JP2017005593A - Imaging apparatus and imaging method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To photograph an image of a clear contour of a cell by irradiating a carrier carrying a cell with illumination light and receiving transmission light that transmits the carrier.SOLUTION: An imaging apparatus for photographing an image of a cell carried by a carrier includes: an illumination part for illuminating the carrier with illumination light; an imaging optical system disposed so as to direct an optical axis toward the carrier; and an imaging part for photographing a cell by receiving transmission light transmitting the carrier that receives irradiation of the illumination light with the imaging optical system to photograph the cell. The numerical aperture of the illumination light is less than or equal to 0.3.SELECTED DRAWING: Figure 7

Description

  The present invention relates to an imaging apparatus and an imaging method for imaging a cell by irradiating a carrier carrying a cell with illumination light and receiving transmitted light transmitted through the carrier.

  In medical and biological science experiments, for example, a cell to be observed is imaged with a CCD camera or the like and converted into data, and various image processing techniques are applied to the image data for observation and analysis. It is becoming. Conventionally, cells have been dyed with an appropriate dye in order to improve the visibility of the cells, but in order to suppress damage to the cells, it may be required to take an image without staining. However, since the cytoplasm of unstained cells is almost colorless and transparent, it is difficult to obtain a high-quality image. In particular, when bright-field imaging is performed with a cell carried on a carrier such as a transparent container as a focus position, it is difficult to obtain a high-contrast image (see, for example, Patent Document 1).

JP 2008-020498 A

  Therefore, in Patent Document 1, the cytoplasm of the unstained cell is substantially transparent, but paying attention to the fact that there is a difference in refractive index inside, the focus position is shifted from the in-focus position. Contrast is improved. However, no consideration is given to the relationship with illumination light. That is, as will be described in detail later, when the numerical aperture (NA) of the illumination light increases, sufficient contrast cannot be obtained.

  The present invention has been made in view of the above problems, and irradiates a carrier carrying a cell with illumination light and receives transmitted light transmitted through the carrier to capture an image with a clear outline of the cell. It is an object to provide an imaging apparatus and an imaging method capable of performing the above.

  1st aspect of this invention is an imaging device which images the image of the cell carry | supported by a support body, Comprising: The illumination part which irradiates illumination light to a support body, and the imaging arrange | positioned toward the support body An imaging unit that has an optical system and receives the transmitted light that passes through the carrier that is irradiated with the illumination light by the imaging optical system and images the cell, and the numerical aperture of the illumination light is 0.3 or less It is characterized by.

  According to a second aspect of the present invention, there is provided an imaging method, the step of irradiating a carrier carrying cells with illumination light, and the transmitted light transmitted through the carrier subjected to illumination light being received to image cells. And a numerical aperture of illumination light is set to 0.3 or less.

  As described above, according to the present invention, the numerical aperture (NA) of the illumination light is used to illuminate the carrier carrying the cell with the illumination light and receive the transmitted light transmitted through the carrier to image the cell. Greatly affects the contrast of the image to be captured. The specific influence will be described in detail later with reference to FIG. 7 and FIG. 8, but by setting the numerical aperture (NA) of illumination light to 0.3 or less, the contrast value is increased and the outline of the cell is clear. It is possible to capture a simple image.

1 is a diagram illustrating a schematic configuration of an imaging apparatus according to the present invention. It is a figure which shows typically the positional relationship of the focus of the objective lens at the time of imaging, and a cell. It is a figure which shows the relationship between a focus position and the contrast value of an image. It is a figure for demonstrating the change of the image by the focus position of an objective lens. It is a flowchart which shows the process for producing a difference image. It is a flowchart which shows 1st Embodiment of the image process for producing a difference image. It is a flowchart which shows 2nd Embodiment of the image process for producing a difference image. It is a figure explaining the difference image creation process in 2nd Embodiment in detail. It is a figure which shows an example of the influence which the numerical aperture (NA) of illumination light has on the contrast value of an image. It is a figure which shows the other example of the influence which the numerical aperture (NA) of illumination light has on the contrast value of an image.

