JP7830887B2 - Imaging device and imaging method - Google Patents

Description

This disclosure relates to an imaging device and an imaging method.

Conventionally, plate-shaped containers with multiple wells are known. Furthermore, it is common practice to inject culture medium into each well of the container and observe the cultured cells. Imaging devices are used when performing such observations.

For example, the imaging device described in Patent Document 1 images the well by illuminating it from above and receiving the light that passes through downwards. However, when the objects to be imaged, such as cells, contained within the well overlap, the light does not reach the objects covered by other objects sufficiently. In this case, imaging of the objects covered by other objects becomes difficult.

International Publication No. 2017/122401

This disclosure provides an imaging apparatus and imaging method capable of capturing multiple objects held within a well without any excess or deficiency.

The imaging device according to this embodiment comprises a container having a well with 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 inside the well from the bottom side of the well. The illumination 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 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 do not intersect the plane constituting the bottom.

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.

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

In the imaging device according to this embodiment, 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-positioned well and the height of the bottom of the highest-positioned well. The depth of field region of the imaging unit may include both the position of the bottom of the highest-positioned well and the position of the bottom of the lowest-positioned well.

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.

In the imaging apparatus according to this embodiment, the stage may further include a barrier member located around the transparent member.

The imaging method according to this embodiment comprises the steps of: preparing a container having a well with 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 within the well from the bottom side of the well. The illumination 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 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 do not intersect the plane constituting the bottom.

According to this embodiment, multiple objects held within the well can be imaged without any excess or deficiency.

Figure 1 is a front view showing an imaging device according to one embodiment. Figure 2 is a perspective view showing the well and light source of an imaging device according to one embodiment. Figure 3 is a cross-sectional view showing the well and light source of an imaging device according to one embodiment. 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(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. Figures 6(a)-6(d) are cross-sectional views showing wells in modified forms, respectively. Figure 7 is a cross-sectional view showing an imaging device according to a modified example. Figure 8 is a cross-sectional view showing a part of the container modified. Figure 9 is a schematic diagram showing the optical axis of the imaging unit in a modified form. Figure 10 shows a method for measuring depth of field. Figure 11 shows the captured image taken when measuring the depth of field, along with graph A. Figure 12 shows graph B of the standard deviation of pixel brightness. Figure 13 is a magnified view of a portion of graph B in Figure 12. Figure 14 shows graph C of the standard deviation of pixel brightness. Figure 15 is a magnified view of a portion of graph C in Figure 14. Figure 16 is a plan view showing a modified stage. Figure 17 is a cross-sectional view showing a modified stage. Figures 18(a) and 18(b) are cross-sectional views showing the stages of modification. Figure 19 is a plan view showing a modified stage.

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 for ease of understanding. Furthermore, modifications can be made 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. Also, 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 shape and geometric conditions, such as parallel, orthogonal, and perpendicular, include not only their strict meanings but also substantially equivalent states. Also, for convenience of explanation, the terms "up" or "down" may be used, but the up and down directions may be reversed.

In this specification, when a component or region is described as being "above (or below)" another component or region, unless otherwise specified, it is not limited to being directly above (or below) the other component. "Above (or below)" a component or region also includes cases where another component is included between the components above (or below) them.

[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.

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 with an opening 22, side walls 23, and a bottom 24. The illumination device 30 irradiates the container 20 with light from the opening 22 side of the well 21. The imaging unit 40 images the container 20 from the bottom 24 side of the well 21. 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. 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 the at least two light sources 31 does not intersect with the plane constituting the bottom 24.

Next, the detailed configuration of the imaging device 10 will be further explained.

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 has an opening 22, side walls 23, and a bottom 24.

The opening 22 is located at the top vertically of the well 21. The planar shape of the opening 22 is circular, but it is not limited to this; it may also be a polygon such as a quadrilateral, or an ellipse. The opening 22 has an outer peripheral edge 22a. The upper plate 25 of the well 21 is formed around the outer peripheral edge 22a of the opening 22.

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 portion 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 portion 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 towards the bottom portion 24 side.

The bottom portion 24 is located at the lower vertical end of the well 21. The bottom portion 24 is the deepest region of the well 21 and is the furthest point from the opening 22 in the direction of the central axis CL. The planar shape of the bottom portion 24 is circular, but it is not limited to this; it may also be a polygon such as a quadrilateral, or an ellipse. The bottom portion 24 has an outer periphery 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 a planar shape; it may also be a point-like portion.

Furthermore, at least the bottom 24 of the well 21 is transparent. This allows observation of the inside of the well 21 using the imaging unit 40. The visible light transmittance of the bottom 24 may be 85% or higher, and preferably 90% or higher. 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 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.).

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 conforming to 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.

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 multiple light sources 31. Each light source 31 emits light at least when the object to be imaged Oj is being imaged. The light emitted from the light sources 31 may be, for example, a single wavelength or a combination of multiple wavelengths (e.g., white light). 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 within 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 between 10 mm and 500 mm.

