Imaging device and imaging method

Abstract

To provide an imaging apparatus and an imaging method, capable of appropriately imaging a plurality of imaged objects held in a well.SOLUTION: An imaging apparatus 10 includes a container 20 which has a well 21 having an opening 22, a side wall 23, and a bottom part 24, an illumination device 30 which irradiates the container 20 with light from the side of the opening 22 of the well 21, and an imaging unit 40 which images an imaged object Oj in the well 21 from the side of the bottom part 24 of the well 21. The illumination device 30 has at least two light sources 31 in a space surrounded by a curved surface Sc connecting the outer peripheral edge 24a of the bottom part 24 of the well 21 and the outer peripheral edge 22a of the opening 22 of the well 21. The curved surface Sc is composed of a straight line connecting the outer peripheral edge 24a of the bottom part 24 and the outer peripheral edge 22a of the opening 22 and a straight line Ls connecting at least two light sources 31 does not cross a plane constituting the bottom part 24.SELECTED DRAWING: Figure 2

Description

Technical Field

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[0001] The present disclosure relates to an imaging device and an imaging method.

Background Art

[0002] Conventionally, a plate-shaped container having a plurality of wells has been known. Also, a culture solution or the like has been injected into each well of the container, and cells or the like cultured therein have been observed. When performing such observation, an imaging device has been used.

[0003] For example, the imaging device described in Patent Document 1 images a well by causing light to enter from above the well and receiving the light transmitted downward. However, when imaging objects such as cells contained in the well overlap, light does not sufficiently reach the imaging object covered by another imaging object. In this case, it becomes difficult to image the imaging object covered by another imaging object.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0005] The present disclosure provides an imaging device and an imaging method capable of imaging a plurality of imaging objects held in a well without excess or deficiency.

Means for Solving the Problems

[0006] The imaging device according to this embodiment comprises a container having a well having an opening, side walls, and a bottom; an illumination device that irradiates the container with light from the opening side of the well; and an imaging unit that images an object to be imaged in the well from the bottom side of the well, wherein the illumination device has at least two light sources in a space surrounded by a curved surface connecting the outer peripheral edge of the bottom of the well and the outer peripheral edge of the opening of the well, the curved surface being composed of straight lines connecting the outer peripheral edge of the bottom and the outer peripheral edge of the opening, and the straight lines connecting the at least two light sources not intersect the plane constituting the bottom.

[0007] In the imaging device according to this embodiment, the at least two light sources are located on a plane perpendicular to the central axis of the well, and when A is the horizontal distance of the region on the plane and surrounded by the curved surface, the distance between the at least two light sources may be A / 2 or less.

[0008] In the imaging device according to this embodiment, a diffuser plate may be placed between the illumination device and the container.

[0009] In the imaging device according to this embodiment, the container has a plurality of wells, the height of the bottom of at least one of the wells is different from the height of the bottom of the other wells, the depth of field of the imaging unit is greater than or equal to the difference between the height of the bottom of the lowest well and the height of the bottom of the highest well, and the depth of field region of the imaging unit may include both the position of the height of the bottom of the highest well and the position of the height of the bottom of the lowest well.

[0010] In the imaging apparatus according to this embodiment, the container is placed on a stage, and a transparent member may be placed in at least the area of ​​the stage where the well is located.

[0011] In the imaging device according to this embodiment, the stage may further include a barrier member located around the transparent member.

[0012] The imaging method according to this embodiment comprises the steps of: preparing a container having a well having an opening, side walls, and a bottom; irradiating the container with light from the opening side of the well using an illumination device; and imaging an object to be imaged in the well from the bottom side of the well, wherein the illumination device has at least two light sources in a space surrounded by a curved surface connecting the outer peripheral edge of the bottom of the well and the outer peripheral edge of the opening of the well, the curved surface being composed of straight lines connecting the outer peripheral edge of the bottom and the outer peripheral edge of the opening, and the straight lines connecting the at least two light sources not intersect the plane constituting the bottom. [Effects of the Invention]

[0013] According to this embodiment, multiple objects held within the well can be imaged without any excess or deficiency. [Brief explanation of the drawing]

[0014] [Figure 1] Figure 1 is a front view showing an imaging device according to one embodiment. [Figure 2] Figure 2 is a perspective view showing the well and light source of an imaging device according to one embodiment. [Figure 3] Figure 3 is a cross-sectional view showing the well and light source of an imaging device according to one embodiment. [Figure 4] Figure 4 is a cross-sectional view showing the arrangement of the well and light source of an imaging device according to one embodiment. [Figure 5] Figure 5(a) is a cross-sectional view showing the arrangement of the well and light source of an imaging device according to one embodiment, and Figure 5(b) is a cross-sectional view showing the arrangement of the well and light source of an imaging device according to a comparative example. [Figure 6] Figures 6(a)-(d) are cross-sectional views showing wells in modified forms, respectively. [Figure 7] Figure 7 is a cross-sectional view showing an imaging device according to a modified example. [Figure 8] Figure 8 is a cross-sectional view showing a part of the container after modification. [Figure 9] FIG. 9 is a schematic diagram showing the optical axis of the imaging unit according to the modified example. [Figure 10] FIG. 10 is a diagram showing a method for measuring the depth of the object field. [Figure 11] FIG. 11 is a diagram showing the captured image captured when measuring the depth of the object field and Graph A. [Figure 12] FIG. 12 is a diagram showing Graph B of the standard deviation of the pixel luminance. [Figure 13] FIG. 13 is a diagram showing a partial enlargement of Graph B in FIG. 12. [Figure 14] FIG. 14 is a diagram showing Graph C of the standard deviation of the pixel luminance. [Figure 15] FIG. 15 is a diagram showing a partial enlargement of Graph C in FIG. 14. [Figure 16] FIG. 16 is a plan view showing the stage according to the modified example. [Figure 17] FIG. 17 is a cross-sectional view showing the stage according to the modified example. [Figure 18] FIGS. 18(a)(b) are cross-sectional views showing the stage according to the modified example. [Figure 19] FIG. 19 is a plan view showing the stage according to the modified example.