  Hereinafter, after describing the overall configuration and operation of the imaging apparatus according to the present invention and the image processing method for the image captured by the imaging apparatus, a specific configuration for clearly capturing the image will be described.

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

  The well plate WP is generally used in the fields of drug discovery and biological science. The well plate WP is formed in a cylindrical shape with a substantially circular cross section on the top surface of a flat plate, and the bottom surface is transparent and flat. Are provided. Although the number of wells W in one well plate WP is arbitrary, for example, 96 (12 × 8 matrix arrangement) can be used. The diameter and depth of each well W is typically about several mm. Note that the size of the well plate, the cross-sectional shape of the well, the number of arrangements, and the like of the well targeted by the imaging apparatus 1 are not limited to these, and may be, for example, 384 holes.

  A predetermined amount of a liquid as a medium is injected into each well W of the well plate WP, and cells, bacteria, and the like cultured in the liquid under predetermined culture conditions are imaging targets of the imaging device 1. The medium may be one to which an appropriate reagent has been added, or it may be in a liquid state and gelled after being placed in the well W. Commonly used liquid volume is about 50 to 200 microliters.

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

  The illumination unit 12 emits illumination light Li toward the well plate WP held by the holder 11. For example, white light can be used as the illumination light Li. For the purpose of imaging unstained cells, which will be described later, the illumination light Li is preferably illumination light having a small numerical aperture (NA), for example, parallel light. The illumination unit 12 illuminates the sample in the well W provided on the well plate WP from above. The numerical aperture (NA) of the illumination light Li can be set to be equal to or less than the numerical aperture of the imaging optical system 130, for example. Note that the numerical aperture (NA) of the illumination light Li is desirably 0.3 or less in order to clarify the captured image, and the reason will be described in detail later.

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

  The imaging unit 13 can be moved 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 imaging unit 13 moves in the horizontal direction with respect to the well W. When the imaging target in one well W is imaged, the mechanical drive unit 146 positions the imaging unit 13 in the horizontal direction so that the well W is included in the imaging field of 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 well W where the sample that is the imaging target is present. 134 is moved up and down integrally.

  The mechanical drive unit 146 moves the illumination unit 12 integrally with the imaging unit 13 in the XY direction when moving the imaging unit 13 in the XY direction. That is, the illumination unit 12 is arranged so that the optical center thereof is substantially coincident with the optical axis OA of the imaging optical system 130. When the imaging unit 13 including the objective lens 131 moves in the XY direction, the illumination unit 12 is interlocked with this. To move in the XY direction. Thereby, in any well W being imaged, the illumination condition can be kept constant and the imaging condition can be maintained satisfactorily. Instead of the configuration in which the well plate WP is fixed and the imaging unit 13 is moved, a configuration in which the imaging unit 13 is fixed and the well plate WP is moved may be employed.

  The imaging unit 13 performs bright field imaging of the sample in the well W. Specifically, light emitted from the illumination unit 12 and incident on the liquid from above the well W illuminates the object to be imaged, and light transmitted downward from the bottom surface of the well W is collected by the objective lens 131 and the aperture stop 132. The image of the imaging object is finally formed on the light receiving surface of the imaging device 134 via the imaging lens 133, and this is received by the light receiving element 1341 of the imaging device 134. The light receiving element 1341 is a 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.

  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. 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.

  Next, the operation of the imaging apparatus 1 configured as described above will be described. The imaging device 1 can take images of cells, bacteria, cell clumps (spheroids) and the like cultured in each well W of the well plate WP, and use the taken images for various observations and analysis. be able to.

  Here, for example, for the purpose of examining the growth state of the cells cultured in the well W, a case is considered in which the cells are imaged without staining, the outline is specified, and the number and size of the cells are observed. Conventionally, for the purpose of optically observing cells that are nearly colorless and transparent, cells have been dyed with an appropriate dye. However, there are cases where it is necessary to perform imaging without damaging the cells for the purpose of observing the cells alive or examining changes over time. In such a case, it is necessary to image the cells without staining.

