity decreases in a portion where there is no influence of meniscus and correction is not necessary. Further, the degree of light quantity reduction due to the influence of the meniscus and the degree of light quantity increase due to aperture position adjustment are both position dependent. For these reasons, the effective region Ra in which the above correction action functions effectively in a combination of a single imaging position and a single aperture position is limited.

  Therefore, it is preferable that the position of the aperture stop 132 at the time of imaging is dynamically set according to the imaging position, that is, the horizontal position of the imaging unit 13. In addition, it is preferable that a plurality of images are captured at different imaging positions, and an area corresponding to each effective area Ra is extracted and combined to form an image of the entire sample.

  FIG. 4 is a diagram showing the relationship between the imaging position and the effective area. According to the knowledge of the inventor of the present application, as shown in FIG. 4, the incident light L is refracted by the meniscus and emitted from the bottom surface of the dish D, and passes through the objective lens 131, the aperture stop 132, and the imaging lens 133 to the imaging device 134. When the inclination angle θ of the principal ray of the light emitted from the bottom surface of the dish D with respect to the optical axis OA coincides with the inclination angle φ of the principal ray incident on the aperture stop 132, the incident light L is incident. A particularly good light quantity correction effect can be obtained at and near the position. In other words, among the images captured at a certain imaging position and a certain aperture position, a position where the above condition is satisfied and its neighboring area are effective areas Ra.

  In this embodiment, a preferable aperture position Zs corresponding to the imaging position and a range of the effective area Ra at that time are obtained in advance, and an aperture stop 131 is arranged at the aperture position Zs corresponding to the imaging position at the time of imaging. Take an image. Then, by extracting a portion corresponding to the effective area Ra from the obtained image, a partial image of the sample from which the influence of the meniscus is eliminated can be obtained. When the bottom shape of the dish D is circular, the imaging position can be represented by a distance Ro from the dish center Cd to the optical axis OA of the imaging optical system 130 due to the rotational symmetry of the meniscus.

  The appearance of the meniscus may vary depending on the viscosity of the liquid, the wettability with the dish wall surface, and the like. Therefore, it is preferable to obtain a correspondence relationship between the throttle position Zs and the effective area Ra for each type of liquid that becomes the medium M and the injection amount. The obtained correspondence relationship can be stored as a reference information in the memory 145 as a table, for example. During imaging, this table can be referred to determine the aperture position Zs and the effective area Ra according to the designated imaging position.

  Further, with respect to the aperture position Zs, the entire imaging unit 13 moves in the Z direction for focusing, and the Z direction position Zf of the focal point FP also moves up and down accordingly, so that in the fixed coordinate system, It is preferably defined as a relative value to the position Zf of the focal point FP. When the appropriate position of the aperture position Zs is defined in the fixed coordinate system, correction corresponding to the variation of the focal position is necessary.

Next, a process in a case where a sample having a larger planar size than the imaging field of the imaging unit 13 is imaged and an image corresponding to the entire sample is created will be described. The basic idea is
(1) Imaging is performed a plurality of times while changing the imaging position, and the sample is divided into a plurality of original images to be captured,
(2) When a meniscus is included in the imaging field of view, a decrease in the amount of light is suppressed by adjusting the position of the aperture stop 132 during imaging.
(3) Extracting and synthesizing a portion not affected by meniscus from each original image by image processing after imaging, and creating an image representing the entire sample.
That's it.

  FIG. 5 is a diagram for explaining the principle of capturing an original image. More specifically, FIG. 5A shows a dividing method when the sample is divided into a plurality of original images. As shown in FIG. 5A, in this example, the inside of the dish D is imaged by dividing it into 29 original images. The imaging ranges of adjacent original images are arranged so as to partially overlap each other in order to prevent missing images, that is, sample regions not included in any original image. In addition, although the actual shape of the imaging range of each original image is a rectangle, in FIG. 5A, it is shown as a rectangle with rounded corners to make it easy to see the overlap of the original images. Further, the number and arrangement of the original images can be appropriately changed depending on the size and shape of the dish.

  The imaging ranges Rc1 (9 places) indicated by the solid line are arranged sufficiently inside the peripheral edge of the original image dish D, and it is not necessary to consider the influence of the meniscus. Therefore, the aperture stop 132 is disposed at the focal position of the objective lens 131 (Zs = Zf). Image information of the central portion of the sample can be obtained by imaging each of these imaging ranges Rc1.

  The imaging ranges Rc2 (16 places) indicated by dotted lines are arranged so as to surround the imaging range Rc1, and each include a part of the periphery of the dish D. During the imaging of these imaging ranges Rc2, the diaphragm position is adjusted according to the magnitude of the meniscus influence.