MODE FOR CARRYING OUT THE INVENTION

[0015] The embodiments will be described in detail below with reference to the drawings. The following figures are schematic representations. Therefore, the size and shape of each part are exaggerated as appropriate to facilitate understanding. Furthermore, it is possible to modify and implement the designs as appropriate without departing from the technical concept. In the following figures, the same parts are denoted by the same reference numerals, and some detailed explanations may be omitted. In addition, the numerical values ​​such as dimensions and material names of each component described in this specification are examples of embodiments and are not limiting; they can be selected and used as appropriate. In this specification, terms that specify shapes and geometric conditions, such as parallel, orthogonal, and perpendicular, include not only their strict meanings but also substantially the same state. Also, for the convenience of explanation, the terms "up" or "down" may be used, but the up and down directions may be reversed.

[0016] In this specification, when a component or region is said to be "above (or below)" another component or region, unless otherwise specified, it is not limited to the case that it is directly above (or below) the other component. When a component or region is said to be "above (or below)" another component or region, it also includes cases where another component is included between the two components above (or below) the other component.

[0017] [Imaging device] First, the configuration of the imaging device according to this embodiment will be described with reference to Figures 1 to 3. Figure 1 is a schematic front view showing the entire imaging device 10 according to this embodiment. Figure 2 is a perspective view showing the well 21 and light source 31 of the imaging device 10 according to this embodiment. Figure 3 is a plan view showing the well 21 and light source 31 of the imaging device 10 according to this embodiment.

[0018] As shown in Figures 1 to 3, the imaging device 10 according to this embodiment comprises a container 20, an illumination device 30, and an imaging unit 40. The container 20 has a well 21 having an opening 22, side walls 23, and a bottom 24. The illumination device 30 irradiates the container 20 with light from the side of the opening 22 of the well 21. The imaging unit 40 images the container 20 from the side of the bottom 24 of the well 21. The illumination device 30 has at least two light sources 31 in a space surrounded by a curved surface Sc connecting the outer peripheral edge 24a of the bottom 24 of the well 21 and the outer peripheral edge 22a of the opening 22 of the well 21. The curved surface Sc is composed of straight lines connecting the outer peripheral edge 24a of the bottom 24 and the outer peripheral edge 22a of the opening 22. The straight line Ls (see Figure 2) connecting at least two light sources 31 does not intersect with the plane constituting the bottom 24.

[0019] Next, we will further describe the detailed configuration of the imaging device 10.

[0020] The container 20 has at least one, preferably multiple, wells 21. Each well 21 is filled with culture medium M. The culture medium M contains the object to be imaged, Oj (see Figure 3), such as cells or biological samples. Each well 21 also has an opening 22, side walls 23, and a bottom 24.

[0021] The opening 22 is located at the top vertical of the well 21. The planar shape of the opening 22 is circular, but it is not limited to this and may be a polygon such as a square or an ellipse. The opening 22 has an outer edge 22a. The top plate 25 of the well 21 is formed around the outer edge 22a of the opening 22.

[0022] The side wall 23 extends vertically downward from the opening 22. The side wall 23 is cylindrical as a whole. The horizontal cross-section of the side wall 23 (cross-section in a plane perpendicular to the central axis CL of the well 21) is circular, but is not limited to this; it may also be a polygon such as a quadrilateral, or an ellipse. The side wall 23 has a first side wall portion 23a on the opening 22 side and a second side wall portion 23b on the bottom 24 side. The first side wall portion 23a is continuous with the opening 22. The first side wall portion 23a is cylindrical, and its horizontal cross-section is uniform along the direction of the central axis CL. The second side wall portion 23b is located vertically below the first side wall portion 23a. The second side wall portion 23b is continuous with the first side wall portion 23a. The bottom 24 is located vertically below the second side wall portion 23b. The second side wall portion 23b is frustoconical in shape. The horizontal cross-section of the second side wall portion 23b gradually decreases along the central axis CL direction from the first side wall portion 23a side toward the bottom portion 24 side.

[0023] The bottom portion 24 is located at the bottom of the well 21 in the vertical direction. The bottom portion 24 is the deepest region of the well 21 and is the furthest from the opening 22 in the direction of the central axis CL. The planar shape of the bottom portion 24 is circular, but is not limited to this and may be a polygon such as a quadrilateral or an ellipse. The bottom portion 24 has an outer edge 24a. In this case, the bottom portion 24 is planar and parallel to a plane perpendicular to the central axis CL of the well 21. However, the bottom portion 24 is not limited to this and may be a point-shaped portion.

[0024] Furthermore, at least the bottom 24 of the well 21 is transparent. This allows the inside of the well 21 to be observed using the imaging unit 40. The visible light transmittance of the bottom 24 may be 85% or more, and preferably 90% or more. There is no particular upper limit to the visible light transmittance of the bottom 24, but it may be, for example, 100% or less. Visible light refers to light rays with a wavelength of 380 nm or more and 780 nm or less. Examples of materials for the well 21 include glass and plastic (polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, acrylic resin, polycarbonate resin, fluororesin, methylpentene resin, vinyl chloride resin, etc.).

[0025] Such a container 20 may be, for example, a microwell plate having multiple wells 21 (e.g., 6, 12, 24, 96, or 384) in accordance with the ANSI / SLAS standard. A microwell plate in accordance with the ANSI / SLAS standard has a roughly rectangular shape. The dimensions of this roughly rectangular microwell plate are: length: 127.76 ± 0.5 mm, width: 85.48 ± 0.5 mm. The distance from the corner of the roughly rectangular shape to the center of the nearest well 21 is: length: 12.7 ± 0.25 mm, width: 12.7 ± 0.25 mm.

[0026] The illumination device 30 is positioned above the container 20. The illumination device 30 irradiates light from above the container 20 toward the well 21. The illumination device 30 has a plurality of light sources 31. The light sources 31 emit light at least when imaging the object to be imaged Oj. The light emitted from the light sources 31 may be, for example, a single wavelength or a combination of multiple wavelengths (white, etc.). Each light source 31 can be, for example, a light-emitting diode (LED) element. As described below, the illumination device 30 has at least two light sources 31 in the space surrounded by the curved surface Sc connecting the outer peripheral edge 24a of the bottom 24 of the well 21 and the outer peripheral edge 22a of the opening 22 of the well 21. The vertical distance between the illumination device 30 and the opening 22 of the well 21 may be 10 mm or more and 500 mm or less.

[0027] As shown in Figures 2 and 3, the outer edge 24a of the bottom 24 of the well 21 is circular in plan view with the central axis CL at its center. Similarly, the outer edge 22a of the opening 22 of the well 21 is circular in plan view with the central axis CL at its center. The outer edge 24a of the bottom 24 is smaller than the outer edge 22a of the opening 22. Therefore, the curved surface Sc connecting the outer edge 24a of the bottom 24 and the outer edge 22a of the opening 22 forms the shape of the side surface of a frustocone. Note that if the bottom 24 of the well 21 is point-shaped, this point-shaped portion is referred to as the outer edge 24a of the bottom 24.