  In bright-field imaging like the imaging device 1, it is difficult to image cells that are nearly transparent with high contrast. For imaging in such a case, for example, a method using a phase contrast microscope is conceivable, but the apparatus configuration is complicated and expensive. In applications where it is sufficient that the outline of the cell can be specified as described above, it is preferable that an image can be acquired with a simpler apparatus configuration. The imaging apparatus 1 can meet such a demand.

  FIG. 2A and FIG. 2B are diagrams for explaining a problem in bright-field imaging of transparent cells. More specifically, FIG. 2A is a diagram schematically showing the positional relationship between the focal point F and the cell C of the objective lens 131 at the time of imaging, and FIG. 2B is a diagram showing the relationship between the focal position and the contrast value of the image. . As shown in FIG. 2A, consider a case where a substantially transparent cell C is imaged while the focal point F of the objective lens 131 is changed in the direction of the optical axis OA (in this case, the Z direction). The Z-direction position of the cell C to be imaged is represented by a symbol Zc, and the Z-direction position of the focal point F of the objective lens 131 is represented by a symbol Zf. The objective lens 131 in the Z direction is positioned below the focal position Zf by the focal length f.

  As shown in FIG. 2B, the contrast value becomes minimal when the focal position Zf in the Z direction substantially coincides with the position Zc of the cell C, and the contrast value becomes high before and after that. Such knowledge is also described in, for example, Patent Document 1 described above. For the following explanation, in the contrast value profile with respect to the focal position shown in FIG. 2B, the values of the focal position Zf giving two local maximum values sandwiching the local minimum value are represented by symbols Za and Zb.

  FIG. 3 is a diagram for explaining the change of the image depending on the focal position of the objective lens. Differences in images due to differences in focal position will be described in the following three cases: (a) Zf> Zc, (b) Zf <Zc, and (c) Zf = Zc.

  First, the case (c) where Zf = Zc will be described. This case corresponds to a focused state where the focal plane FP of the objective lens 131 is on the cell C. In this case, as shown in the image example, the image of the cell C is clear even to a fine structure, but the difference in density is small and the contrast value of the image is minimized. When the luminance histogram for each pixel is taken, the variation is small. For this reason, for example, it is not suitable for an application that automatically performs binarization of an image based on pixel values or extraction of a cell contour.

  In the case (a), the focal plane FP of the objective lens 131 is positioned in the (+ Z) direction (upward) from the cell C, that is, the cell C is closer to the objective lens 131 than the focal length f of the objective lens 131. Corresponds to the case. In this case, the objective lens 131 sees the focal plane FP through the cell C. In the case (b), the focal plane FP of the objective lens 131 is located in the (−Z) direction (downward) with respect to the cell C, that is, the cell C is at a position farther than the focal length f of the objective lens 131. It corresponds to. In these cases, due to the lens action by the transparent cell C, a change in luminance appears particularly at the peripheral edge of the cell C.

  That is, in the case (a) in which the cell C is closer to the focal length f of the objective lens 131, the luminance is higher (brighter) at the periphery of the cell C, while the cell C is farther than the focal length f of the objective lens 131. In the case (b), the luminance is low (darker) at the periphery of the cell C. For this reason, the histogram of pixel values spreads to the high luminance side in case (a), and spreads to the low luminance side in case (b). Thus, in the case (a) and the case (b), opposite luminance changes appear in the peripheral portion of the cell C. In these cases, since the focal point F of the objective lens 131 does not match the cell C, the sharpness of the image of the cell C is lower than the image of the case (c).

  A difference in units of pixels is obtained between an image obtained by imaging in case (a) and an image obtained by imaging in case (b). In a difference image in which the difference between pixel values is a new pixel value of each pixel, the distribution of luminance values in the histogram is widened, and the contrast value of the image is increased. In particular, the difference in brightness is emphasized at the peripheral portion of the cell C where the difference in luminance is large. Therefore, although the difference image is not always clear about the internal structure of the cell C, the difference image is an image showing the outline portion with high contrast.

The pixel value Pd of each pixel of the difference image is expressed by the following equation using pixel values Pa and Pb of the same pixel of the image obtained by imaging in case (a) and case (b) and a constant K:
Pd = Pa-Pb + K
Can be represented by The constant K is an offset value for avoiding the pixel value Pd from becoming a negative value depending on the content of the image. The constant K may be determined in advance based on an expected distribution of pixel values, or may be determined dynamically according to the calculation result of the difference (Pa−Pb) in pixel values. However, a single constant K is applied to each pixel in one difference image. In the above equation, at least one of the pixel values Pa and Pb may be multiplied by an appropriate coefficient.