As shown in Figures 2 and 3, the outer edge 24a of the bottom 24 of the well 21 is circular in plan view, centered on the central axis CL. Similarly, the outer edge 22a of the opening 22 of the well 21 is also circular in plan view, centered on the central axis CL. 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.

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.

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 the region on the plane Sf and surrounded by the curved surface Sc. Note that three or more light sources 31 may be arranged within the 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.

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. Furthermore, t indicates 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 parallel to the horizontal plane.

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

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

At this time, 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(x-y)/z...(4)
This is the result.

In other words, the positions of the light sources 31 are set so that at least two light sources 31 are arranged within a region of horizontal distance A on the plane Sf. Note that if the horizontal cross-section of the curved surface Sc is a circle, the at least two light sources 31 are arranged within a circle with diameter A centered on the central axis CL.

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 for complete and accurate imaging of all objects Oj within the well 21. Alternatively, three or more light sources 31 may be arranged at equal intervals of A/2 or less.

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, allowing imaging by the imaging unit 40. The transparent member 51 may be, for example, a glass plate.

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 passing through the container 20 downwards. That is, when imaging the object 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 Oj inside the container 20 as digital data. The imaging unit 40 includes a camera 41 and a lens 42 attached to the camera 41.

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, the presence of lines connecting the images can hinder the extraction of cells and other objects. According to this embodiment, such lines connecting images are eliminated. Furthermore, the number of images taken and the processing time can be reduced compared to methods that combine multiple images. Therefore, when the object being imaged Oj is a cell, the reduced observation time can minimize the impact on the cell.

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, for example, have 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 be an area scan camera (a camera with image sensors arranged in a two-dimensional matrix). Compared to a line sensor (one-dimensionally arranged image sensors), an area scan camera can capture an image at the moment the shutter is released, resulting in less image blur and faster image acquisition. Furthermore, area scan cameras are inexpensive and easy to implement. Additionally, shadows and distortions originating from line sensors can be eliminated, allowing for the acquisition of, for example, more desirable images of cells.

For lens 42, a telecentric lens, for example, may be used. By attaching lens 42 to camera 41, the imaging range and depth of field can be adjusted. The camera 41 and lens 42 can be arranged as shown in Figure 1, for example. Depending on the application, an optical jig such as a mirror may be placed between camera 41 and lens 42.

The imaging unit 40 is attached to the imaging unit movement mechanism 43. The imaging unit movement mechanism 43 moves the imaging unit 40 horizontally and vertically below the stage 50. The imaging unit movement mechanism 43 changes the height and horizontal position of the camera 41 while maintaining the camera's orientation. The imaging unit movement mechanism 43 includes a lifting/lowering mechanism 44 and a horizontal movement mechanism 45.

The lifting mechanism 44 is a mechanism that moves 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.

The horizontal movement mechanism 45 is a mechanism that moves the imaging unit 40 and the lifting/lowering 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.

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

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.

The imaging device 10 further includes a control unit 70. The control unit 70 receives the captured image acquired by the imaging unit 40. The control unit 70 is, for example, configured 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 signals input from the imaging unit 40. The control unit 70 may also include 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.

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

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

First, a container 20 having a well 21 is prepared, and the container 20 is placed on the stage 50. The well 21 contains a culture medium M and the object to be imaged, such as cells or a biological sample Oj. 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.

Next, the illumination device 30 irradiates light into the container 20 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.

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 exist 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 in 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, the 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 the space surrounded by the curved surface Sc. As a result, the light from the second light source 31B reaches the second object to be imaged Oj2. As a result, the 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, no matter where the objects to be imaged Oj1 and Oj2 are located in the well 21, the 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. Furthermore, it is more preferable that the first light source 31A and the second light source 31B are located within the space surrounded by the curved surface Sd, which includes the inner surface of the first side wall portion 23a. In this case, the multiple objects to be imaged Oj1, Oj2 housed within the second side wall portion 23b of the well 21 can be imaged more efficiently and without excess or deficiency.

On the other hand, as a comparative example, if, for example, as shown in Figure 5(b), only one light source 31 exists within 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.

[Variations]
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.

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

In Figure 6(a), the side wall 23 of the well 21 is cylindrical with a uniform horizontal cross-section. 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. While the horizontal cross-section of the side wall 23 is circular, it is not limited to this; it may also 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. At least two light sources 31 are arranged within the space on the plane Sf and surrounded by the curved surface Sc.

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 on the plane Sf and within the space surrounded by the curved surface Sc.

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 within a space on a plane Sf and surrounded by a curved surface Sc. More preferably, the at least two light sources 31 are located within a space surrounded by a curved surface Sd, including 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 completely. In particular, multiple objects Oj housed within the second side wall portion 23b can be imaged more efficiently and completely.

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 within a space on a plane Sf and surrounded by a curved surface Sc. More preferably, the at least two light sources 31 are located within a space surrounded by a curved surface Sd, including 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 completely. In particular, multiple objects Oj housed within the second side wall portion 23b can be imaged more efficiently and completely.