[0028] In this specification, "the curved surface Sc connecting the outer peripheral edge 24a of the bottom 24 and the outer peripheral edge 22a of the opening 22" refers to the trajectory of the straight line connecting the outer peripheral edge 24a of the bottom 24 and the outer peripheral edge 22a of the opening 22 in all cross-sections including the central axis CL of the well 21.

[0029] In this embodiment, the light sources 31 of the lighting device 30 are located on the same plane Sf. This plane Sf is parallel to the horizontal plane (a plane perpendicular to the central axis CL of the well 21). In this case, two light sources 31 are arranged within a region on the plane Sf and surrounded by the curved surface Sc. Note that three or more light sources 31 may be arranged within a region on the plane Sf and surrounded by the curved surface Sc. Furthermore, at least two light sources 31 may be located on different horizontal planes. The straight line Ls (see Figure 2) connecting at least two light sources 31 does not intersect with the plane constituting the bottom 24. Note that if the container 20 has multiple wells 21, at least two light sources 31 may be arranged within the space surrounded by the curved surface Sc for all of the wells 21.

[0030] Figure 3 shows a cross-section of the well 21 in a plane containing the central axis CL. In the cross-section shown in Figure 3, θ is the angle between the curved surface Sc and the horizontal plane (a plane perpendicular to the central axis CL), x is the horizontal distance of the opening 22, y is the horizontal distance of the bottom 24, z is the height distance between the bottom 24 and the opening 22, and h is the height distance between the opening 22 and the plane Sf. Also, t is the horizontal distance between the intersection point P1 of the plane Sf and the curved surface Sc and the outer edge 22a of the opening 22. The arrangement region Ra is the region where at least two light sources 31 are arranged. This arrangement region Ra is located on the plane Sf, which is parallel to the horizontal plane.

[0031] At this time, the following equations (1) and (2) hold true. tanθ = z / {(xy) / 2} ···(1) t = h / tanθ ... (2)

[0032] From equations (1) and (2), t = h(xy) / 2z···(3) This is the result.

[0033] In this case, the horizontal distance A of the arrangement region Ra, which is on a plane Sf and surrounded by a curved surface Sc, is A = x + 2t = x + h(xy) / z ... (4) This is the result.

[0034] In other words, the positions of the light sources 31 are set such that at least two light sources 31 are placed within a region of horizontal distance A on the plane Sf. If the horizontal cross-section of the curved surface Sc is a circle, then at least two light sources 31 are placed within a circle with diameter A centered on the central axis CL.

[0035] As shown in Figure 4, it is preferable that the spacing between the multiple light sources 31 be A / 2 or less. In this case, regardless of the horizontal positional relationship between the well 21 and the illumination device 30, the light from the illumination device 30 can reach the object to be imaged Oj. This allows all objects Oj in the well 21 to be imaged without any excess or deficiency. Alternatively, three or more light sources 31 may be arranged at equal intervals of A / 2 or less.

[0036] Referring again to Figure 1, the container 20 is positioned on the stage 50. A transparent member 51 is positioned in at least the area of ​​the stage 50 where the well 21 is located, so that imaging can be performed by the imaging unit 40. The transparent member 51 may be, for example, a glass plate.

[0037] The imaging unit 40 is positioned below the container 20. The imaging unit 40 images the inside of the container 20 from below via the stage 50. The imaging unit 40 acquires an image of the container 20 by receiving transmitted light emitted from the illumination device 30 and transmitted through the container 20 toward the bottom. That is, when imaging the object to be imaged Oj, the imaging unit 40 images the inside of the well 21 while illuminating the well 21 with light from the illumination device 30. This acquires an image of the object to be imaged Oj inside the container 20 as digital data. The imaging unit 40 has a camera 41 and a lens 42 attached to the camera 41.

[0038] The imaging range of the imaging unit 40 is preferably set to encompass the entirety of one well 21. This allows for complete and accurate observation of the area within the well 21. This configuration offers advantages over methods that combine multiple images in the following ways: When images are combined, lines connecting the images may interfere with the extraction of cells and other objects. In this embodiment, such lines connecting the images are not generated. Furthermore, the number of images taken and the processing time can be reduced compared to methods that combine multiple images. Therefore, when the object to be imaged Oj is a cell, the impact on the cell can be reduced by shortening the observation time.

[0039] Camera 41 has an image sensor such as a CCD or CMOS. Camera 41 may have multiple image sensors arranged in a row. Camera 41 may have, for example, image sensors arranged in one dimension or image sensors arranged in a two-dimensional matrix. Considering preferred image acquisition, it is preferable that camera 41 is an area scan camera (a camera having image sensors arranged in a two-dimensional matrix). Compared to a line sensor (image sensors arranged in one dimension), an area scan camera can capture an image at the moment the shutter is released, resulting in less blurred images and faster image acquisition. Furthermore, area scan cameras are inexpensive and easy to introduce. In addition, shadows and distortions originating from line sensors can be eliminated, allowing for the acquisition of, for example, more desirable images of cells.

[0040] For lens 42, for example, a telecentric lens may be used. By attaching lens 42 to camera 41, the imaging range and depth of field can be adjusted. Camera 41 and lens 42 can be arranged as shown in Figure 1, for example. Depending on the application, optical fixtures such as mirrors may be placed between camera 41 and lens 42.

[0041] The imaging unit 40 is attached to the imaging unit moving mechanism 43. The imaging unit moving mechanism 43 moves the imaging unit 40 horizontally and vertically below the stage 50. The imaging unit moving mechanism 43 changes the height and horizontal position of the camera 41 while maintaining the posture of the camera 41. The imaging unit moving mechanism 43 has a lifting mechanism 44 and a horizontal movement mechanism 45.

[0042] The lifting mechanism 44 is a mechanism for moving the imaging unit 40 up and down. The lifting mechanism 44 includes, for example, a mechanism that converts the rotational motion of a motor into linear motion via a ball screw. When the lifting mechanism 44 is operated, the height of the imaging unit 40 changes. This changes the distance between the container 20 and the imaging unit 40 (the imaging distance between the object to be imaged Oj and the lens 42). The camera 41 of the imaging unit 40 has a constant focal length. Therefore, when the position of the imaging unit 40 moves up and down, the focal position of the camera 41 also moves up and down along the optical axis.