  In this way, in order to obtain an image with a clear outline of the cell C, a difference image between the two images picked up with different focal positions may be created. However, for the purpose of emphasizing the contour, the two images need to be respectively captured at two focal positions sandwiching the focal position (Zf = Zc) that takes the minimum value in the contrast value profile with respect to the focal position. is there. In order to maximize the contour enhancement effect, it is more preferable that two images are captured at a focal position close to the two focal positions Za and Zb that take the maximum contrast value in the profile shown in FIG. 2B. preferable. Hereinafter, specific processing contents for achieving such an object will be described.

  FIG. 4 is a flowchart showing a process for creating a difference image. This process is realized by the CPU 141 executing a control program stored in advance in the memory 145, for example, and controlling each part of the apparatus based on the control program. First, a well plate WP including a well W in which cells to be imaged are cultured is set in the holder 11 (step S101). Next, the imaging unit 13 is set to a predetermined initial position (step S102). The initial position of the imaging unit 13 in the horizontal direction is a position where the center of the well W to be imaged and the optical axis OA of the imaging optical system 130 coincide. The initial position in the vertical direction is the position closest to the bottom surface of the well plate WP in the variable range of the imaging unit 13 in the vertical direction.

  Next, the well 13 is picked up by the image pickup unit 13 to obtain one original image (step S103), the image pickup unit 13 is moved downward one step at a predetermined interval (step S105), and image pickup is performed again (step S105). Step S103). Such movement and imaging of the imaging unit 13 step by step are repeated until the imaging unit 13 reaches the imaging end position, that is, the lowest position in the variable range and the farthest from the bottom surface of the well plate WP (see FIG. Step S104). As a result, a plurality of original images captured with the focal positions of the imaging optical system 130 different from each other in the vertical direction are acquired. These original images are stored and saved in the image memory 144.

  The size of the cell C to be imaged is generally about several μm to several tens of μm, and the movement of the imaging unit 13 corresponding to this is about 2 μm to 10 μm per step, for example.

  For each original image obtained in this way, the CPU 141 calculates the contrast value of the image (step S106). Several methods are known for calculating the contrast value, but any method can be applied as long as the same method is used for each original image. For example, the contrast value of the original image can be determined by the difference between the pixel value of the highest luminance pixel in the original image and the pixel value of the lowest luminance pixel. The same applies to “contrast value” in FIGS. 7 and 8 described later.

  Subsequently, based on the two original images extracted from the plurality of captured original images, the CPU 141 creates a difference image by predetermined image processing (step S107). There are two embodiments of the difference image creation method based on mutually different principles, which will be described later. The created difference image is an image in which the outline of the cell C is emphasized, and is stored and saved in the image memory 144, and is output as an output image via the interface 142 as necessary (step S108). As an image output mode, the difference image may be displayed on a display provided as a part of the interface 142, and the image data of the difference image is output to an external device or an external storage medium. It may be a thing.

  FIG. 5A and FIG. 5B are flowcharts showing two modes of difference image creation. More specifically, FIG. 5A shows a first embodiment of image processing for creating a difference image, and FIG. 5B shows a second embodiment of image processing for creating a difference image. In these embodiments, the contrast value of each original image calculated as described above is a maximum value or a minimum value in a virtual profile in which the contrast values are arranged along the set value of the focal position when the original image is captured. Based on the value, the original image that is the basis of the difference image is extracted. Note that it is sufficient that the maximum value or the minimum value in the profile can be obtained by any method, and processing for actually creating the profile is not required.

  In the process of the first embodiment shown in FIG. 5A, two maximum values of the contrast value in the profile are detected (step S201). The maximum value can be detected, for example, by obtaining a point in the profile that changes from a phase where the contrast value increases to a phase where the contrast value decreases as the focus position setting value changes. Then, the set values of the two focal positions that give these maximum values are obtained (step S202). These two set values should be close to the maximum values at the symbols Za and Zb in FIG. 2B.