  In addition, imaging ranges Rc3 (four locations) indicated by broken lines are ranges of original images that are supplementarily captured for improving image quality. Although the influence of the meniscus does not appear in the imaging range Rc1 set in the center portion of the dish D, the image quality may deteriorate at the peripheral portion of the original image due to, for example, vignetting or aberration in the observation optical system 130. In order to use the image information of the central portion of the imaging range with better image quality, the imaging range Rc3 is set so as to cover the boundary portion between the imaging ranges Rc1 in the center of the dish D. The aperture stop 132 is disposed at the focal position of the objective lens 131 even during imaging of the imaging range Rc3.

  FIG. 5B shows an example of the data structure of a reference table that is referred to during imaging. In order to divide and sample the sample in one dish D into a plurality of original images as described above, for example, a table shown in FIG. 5B is referred to. Image numbers (1 to 29) are assigned to the original images corresponding to the above-described 29 imaging ranges in an appropriate order. For each original image specified by the image number, information indicating the imaging position (XY coordinates) for obtaining the original image, the set value of the aperture position Zs, and the effective area Ra of the original image (for example, Are coordinated with each other in a table. When an original image having a certain image number is captured, a set value of the aperture position Zs obtained by referring to the table is applied, and a partial image corresponding to the effective area Ra is extracted from the captured original image. By combining and reconstructing the partial images extracted from the original images, an image corresponding to the entire sample can be obtained.

  The effective area Ra in each original image is set so that the entire area in the dish D can be covered by connecting the effective areas extracted from each original image.

  FIG. 6 is a flowchart showing image creation processing in this embodiment. This image creation process is realized by the CPU 141 executing a control program stored in advance in the memory 145 and causing each unit of the apparatus to perform a predetermined operation.

  First, the dish D carrying a sample from the outside is carried into the imaging apparatus 1 and set in the holder 11 (step S101). Next, the information regarding the sample given from a user or an external host apparatus is received via the interface 142 (step S102). The sample information includes, for example, the size of the dish D, the type and amount of medium M to be injected, the type of cell C to be cultured, and the like.

  Based on this information and information stored in advance in the memory 145 as a reference table, the CPU 141 captures an imaging schedule including a combination of imaging position allocation and imaging sequence, aperture stop 132 position setting, imaging resolution, illumination conditions, and the like. Is created (step S103). Then, based on the imaging schedule, the imaging unit 13 executes imaging a plurality of times while sequentially changing and setting the imaging position, the aperture position, and the like (step S104), thereby acquiring a plurality of original images.

  Based on the plurality of original images thus obtained, the CPU 141 performs image processing, thereby creating an image corresponding to the entire image of the sample. Specifically, partial images corresponding to the effective area Ra are extracted from each original image (step S105), and a composite image obtained by synthesizing them is created (step S106). In the obtained composite image, the continuity of image elements may be impaired at the joint between partial images extracted from different original images. Therefore, the discontinuity of the seam is eliminated by the smoothing process (step S107). Then, the composite image created in this way is stored in the image memory 144 and is output to the external device as an output image (step S108).

  In the created output image, the decrease in the amount of light at the peripheral edge due to the influence of the meniscus is corrected by the aperture position adjustment, and an image with little unevenness in the amount of light in the entire dish D is obtained by the image creation process described above.

  As described above, in this embodiment, the dish D corresponds to the “sample container” of the present invention, and the holder 11 functions as the “holding means” of the present invention. The illumination unit 12 functions as the “illuminating unit” of the present invention, while the imaging device 134 functions as the “imaging unit” of the present invention. Further, the mechanical drive unit 146 has functions as “positioning means” and “aperture position changing means” of the present invention. The CPU 141 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 above embodiment, an imaging lens is provided in the observation optical system. However, even in an observation optical system that does not use an imaging lens, the present invention is applied to the arrangement of the objective lens and the aperture stop. It is possible to obtain the same effects as those of the above embodiment.

  In the imaging unit 13 of the above embodiment, imaging is performed using the light receiving element 1341 that is a two-dimensional image sensor. However, the linear image sensor extending in one direction is scanned and moved in a direction orthogonal to the two-dimensional image. Even if an image pickup unit that picks up an image is used, the same effect as described above can be obtained.

  Moreover, although the illumination part 12 of the said embodiment makes diffused light enter into the dish D as illumination light, depending on the kind of cell in a sample, illumination light with a lower diffusivity, for example, parallel light, is made incident. In some cases, a clearer image can be obtained. In order to cope with such a case, for example, the following configuration may be adopted.

  FIG. 7 is a diagram illustrating another configuration example of the illumination unit. In this modification, only the configuration of the illuminating unit is different from that in the above embodiment. Therefore, the description of the same configuration as that in the above embodiment is omitted or the same reference numerals are given and the description thereof is omitted. The illumination unit 22 of this modified example generates illumination light including many components close to parallel to the optical axis OA of the observation optical system 130 by the combination of the light source 221 and the diaphragm 222 and irradiates the dish D. In this case, the diaphragm 222 corresponds to the “reducing portion” of the present invention.