[0043] The horizontal movement mechanism 45 is a mechanism that moves the imaging unit 40 and the lifting movement mechanism 44 together horizontally. The horizontal movement mechanism 45 includes a mechanism that converts the rotational motion of the motor into linear motion via a ball screw. By operating this horizontal movement mechanism 45, the camera 41 can be positioned below each well 21.

[0044] The lighting device 30 is also attached to the lighting relocation mechanism 33. The lighting relocation mechanism 33 moves the lighting device 30 horizontally above the container 20. The lighting relocation mechanism 33 includes, for example, a mechanism that converts the rotational motion of a motor into linear motion via a ball screw. By driving the lighting relocation mechanism 33, the lighting device 30 may be positioned above each well 21.

[0045] The illumination movement mechanism 33 and the horizontal movement mechanism 45 may be driven synchronously. In this case, the illumination device 30 and the imaging unit 40 are always positioned in the same relative position in a plan view. That is, the illumination device 30 and the imaging unit 40 move in the same direction and by the same distance.

[0046] The imaging device 10 further includes a control unit 70. The control unit 70 receives the captured image acquired by the imaging unit 40 as input. The control unit 70 is configured, for example, by a computer. The control unit 70 has the function of controlling the operation of each element within the imaging device 10, and the function of generating the captured image based on the signal input from the imaging unit 40. The control unit 70 may also have a processor such as a CPU, memory such as RAM, and a storage unit such as a hard disk drive. The control unit 70 stores a control program for controlling the operation of each part within the imaging device 10.

[0047] The control unit 70 may be connected to the above-mentioned lighting device 30, lighting movement mechanism 33, imaging unit 40, lifting movement mechanism 44, and horizontal movement mechanism 45 in a communication manner. The control unit 70 controls the operation of each of the above-mentioned parts according to the control program. This enables imaging of the object Oj to be imaged in each well 21 of the container 20.

[0048] Next, the operation of this embodiment, which has the above configuration, will be described.

[0049] First, a container 20 having a well 21 is prepared and placed on the stage 50. The well 21 contains a culture medium M and an object to be imaged Oj, such as cells or a biological sample. Next, the control unit 70 moves the illumination device 30 and the imaging unit 40 to predetermined positions. This positions the illumination device 30 and the imaging unit 40 so that the target well 21 can be imaged.

[0050] Next, the illumination device 30 irradiates the container 20 with light from the opening 22 side of the well 21. At this time, the light from the light source 31 of the illumination device 30 reaches the object Oj to be imaged inside the container 20. Next, the imaging unit 40 images the object Oj inside the well 21 from the bottom 24 side of the well 21. This allows the object Oj to be imaged to be captured.

[0051] In this embodiment, the illumination device 30 has at least two light sources 31 within a space surrounded by a curved surface Sc connecting the outer peripheral edge 24a of the bottom 24 of the well 21 and the outer peripheral edge 22a of the opening 22 of the well 21. For example, as shown in Figure 5(a), a first light source 31A and a second light source 31B are located within the space surrounded by the curved surface Sc. The first light source 31A and the second light source 31B are located on the same plane Sf. A first object to be imaged Oj1 and a second object to be imaged Oj2 are housed within the well 21. The first object to be imaged Oj1 is positioned to cover the second object to be imaged Oj2. In this case, light from the first light source 31A reaches the first object to be imaged Oj1, so the first object to be imaged Oj1 can be imaged. On the other hand, the second object to be imaged Oj2 is hidden in the shadow of the first object to be imaged Oj1. Therefore, light from the first light source 31A does not directly reach the second object to be imaged Oj2. In contrast, in this embodiment, the second light source 31B is provided in a space surrounded by a curved surface Sc. As a result, light from the second light source 31B reaches the second object to be imaged Oj2. As a result, light reaches the second object to be imaged Oj2, which is covered by the first object to be imaged Oj1, and the second object to be imaged Oj2 can be imaged. Thus, according to this embodiment, regardless of the position of the objects to be imaged Oj1 and Oj2 in the well 21, light from the illumination device 30 can reach the objects to be imaged Oj1 and Oj2. As a result, all objects to be imaged Oj1 and Oj2 in the well 21 can be imaged evenly. It is more preferable that the first light source 31A and the second light source 31B are located in a space surrounded by a curved surface Sd that includes the inner surface of the first side wall portion 23a. In this case, multiple objects to be imaged, Oj1 and Oj2, housed within the second side wall portion 23b of the well 21 can be imaged more efficiently and without excess or deficiency.

[0052] On the other hand, as a comparative example, for example as shown in Figure 5(b), if there is only one light source 31 in the space surrounded by the curved surface Sc, there is a risk that light will not reach the second object to be imaged Oj2, which is covered by the first object to be imaged Oj1. In this case, there is a risk that the imaging unit 40 will not be able to image the second object to be imaged Oj2.

[0053] [Differentiation] Next, various modifications of this embodiment will be described with reference to Figures 6 to 19. Figures 6 to 19 are diagrams showing modifications of this embodiment. In Figures 6 to 19, the same reference numerals are used for parts that are the same as those shown in Figures 1 to 5, and detailed descriptions are omitted.

[0054] (A variation of Well's work) Figures 6(a)-(d) show various modified examples of well 21.

[0055] In Figure 6(a), the side wall 23 of the well 21 is cylindrical with a uniform horizontal cross-section overall. An opening 22 is located at the upper end of the side wall 23. A bottom 24 is located at the lower end of the side wall 23. The horizontal cross-section of the side wall 23 is circular, but is not limited to this; it may be a polygon such as a quadrilateral, or an ellipse. In this case, the curved surface Sc connecting the outer edge 24a of the bottom 24 and the outer edge 22a of the opening 22 is cylindrical in shape. At least two light sources 31 are arranged in the space on the plane Sf and surrounded by the curved surface Sc.

[0056] In Figure 6(b), the side wall 23 of the well 21 has a shape in which the horizontal cross-section gradually decreases from the opening 22 side to the bottom 24 side. The opening 22 is located at the upper end of the side wall 23. The bottom 24 is located at the lower end of the side wall 23. The horizontal cross-section of the side wall 23 is circular, but it is not limited to this and may be a polygon such as a quadrilateral or an ellipse. Also, the cross-section of the side wall 23 is straight, but it is not limited to this and may be curved. In this case, the curved surface Sc connecting the outer edge 24a of the bottom 24 and the outer edge 22a of the opening 22 forms the shape of the side surface of a frustocone. At least two light sources 31 are arranged in the space on the plane Sf and surrounded by the curved surface Sc.