  By extracting two original images corresponding to the two maximum values obtained in this way, obtaining a difference between the original images for each pixel, and adding a constant K as an offset value. Then, the pixel value of each pixel constituting the difference image is obtained (step S203). A difference image is obtained by arranging the pixels having the obtained pixel values in a predetermined arrangement and reconstructing the image (step S204).

  As described above, the maximum value of the contrast value in the profile is not used for the subsequent calculation. Therefore, strictly speaking, the processing for obtaining the maximum value of the contrast value in step S201 is not necessarily required, and it is only necessary to know the set value of the focal position corresponding to the maximum value.

  As described above, in this embodiment, two maximum values in the profile of the contrast value of the image with respect to the setting value of the focal position among the plurality of original images captured while changing the setting value of the focal position in multiple stages are supported. A difference image is created based on the two original images. In this case, it is only necessary to obtain the contrast value directly from the captured original image, find the two peak portions in the profile, and obtain the maximum value thereof, so it is not necessary to know the in-focus position of the imaging optical system 130 with respect to the cell C. Further, since the difference image is created from the captured original image, it is not necessary to acquire an image after specifying the in-focus position. Therefore, processing is simple.

  Since imaging is performed by discretely changing the focal position, the detected maximum value does not necessarily match the contrast values corresponding to the positions Za and Zb shown in FIG. 2B. However, for the purpose of obtaining an image that clearly shows the outline of the cell, if the two original images used to create the difference image are respectively extracted from the two peak portions sandwiching the minimum value in the profile, Good. Therefore, it does not matter whether or not the detected maximum value matches the actual maximum value unless the increment of the focal position is set to be extremely large. That is, a sufficient contour emphasis effect can be obtained by the above method.

  On the other hand, in the process of the second embodiment shown in FIG. 5B, two minimum values of the contrast value in the profile are detected (step S301). Also in this case, it is not necessary to obtain the local minimum value, and it is sufficient to know the set value of the focal position that provides the local minimum value. Two set values separated by a predetermined distance are specified with respect to the set value of the focal position thus obtained. A difference image is created from two original images corresponding to these two setting values (step S302). The processing for obtaining the difference image (steps S303 and S304) is the same as that in the first embodiment (steps S203 and S204).

  FIG. 6 is a diagram for explaining the difference image creation processing in the second embodiment in more detail. In this embodiment, as shown in FIG. 6, in the profile of the contrast value of the image with respect to the setting value of the focal position, a setting value Zm that gives a minimum value is obtained. Although this value Zm is estimated to be a position near the focal position Zc shown in FIG. 2B, the two may not coincide with each other. Then, using this position Zm as a reference, two original images corresponding to positions separated by a distance ΔZ in the (+) direction and the (−) direction are used as the original images that are the basis of the difference image.

  According to the knowledge of the inventors of the present application, in the profile of the contrast value with respect to the focal position, the distance between the focal position that gives the minimum value and the focal position that gives the maximum value depends on the type of the cell C, and If the type of cell C is known, this distance is generally predictable. Therefore, if this distance ΔZ is set in advance according to the type of the cell C and the setting value Zm of the focal position corresponding to the minimum value is obtained, two original images used for creating the difference image are immediately selected. It is possible.

  Further, according to the knowledge of the inventors of the present application, the distance between the focal position that gives the minimum value and the focal position that gives the maximum value in the profile varies depending on the numerical aperture (NA) of the imaging optical system 130 even for the same cell type. Therefore, when the imaging optical system 130 is configured to be exchangeable and the numerical aperture (NA) may be changed, the distance ΔZ is preferably set according to the numerical aperture (NA).

  For example, the relationship between the cell type and the NA of the imaging optical system 130 and the distance ΔZ can be obtained in advance and tabulated and stored in the memory 145. In the process of step S302, the distance ΔZ is obtained by referring to the table from the cell type information given in advance and the numerical aperture (NA) of the imaging optical system 130, and is separated from the set value Zm corresponding to the minimum value by this distance ΔZ. Two original images may be selected.