  Moreover, although the imaging device 1 of the said embodiment is a structure which moves the imaging part 13 with respect to the dish D hold | maintained at the holder 11, even if it fixes the imaging part 13 and moves the dish D, it is technical. Is equivalent to. Also, as a focusing method, instead of the configuration in which the entire imaging unit 13 is moved up and down as described above, for example, a method for adjusting the distance between lenses of an optical system including a plurality of lenses may be applied.

  The present invention can be particularly suitably applied to a field that requires imaging of a sample including cells, such as a dish used in the medical / biological science field, but the application field thereof is the medical / biological field. It is not limited to.

DESCRIPTION OF SYMBOLS 1 Imaging device 11 Holder (holding means)
12 Illumination part (illumination means)
DESCRIPTION OF SYMBOLS 13 Imaging part 14 Control part 131 Objective lens 132 Aperture stop 133 Imaging lens 134 Imaging device (imaging means)
141 CPU (image processing means)
146 Mechanical drive (positioning means, aperture position changing means)
D dish (sample container)
FP Focus Ra Effective area Rz Variable range (of aperture position Zs) Zf Focus position Zs Aperture position

Claims (12)

In an imaging device for imaging a sample in which a liquid is injected into a recess having a flat bottom surface provided in a sample container,
Holding means for holding the sample container so that the bottom surface of the recess is horizontal;
Illuminating means for allowing illumination light to enter the recess from above the sample container;
Imaging means for receiving light transmitted from the bottom surface of the recess and imaging the sample;
An observation optical system that is provided between the sample container held by the holding unit and the imaging unit, and that makes the light emitted from the bottom surface of the recess enter the imaging unit;
Positioning means for positioning the imaging means with respect to the depression by moving the imaging means and the observation optical system integrally relative to the sample container in a direction parallel to the bottom surface of the depression. ,
The observation optical system includes an objective lens provided on an optical path between the holding unit and the imaging unit, an aperture stop provided on an optical path between the objective lens and the imaging unit, and the aperture An aperture position changing unit that moves the aperture in the optical axis direction of the objective lens;
An imaging apparatus in which the aperture position changing unit changes the position of the aperture stop in accordance with a relative position between the imaging means and the observation optical system and the sample container.   The imaging apparatus according to claim 1, wherein the movable range of the aperture stop includes a focal position of the objective lens and a position closer to the imaging means than the focal position.   The imaging apparatus according to claim 1, further comprising: an image processing unit that synthesizes a plurality of images captured by the imaging unit with different relative positions of the imaging unit, the observation optical system, and the sample container.   The throttling position changing unit changes the position of the aperture stop based on information in which a relative position between the imaging unit and the observation optical system and the sample container and a position of the aperture stop are associated in advance. 4. The imaging device according to any one of 3.   The said observation optical system is provided with the imaging lens which converges the light which passed the said aperture stop on the said imaging means on the optical path between the said aperture stop and the said imaging means. Imaging device.   The imaging device according to claim 1, wherein the illuminating unit causes diffused light to enter the recess.   The imaging device according to claim 1, wherein the illuminating unit includes a light source and a reduction unit that reduces a diffusion degree of light incident on the depression from the light source. Holding a sample container carrying a sample in which a liquid is injected into a recess having a flat bottom so that the bottom of the recess is horizontal; and
An imaging means for imaging the sample and an observation optical system for allowing light transmitted from the bottom surface of the recess to enter the imaging means, and a direction parallel to the bottom surface of the recess with respect to the sample container To position the imaging means relative to the recess,
A step of causing illumination light to enter the recess from above the sample container;
Imaging the sample by allowing the light transmitted from the bottom surface of the recess to enter the imaging means by the observation optical system,
The observation optical system includes an objective lens provided on an optical path between the sample container and the imaging unit, an aperture stop provided on an optical path between the objective lens and the imaging unit, and the aperture An aperture position changing unit that moves the aperture in the optical axis direction of the objective lens;
An imaging method in which the position of the aperture stop is changed by the stop position changing unit in accordance with a relative position between the image pickup means and the observation optical system and the sample container.   The imaging method according to claim 8, wherein the imaging unit is positioned at different positions with respect to the sample container a plurality of times, and imaging is performed each time.   The imaging method according to claim 9, wherein in one imaging, a part of the sample is captured in an imaging field of view of the imaging unit.   The imaging method according to claim 10, wherein an imaging field of view of the imaging unit is partially overlapped on the sample in two different imaging operations.   The imaging method according to claim 9, further comprising a step of synthesizing a plurality of images obtained by imaging a plurality of times by image processing.

Images