[0057] In Figure 6(c), the side wall 23 of the well 21 has a first side wall portion 23a and a second side wall portion 23b. The first side wall portion 23a has a shape in which the horizontal cross-section gradually decreases from the opening 22 side toward the second side wall portion 23b side. The second side wall portion 23b has a shape in which the horizontal cross-section gradually decreases from the first side wall portion 23a side toward the bottom portion 24 side. The inclination angle of the first side wall portion 23a with respect to the central axis CL and the inclination angle of the second side wall portion 23b with respect to the central axis CL are different from each other. In addition, although the horizontal cross-section of the side wall 23 is circular, it is not limited to this and may be a polygon such as a quadrilateral or an ellipse. The bottom portion 24 is point-shaped, but may be planar. In this case, the curved surface Sc connecting the outer peripheral edge 24a of the bottom portion 24 and the outer peripheral edge 22a of the opening 22 has a shape that constitutes the side surface of a cone. At least two light sources 31 are arranged in a space on a plane Sf and surrounded by a curved surface Sc. More preferably, at least two light sources 31 are located in a space surrounded by a curved surface Sd that includes the inner surface of the first side wall portion 23a. In this case, multiple objects Oj housed in the well 21 can be imaged more efficiently and without excess or deficiency. In particular, multiple objects Oj housed in the second side wall portion 23b can be imaged more efficiently and without excess or deficiency.

[0058] In Figure 6(d), the side wall 23 of the well 21 has a first side wall portion 23a and a second side wall portion 23b. The first side wall portion 23a has a shape in which the horizontal cross-section gradually decreases from the opening 22 side toward the second side wall portion 23b side. The second side wall portion 23b has a shape in which the horizontal cross-section gradually decreases from the first side wall portion 23a side toward the bottom portion 24 side. The second side wall portion 23b includes a curved surface. That is, in a cross-section including the central axis CL, the second side wall portion 23b is configured in a curved shape. In addition, although the horizontal cross-section of the side wall 23 is circular, it is not limited to this and may be a polygon such as a quadrilateral or an ellipse. The bottom portion 24 is point-shaped, but may be planar. In this case, the curved surface Sc connecting the outer peripheral edge 24a of the bottom portion 24 and the outer peripheral edge 22a of the opening 22 has a shape that constitutes the side surface of a cone. At least two light sources 31 are arranged in a space on a plane Sf and surrounded by a curved surface Sc. More preferably, at least two light sources 31 are located in a space surrounded by a curved surface Sd that includes the inner surface of the first side wall portion 23a. In this case, multiple objects Oj housed in the well 21 can be imaged more efficiently and without excess or deficiency. In particular, multiple objects Oj housed in the second side wall portion 23b can be imaged more efficiently and without excess or deficiency.

[0059] (Variations of lighting) Figure 7 shows an imaging device 10 according to a modified example. In the imaging device 10 shown in Figure 7, a diffuser plate 35 is placed between the illumination device 30 and the container 20. The diffuser plate 35 has the function of diffusing the light emitted from the light source 31 in various directions. By placing the diffuser plate 35, the light emitted from the light source 31 is incident on the object to be imaged Oj from various directions, not just one direction. This makes it possible to image multiple objects to be imaged Oj without excess or deficiency, and with greater clarity. Furthermore, by placing the diffuser plate 35, the direction in which the light emitted from the light source 31 is emitted can be adjusted in various directions. The diffuser plate 35 may have a total light transmittance of 50% or more and 99% or less. The material of the diffuser plate 35 may be, for example, acrylic resin, polycarbonate resin, or other resins.

[0060] (Modified examples of wells and imaging units) Figure 8 shows a modified example of the container 20. In Figure 8, the container 20 has a plurality of wells 21 arranged side by side. With the container 20 placed on the stage 50, the height of the bottom 24 (vertical distance from the top surface of the stage 50) of at least one well 21 is different from the height of the bottom 24 of the other wells 21. For example, in Figure 8, the height H1 of the bottom 24 of the well 21A located in the center is the highest. Also, the height H2 of the bottom 24 of the well 21B located at the end is the lowest. Hereafter, the height of the bottom 24 of the well 21A, which is at the highest position, will be referred to as the first height H1, and the height of the bottom 24 of the well 21B, which is at the lowest position, will be referred to as the second height H2.

[0061] At this time, the depth of field of the imaging unit 40 is greater than or equal to the difference between the first height H1 and the second height H2 (WBEV (Well Bottom Elevation Variation)). Furthermore, the depth of field region (DOF) of the imaging unit 40 includes both the position at the first height H1 and the position at the second height H2.

[0062] When container 20 is a microwell plate, the ANSI / SLAS standard generally defines the difference between the first height H1 and the second height H2 as the Well Bottom Elevation Variation (WBEV). This WBEV value is approximately 0.05 mm to 0.3 mm. The ANSI / SLAS standard also states that the total height of container 20, measured from a stationary plane (e.g., the top surface of stage 50) to the maximum protrusion of container 20, is 14.35 mm ± 0.76 mm.

[0063] In the example shown in Figure 8, the depth of field of the imaging unit 40 is set to be greater than or equal to the WBEV, and the depth of field region (DOF) of the imaging unit 40 includes both the position at the first height H1 and the position at the second height H2. As a result, no matter which well 21 is imaged by the imaging unit 40, the imaging unit 40 will be in focus, so all wells 21 can be imaged without blurring. Furthermore, the internal space of all wells 21 can be imaged, and the entire internal space of each well 21 can be imaged without any excess or deficiency.

[0064] Furthermore, it is preferable that the lowest position of the depth of field (DOF2) coincides with the second height H2, or that it is shifted downward from the second height H2 by a value smaller than the thickness of the bottom 24 of the well 21. The depth of field varies depending on the sensor size and optical components of the camera 41. Therefore, by setting the lowest position of the depth of field (DOF2) to the above position, objects located on the back side of the bottom 24 of the well 21 (opposite side of the light source 31) can be avoided from being observed.