  By doing so, the original image can be selected one by one from the two peak portions sandwiching the minimum value in the profile, as in the first embodiment. By taking the difference between these two original images, a difference image with a clear cell outline is obtained. Also in this case, it is not necessary to obtain the in-focus position, and two original images that are the basis of the difference image can be easily selected based on the contrast value obtained directly from the original image.

  In any of these embodiments, a difference image can be created from an original image obtained by general bright field imaging. Therefore, a complicated configuration such as a phase difference observation optical system is not required, and an image with a clear cell outline can be obtained with a relatively simple apparatus configuration and simple processing. However, as will be described below, in the imaging apparatus 1 having the configuration shown in FIG. 1, the contrast value of the original image obtained by bright field imaging is closely related to the numerical aperture (NA) of the illumination light Li. . Therefore, in order to clarify the outline of a cell by the above image processing, it is first necessary to clarify the original image by optimizing the numerical aperture (NA) of the illumination light Li. Hereinafter, a specific configuration for clearly capturing an image in the image capturing apparatus 1 will be described with reference to FIGS. 7 and 8.

  As described above, in the imaging apparatus 1, the contrast value changes in accordance with the change in the focal position. This is because the refractive index is different between the cell C and the peripheral portion of the cell C. That is, when the cell C is irradiated with the illumination light Li, for example, as shown in a “schematic diagram” in FIG. 3, a part of the illumination light Li is refracted by the lens effect at the outer edge portion of the cell C. Here, when the illumination light Li is close to parallel light, that is, when the numerical aperture (NA) of the illumination light Li is small, the following effects can be obtained. That is, if there is no cell C, the captured light does not enter the lens constituting the imaging optical system 130 due to the lens effect of the cell C, and the cell outline becomes dark, or the light that cannot be captured without the cell C is the lens effect of the cell C. As a result, the lens constituting the imaging optical system 130 enters and the cell outline appears bright. Thus, it becomes possible to clearly capture the outline of the cell C by adjusting the illumination direction.

  Therefore, the inventor of the present application executes steps S101 to S106 shown in FIG. 4 for each numerical aperture (NA) while changing the numerical aperture (NA) of the illumination light Li in multiple stages, and profile of the contrast value with respect to the focal position. And the effect of the numerical aperture (NA) on the captured image was verified.

  More specifically, the above-mentioned verification was performed on two different types of cells C, and the above-mentioned profiles for each numerical aperture (NA) were obtained as shown in the column (a) of FIG. 7 and the column (a) of FIG. . Here, the numerical aperture (NA) is set to “0.02”, “0.05”, “0.10”, “0.20”, “0.29”, “0.37”, “0.43”. The profile acquisition (steps S101 to S106) was performed while changing and setting the seven levels.

  Further, based on these profiles, the contrast value for each numerical aperture (NA) at the so-called just focus position where the focal position Zf (FIG. 2A) substantially coincides with the position Zc (FIG. 2A) of the cell C is obtained. These are graphs shown in the (b) column of FIG. 7 and the (b) column of FIG. Further, the graphs shown in the column (c) of FIG. 7 and the column (c) of FIG. 8 show the maximum contrast value for each numerical aperture (NA) based on the same profile.

  As shown in FIGS. 7 and 8, the contrast value increases as the numerical aperture (NA) of the illumination light Li decreases, but the contrast value increases by setting the numerical aperture (NA) to 0.3 or less. The ratio increases. Therefore, from the verification, in order to image the outline of the cell C, the illumination unit 12 needs to be configured so that the numerical aperture (NA) is 0.3 or less. Further, it is preferable to configure the illumination unit 12 so that the numerical aperture (NA) is 0.2 or less, and more specifically, the illumination unit 12 is configured so that the numerical aperture (NA) is 0.1 or less. It is desirable to configure.

  As described above, in the above-described embodiment, the well plate WP corresponds to an example of the “carrier” of the present invention. The mechanical drive unit 146 functions as the “position changing unit” of the present invention, while the CPU 141 functions as the “control unit” of the present invention.

  In the above embodiment, the focal position Zf corresponds to the “front focal position” of the present invention, and the focal positions Za and Zb correspond to examples of the “first position” and the “second position” of the present invention, respectively. Yes.