[0065] Furthermore, it is preferable to set the uppermost position of the depth of field (DOF1) at a position 10 μm or more higher than the first height H1. Since the size of a typical cell is about 10 μm, when the object Oj to be imaged is a cell, the object Oj can be imaged without excess or deficiency. Also, it is preferable to set the uppermost position of the depth of field (DOF1) at a position 1 mm or more higher than the first height H1. Since large cells such as fertilized eggs are about 1 mm or larger in size, when the object Oj to be imaged is a large cell, the object Oj can be imaged without excess or deficiency. More preferably, it is preferable to set the uppermost position of the depth of field (DOF1) at a position higher than the height of the liquid level filled in the well 21A where the bottom 24 is at the highest position. This allows the liquid filled in all wells 21 to be imaged without excess or deficiency.

[0066] Thus, the depth of field of the imaging unit 40 is greater than or equal to the WBEV difference between the first height H1 and the second height H2. Furthermore, the depth of field region DOF of the imaging unit 40 includes both the position at the first height H1 and the position at the second height H2. In this case, the number of images taken and the processing time can be reduced compared to the method of combining images taken from multiple wells 21. In addition, if the object to be imaged Oj is a cell, the impact on the cell can be reduced by shortening the cell observation time.

[0067] As an example, an area sensor camera (resolution: 20.2MP, sensor dimensions: 12.3mm x 12.3mm, horizontal / vertical resolution: 4504px x 4504px, horizontal / vertical pixel size: 2.74μm x 2.74μm) may be used as the camera 41 of the imaging unit 40. Alternatively, a telecentric lens (with aperture) may be used as the lens 42 of the imaging unit 40. For example, the aperture can be adjusted so that the depth of field is 1.0mm, 3.6mm, 5.7mm, and 12.5mm. A well plate containing 96 wells 21 may be used as the container 20. The entire well 21 of this well plate may be imaged in one shot with a resolution of 2.74μm / pix (imaging conditions: lens magnification set to 1.0x, work distance (distance from lens to inner surface of bottom of well) approximately 112mm). This allows for complete and accurate imaging of the object Oj in all wells 21.

[0068] As a further method to widen the depth of field, the optical axis between the object Oj and the imaging unit 40 may be divided into multiple sections using an optical element such as a half-mirror or beam splitter. By intentionally changing the optical distance from the imaging unit 40 to the object Oj for each of the divided optical axes, the depth of field can be widened. Methods for changing the optical distance include extending the distance of some of the optical axes as shown in Figure 9, or inserting materials with different refractive indices, such as glass, between some of the optical axes.

[0069] For example, in Figure 9, a first optical element 61, a second optical element 62, a first mirror 63, and a second mirror 64 may be placed between the imaging unit 40 and the object to be imaged Oj. The first optical element 61 and the second optical element 62 may each be half mirrors or beam splitters, etc. In this case, the optical axis Oa between the object to be imaged Oj and the imaging unit 40 is divided into a first optical axis Oa1 and a second optical axis Oa2. The first optical axis Oa1 passes from the imaging unit 40 through the first optical element 61 and the second optical element 62, respectively, and reaches the object to be imaged Oj. The second optical axis Oa2 is sequentially reflected from the imaging unit 40 by the first optical element 61, the first mirror 63, the second mirror 64, and the second optical element 62, and reaches the object to be imaged Oj. At the position of the object to be imaged Oj, the first optical axis Oa1 and the second optical axis Oa2 overlap each other.

[0070] In this case, the optical path length of the second optical axis Oa2 is shifted relative to the first optical axis Oa1 by twice the distance Lx between the first optical element 61 (second optical element 62) and the first mirror 63 (second mirror 64). In this case, the depth of field DOFa of the first optical axis Oa1 and the depth of field DOFb of the second optical axis Oa2 are overlapped. This makes it possible to increase the overall depth of field DOFt.

[0071] For example, it is conceivable to divide the optical axis Oa into two by applying the configuration shown in Figure 9 to an imaging unit 40 capable of imaging with a depth of field of 4.5 mm. In this case, the distance Lx between the first optical element 61 (second optical element 62) and the first mirror 63 (second mirror 64) is set to 2.0 mm. At this time, the second optical axis Oa2 is shifted by 4.0 mm, so the overlap amount DOFc between the depth of field DOFa of the first optical axis Oa1 and the depth of field DOFb of the second optical axis Oa2 becomes 0.5 mm. Therefore, the image synthesized by the imaging unit 40 will be an image with a depth of field of 8.5 mm. Note that a smaller overlap amount DOFc simplifies various processing such as light intensity adjustment when using the acquired synthesized image. However, if the overlap amount DOFc is extremely small, there is a possibility that the depth of field DOFt will become discontinuous if there are discrepancies in various settings. Furthermore, the depth of field can be increased by dividing the optical axis Oa between the object to be imaged Oj and the imaging unit 40 into three or more sections.

[0072] Depth of field can be measured as follows:

[0073] First, prepare a DOF (depth of field) measurement chart 81 (for example, product code: #54-440, distributor: Edmund Optics Japan Co., Ltd.) as shown in Figure 10. Next, position the test pattern on this measurement chart 81 at a 45° angle to the optical axis Oa of the lens 42 of the imaging unit 40, and take an image with the imaging unit 40. This will obtain an image IC including a spatial frequency pattern SP (15 lines / mm) and a direct-reading scale DS for depth measurement, as shown in Figure 11.

[0074] Next, based on the captured image IC, the pixel brightness is displayed as a two-dimensional graph (hereinafter also referred to as graph A) along a straight line Ld drawn within the spatial frequency pattern SP (cross-sectional plot diagram) (see Figure 11). Note that the straight line Ld is located perpendicular to the longitudinal direction of the spatial frequency pattern SP. In graph A shown in Figure 11, the x-axis represents the distance along the straight line (pixels), and the y-axis represents the pixel brightness (256 levels (8-bit data, resulting in values ​​from 0 to 255)). This operation can be performed, for example, using ImageJ software.

[0075] Next, in graph A, select the PL region where the brightness begins to change significantly from the left, and the PR region where the brightness begins to change significantly from the right. Then, apply the distance between the selected PL and PR regions to the image IC, and read the distance between the PL and PR regions using the direct-reading scale DS. This distance between the PL and PR regions is defined as the depth of field (DOF).

[0076] Next, I will explain how to select a polarizing filter (PL) for the area where the brightness starts to change significantly from the left.