  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 each of the above-described embodiments, the aperture stop and the imaging lens are provided in the imaging optical system. However, even if the imaging optical system does not use at least one of these, by applying the present invention, the above-described embodiment It is possible to obtain the same effect as the above. In the above embodiment, the focus position is changed by moving the entire imaging optical system 130 up and down. For example, this is an aspect in which the focus position is changed by changing the distance between a plurality of lenses constituting the imaging optical system. May be.

  In the first embodiment, two original images corresponding to two maximum values in the profile are selected. That is, the “first position” of the present invention is a position corresponding to the illumination-side maximum region of the profile, and the “second position” of the present invention is a position corresponding to the imaging optical system-side maximum region of the profile. . Thus, the original image obtained at the position where the maximum value is shown is used. On the other hand, in the second embodiment, two original images corresponding to a position separated by a predetermined distance ΔZ from the focal position Zm corresponding to the minimum value are selected, and the original image is picked up regardless of whether it is a maximum value or not. ing.

  In the above embodiment, the original images selected one by one from the two peak portions sandwiching the minimum value in the profile are selected. However, only the original image selected from one peak portion may be used. That is, an image captured by positioning the front focal point of the imaging optical system at at least one of the “first position” and the “second position” of the present invention may be used.

  The above-described imaging apparatus 1 uses the well plate WP provided with a number of wells W as the “supporting body” of the present invention, but the shape of the “supporting body” is not limited to this and is arbitrary. . For example, the present invention can also be applied to the purpose of imaging cells cultured in a container called “dish” having a larger diameter and a shallow dish type, or cells carried on a slide glass.

  In the imaging device 1 of the above embodiment, the configuration for imaging a cell and the configuration for image processing are integrated, but these are configured as separate units and connected to each other. It may be a thing.

  The present invention can be particularly suitably applied to a field that requires imaging of a cell carried on a carrier such as a well on a well plate used in the medical / biological science field. It is not limited to the medical / biological science field.

DESCRIPTION OF SYMBOLS 1 Imaging device 12 Illumination part 13 Imaging part 14 Control part 141 CPU (control part)
130 Imaging Optical System 146 Mechanical Drive Unit (Position Changing Unit)
C cell W well WP well plate (support)

Claims (6)

An imaging device that captures an image of a cell carried on a carrier,
An illumination unit for illuminating the carrier with illumination light;
An imaging unit having an imaging optical system arranged with an optical axis directed toward the carrier, and receiving the transmitted light transmitted through the carrier that is irradiated with the illumination light by the imaging optical system and imaging the cells And
An imaging apparatus having a numerical aperture of the illumination light of 0.3 or less. The imaging apparatus according to claim 1,
A position changing unit that changes the front focal position of the imaging optical system in a direction along the optical axis of the imaging optical system;
A control unit that sets a front focal position of the imaging optical system,
The control unit positions the front focal point of the imaging optical system at a first position away from the cell carried by the carrier toward the illumination unit or a second position away from the imaging optical system. An imaging device that causes an imaging unit to perform imaging. The imaging apparatus according to claim 2,
The controller is
The setting value of the front focal position by the position changing unit is changed and set in multiple stages, and the imaging unit is imaged with each setting value to obtain a plurality of original images,
Obtaining a contrast value profile for the set value by calculating a contrast value for each original image;
An imaging apparatus having a setting value on the illumination unit side as the first position and a setting value on the imaging optical system side as the second position with a minimum value in the profile interposed therebetween. The imaging apparatus according to claim 3,
The said control part is an imaging device which makes the setting value corresponding to the maximum area | region on the said illumination part side in the said profile a said 1st position. The imaging apparatus according to claim 3 or 4,
The said control part is an imaging device which makes the setting value corresponding to the maximum area | region by the side of the said imaging optical system in the said profile the said 2nd position. Irradiating a carrier carrying cells with illumination light;
Receiving light transmitted through the carrier that is irradiated with the illumination light and imaging the cells;
An imaging method, wherein the numerical aperture of the illumination light is set to 0.3 or less.

Images