[0077] First, set the group size to 2% or more of the total number of data points on the X-axis (distance (pixels)) of graph A (Figure 11). For example, if the captured image IC is a 4504 pixels x 4504 pixels image, the total number of data points on the X-axis (distance (pixels)) of graph A is 4504. In this case, setting the group size to 2.2% of the total number of data points will result in 100 groups.

[0078] Next, for the x-axis (distance (pixels)) of graph A, the consecutive portion corresponding to the number of groups set above is designated as group No. For example, for the x-axis (distance (pixels)) of graph A, pixels 0 to 99 (a consecutive portion of 100 groups) is designated as group No. 0. Similarly, for the x-axis (distance (pixels)) of graph A, pixels 1 to 100 (a consecutive portion of 100 groups) is designated as group No. 1. In this way, for the x-axis (distance (pixels)) of graph A, pixels n to n+99 (a consecutive portion of 100 groups) is designated as group No. n. Graph B is created from this group No. n and the standard deviation of the y-axis (pixel brightness) of graph A in the range n to n+99 pixels for group No. n (calculated, for example, using the STDEV function in Excel). Graph B is shown in Figure 12. Then, from graph B, find the portion where the standard deviation of pixel brightness is increasing.

[0079] In this case, first, for group numbers smaller than the upward-sloping portion of graph B, the mean of the standard deviation of pixel brightness (1.6 in this case) is taken for any consecutive group numbers. The number of any consecutive group numbers is set to be 4% or more of the total number of data points on the X axis (distance (pixels)) of graph A. For example, in Figure 13, the total number of data points on the X axis (distance (pixels)) is 4504, and since it is set to 4.4%, the number of any consecutive group numbers is 200.

[0080] Next, we calculate 1.5 times the mean of the above standard deviation (here, the mean is 1.6 × 1.5 = 2.4). Then, for the part where the standard deviation of pixel brightness is increasing, we find the group No. of the starting point where the value is continuously larger than that value (here, 2.4) (in Figure 13, this is group No. 1800). This calculated starting group No. 1800 corresponds to pixels 1800 to 1899 on the X axis (distance (pixels)) of graph A. In other words, it can be said that the brightness starts to change significantly from the left at the 1899th pixel on the X axis (distance (pixels)). Therefore, we define the 1899th pixel on the X axis (distance (pixels)) of graph A as the part PL where the brightness change starts to change significantly from the left.

[0081] Next, I will explain how to select the PR (Precision Reduction) area where the brightness starts to change significantly from the right side.

[0082] First, set the group size to 2% or more of the total number of data points on the X-axis (distance (pixels)) of graph A (Figure 11). For example, if the captured image IC is a 4504 pixels x 4504 pixels image, the total number of data points on the X-axis (distance (pixels)) of graph A is 4504. In this case, setting the group size to 2.2% of the total number of data points will result in 100 groups.

[0083] Next, for the x-axis (distance (pixels)) of graph A, the consecutive portion corresponding to the number of groups set above is designated as group No. For example, for the x-axis (distance (pixels)) of graph A, pixels 4503 to 4404 (a consecutive portion corresponding to 100 groups) is designated as group No. 4503. Similarly, for the x-axis (distance (pixels)) of graph A, pixels 4502 to 4403 (a consecutive portion corresponding to 100 groups) is designated as group No. 4502. In this way, for the x-axis (distance (pixels)) of graph A, pixels n to n-99 (a consecutive portion corresponding to 100 groups) is designated as group No. n. Graph C is created from this group No. n and the standard deviation of the y-axis (pixel brightness) of graph A in the range n to n-99 pixels for group No. n (calculated, for example, using the STDEV function in Excel). This graph C of the standard deviation is shown in Figure 14. Then, from graph C, find the portion where the standard deviation of pixel brightness is sloping upwards from left to right.

[0084] In this case, first, for group numbers larger than the upward-sloping portion of graph C, the mean of the standard deviation of pixel brightness (1.45 in this case) is taken for any consecutive group numbers. The number of any consecutive group numbers is set to be 4% or more of the total number of data points on the X axis (distance (pixels)) of graph A. For example, in Figure 15, the total number of data points on the X axis (distance (pixels)) is 4504, and since it is set to 4.4%, the number of any consecutive group numbers is 200.

[0085] Next, we calculate 1.5 times the mean of the above standard deviation (here, the mean is 1.45 × 1.5 = 2.2). Then, for the part where the standard deviation of pixel brightness is sloping upwards to the left, we find the group No. of the starting point where the value is continuously larger than that value (here, 2.2) (in Figure 15, this is group No. 2400). This calculated starting group No. 2400 corresponds to pixels 2400 to 2301 on the X-axis (distance (pixels)) of graph A. In other words, it can be said that the brightness starts to change significantly from the right at pixel 2301 on the X-axis (distance (pixels)). Therefore, pixel 2301 on the X-axis (distance (pixels)) of graph A is defined as the part PR where the brightness change starts to change significantly from the right.

[0086] If the PL (partial PL) is unavailable (i.e., there is no region where the brightness increases significantly from the left side), the leftmost value readable from the direct-reading scale DS will be used as the PL reading for depth of field. If the PR (partial PR) is unavailable (i.e., there is no region where the brightness increases significantly from the right side), the rightmost value readable from the direct-reading scale DS will be used as the PR reading for depth of field. If neither the PL nor the PR can be selected, the value read from the entire direct-reading scale DS (the entire captured area) will be used as the depth of field.

[0087] (Stage torture) Figures 16 and 17 show a modified stage 50. The stage 50 has a transparent member 51 on which the container 20 is placed, and a barrier member 52 located around the transparent member 51. The example shown in Figures 16 and 17 is preferably used when the imaging unit 40 does not move horizontally.

[0088] The transparent member 51 transmits light that comes through from above the container 20. Preferably, the transparent member 51 is larger than the entire well 21 so that an image including the entire well 21 can be captured. It is even more preferable that the transparent member 51 is larger than the entire container 20. The transparent member 51 is a plate-shaped member. The visible light transmittance of the transparent member 51 may be 85% or more, and preferably 90% or more. There is no particular upper limit to the visible light transmittance of the transparent member 51, but it may be, for example, 100% or less. Examples of materials for the transparent member 51 include glass and plastic (polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, acrylic resin, polycarbonate resin, fluororesin, methylpentene resin, vinyl chloride resin, etc.).

[0089] The barrier member 52 is provided around the transparent member 51 and is a member that supports the transparent member 51. As shown in Figure 17, the barrier member 52 may be composed of two members. In this case, the barrier member 52 has a plate-shaped first member 53 and a plate-shaped second member 54 laminated on the first member 53. The first member 53 and the second member 54 may each be metal plates. The first member 53 has a first opening 55 that is smaller than the transparent member 51. The second member 54 has a second opening 56 that accommodates the transparent member 51. The second opening 56 is larger than the first opening 55. The second member 54 is thicker than the transparent member 51. This prevents the container 20 from falling off the transparent member 51 when the container 20 is slid on the transparent member 51. Preferably, when one corner 20a1 of the container 20 contacts each of the four corners 56a of the second opening 56, the well 21 closest to the corner 20a2 that is in a vertex relationship with the corner 20a1 is positioned so that the imaging unit 40 can image it.

[0090] As shown in Figure 18(a), the barrier member 52 may be composed of a single member. In Figure 18(a), the barrier member 52 has a first portion 53A and a second portion 54A formed on the first portion 53A. The first portion 53A has a first opening 55A that is smaller than the transparent member 51. The second portion 54A has a second opening 56A that accommodates the transparent member 51. The second opening 56A is larger than the first opening 55A. The thickness of the second portion 54A is greater than that of the transparent member 51.

[0091] As shown in Figure 18(b), the barrier member 52 may be composed of a single member. In Figure 18(b), the barrier member 52 has a first portion 53B and a second portion 54B that protrudes from the first portion 53B. The outer circumference of the second portion 54B is smaller than the outer circumference of the first portion 53B. The first portion 53B has a first opening 55B that is smaller than the transparent member 51. The second portion 54B has a second opening 56B that accommodates the transparent member 51. The second opening 56B is larger than the first opening 55B. The thickness of the second portion 54B is thicker than the transparent member 51.

[0092] Furthermore, as shown in Figure 19, a ruler 57 with markings may be fixed along the X and Y axes of the stage 50. Alternatively, a pole 58 may be connected to each ruler 57. Each pole 58 is movable along the X and Y axes. This makes it easy to position the well 21 to be imaged at the imaging location. By moving the two poles 58 and aligning the corner 20a of the container 20 with the intersection of the two poles 58, the inner wells 21 can also be easily aligned. It is also preferable to align the markings on the ruler 57 with the intervals of each well 21. This makes it possible to confirm which well 21 is being imaged using the markings on the ruler 57.

[0093] It is also possible to combine the multiple components disclosed in each of the above embodiments and variations as needed. Alternatively, some components may be removed from all the components shown in each of the above embodiments and variations. [Explanation of symbols]

[0094] 10 Imaging device 20 containers 21 wells 22 Opening 22a Outer edge 23 Side wall 23a 1st side wall part 23b 2nd side wall part 24 Bottom 24a Outer edge 25 Top board 30 Lighting devices 31 Light source 40 Imaging Units 41 Cameras 42 lenses 50 stages

Claims

1. A container having a well with an opening, side walls and a bottom, A lighting device that irradiates light into the container from the opening side of the well, The system includes an imaging unit that images an object to be imaged inside the well from the bottom side of the well, The lighting device has at least two light sources within a space surrounded by a curved surface connecting the outer peripheral edge of the bottom of the well and the outer peripheral edge of the opening of the well. The curved surface is composed of a straight line connecting the outer edge of the bottom and the outer edge of the opening, The straight line connecting the at least two light sources does not intersect the plane constituting the bottom. The side wall of the well has a first side wall portion and a second side wall portion, and the horizontal cross-section of the first side wall portion gradually decreases from the opening toward the second side wall portion located toward the bottom side of the side wall. The second side wall portion has a gradually decreasing horizontal cross-section from the first side wall portion side toward the bottom side. The inclination angle of the first side wall portion with respect to the central axis of the well and the inclination angle of the second side wall portion with respect to the central axis are different from each other, or the second side wall portion includes a curved surface. The container is placed on a stage, and a transparent member is placed in at least the area of ​​the stage where the well is located. The stage further includes a barrier member located around the transparent member, The barrier member comprises a first member and a second member laminated on the first member. The first member has a first opening smaller than the transparent member, the second member has a second opening for housing the transparent member, the second opening is larger than the first opening, and the second member is thicker than the transparent member. Imaging device.

2. The imaging apparatus according to claim 1, wherein the at least two light sources are located on a plane perpendicular to the central axis of the well, and when A is the horizontal distance of the region on the plane and surrounded by the curved surface, the distance between the at least two light sources is A / 2 or less.

3. The imaging apparatus according to claim 1 or 2, wherein a diffuser plate is arranged between the lighting device and the container.

4. The container has a plurality of wells, and the height of the bottom of at least one of the wells is different from the height of the bottom of the other wells. The depth of field of the imaging unit is greater than or equal to the difference between the height of the bottom of the lowest well and the height of the bottom of the highest well. The imaging apparatus according to any one of claims 1 to 3, wherein the depth of field region of the imaging unit includes both the height of the bottom of the highest-positioned well and the height of the bottom of the lowest-positioned well.

5. A step of preparing a container having a well with an opening, side walls and a bottom, A lighting device is used to irradiate the container with light from the opening side of the well, The process includes the step of imaging an object to be imaged in the well from the bottom side of the well, The lighting device has at least two light sources within a space surrounded by a curved surface connecting the outer peripheral edge of the bottom of the well and the outer peripheral edge of the opening of the well. The curved surface is composed of a straight line connecting the outer edge of the bottom and the outer edge of the opening, The straight line connecting the at least two light sources does not intersect the plane constituting the bottom. The side wall of the well has a first side wall portion and a second side wall portion, and the horizontal cross-section of the first side wall portion gradually decreases from the opening toward the second side wall portion located toward the bottom side of the side wall. The second side wall portion has a gradually decreasing horizontal cross-section from the first side wall portion side toward the bottom side. The inclination angle of the first side wall portion with respect to the central axis of the well and the inclination angle of the second side wall portion with respect to the central axis are different from each other, or the second side wall portion includes a curved surface. The container is placed on a stage, and a transparent member is placed in at least the area of ​​the stage where the well is located. The stage further includes a barrier member located around the transparent member, The barrier member comprises a first member and a second member laminated on the first member. An imaging method comprising: a first member having a first opening smaller than the transparent member; a second member having a second opening for housing the transparent member, the second opening being larger than the first opening; and the second member being thicker than the transparent member.

Images