(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.

(Variations of wells and imaging units)
Figure 8 shows a modified version 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. 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.

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.

When the 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. Furthermore, the ANSI/SLAS standard specifies that the total height of the container 20, measured from the stationary plane (e.g., the top surface of the stage 50) to the maximum protrusion of the container 20, is 14.35 mm ± 0.76 mm.

In the example shown in Figure 8, the depth of field of the imaging unit 40 is set to WBEV or greater, 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, allowing all wells 21 to 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 over- or under-images.

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 the light source 31) can be avoided from being observed.

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 approximately 10 μm, when the object Oj is a cell, the object Oj can be imaged completely and without excess. 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 approximately 1 mm or larger, when the object Oj is a large cell, the object Oj can be imaged completely and without excess. 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 in the well 21A where the bottom 24 is at its highest position. This allows for complete and complete imaging of the liquid filling all the wells 21.

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, compared to a method that combines images captured from multiple wells 21, the number of images taken and the processing time can be reduced. Also, if the object being imaged Oj is a cell, the impact on the cell can be reduced by shortening the cell observation time.

As an example, an area sensor camera (resolution: 20.2 MP, sensor dimensions: 12.3 mm × 12.3 mm, horizontal/vertical resolution: 4504 px × 4504 px, horizontal/vertical pixel size: 2.74 μm × 2.74 μm) may be used as the camera 41 of the imaging unit 40. A telecentric lens (with aperture) may also 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 1.0 mm, 3.6 mm, 5.7 mm, and 12.5 mm. 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 a single shot with a resolution of 2.74 μm/px (imaging conditions: lens magnification set to 1.0x, work distance (distance from lens to inner surface of well bottom) approximately 112 mm). This allows for complete and accurate imaging of all objects Oj within all wells 21.

Furthermore, to further widen the depth of field, the optical axis between the object Oj and the imaging unit 40 may be divided into multiple sections using optical elements 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.

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 Oj being imaged, the first optical axis Oa1 and the second optical axis Oa2 overlap each other.

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 overlap. This allows for an increase in the overall depth of field DOFt.

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.

Depth of field can be measured as follows:

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 image it using the imaging unit 40. This will acquire an image IC, as shown in Figure 11, which includes the spatial frequency pattern SP (15 lines/mm) and the direct-reading scale DS for depth measurement.

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 positioned 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.

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

Next, we will explain how to select the PL (Photovoltaic Line) for the area where the brightness starts to change significantly from the left side.

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 4504 pixels x 4504 pixels in size, 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 data points results in 100 groups.

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. 1. 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 then created using this group No. n and the standard deviation of the y-axis (pixel brightness) of graph A for the range n to n+99 pixels (calculated, for example, using the STDEV function in Excel). This graph B is shown in Figure 12. Then, from graph B, find the portion where the standard deviation of pixel brightness is increasing.

In this case, first, for group numbers smaller than the upward-sloping portion of Graph B, the average value of the standard deviation of pixel brightness (here, 1.6) is taken for any consecutive group numbers. The number of any consecutive group numbers is set to be at least 4% 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 the setting is 4.4%, the number of any consecutive group numbers is 200.

Next, calculate 1.5 times the mean of the above standard deviation (here, the mean is 1.6 × 1.5 = 2.4). Then, for the portion where the standard deviation of pixel brightness is increasing, find the starting group No. of the portion 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, the brightness starts to change significantly from the left at the 1899th pixel on the X-axis (distance (pixels)). Therefore, the 1899th pixel on the X-axis (distance (pixels)) of graph A is defined as PL, the portion where the brightness change begins to change significantly from the left.

Next, we will explain how to select the PR (Pressure Level) area where the brightness starts to change significantly from the right side.

First, set the group size to at least 2% 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 4504 pixels x 4504 pixels in size, 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 data points results in 100 groups.

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. A graph C is created consisting of 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, we find the region where the standard deviation of pixel brightness slopes upward to the left.

In this case, first, for group numbers larger than the upward-sloping portion of graph C, the average of the standard deviations 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 at least 4% 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 the setting is 4.4%, the number of any consecutive group numbers is 200.

Next, calculate 1.5 times the mean of the above standard deviation (here, mean: 1.45 × 1.5 = 2.2). Then, for the portion where the standard deviation of pixel brightness is increasing from left to right, find the starting group No. of the portion 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, the brightness starts to change significantly from the right at the 2301st pixel on the X-axis (distance (pixels)). Therefore, the 2301st pixel on the X-axis (distance (pixels)) of graph A is defined as PR, the portion where the brightness change begins to change significantly from the right.

Furthermore, if the PL (partial brightness) setting, where brightness begins to change significantly from the left, cannot be selected (i.e., there is no continuous increase relative to 1.5 times the average standard deviation), 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 brightness) setting, where brightness begins to change significantly from the right, cannot be selected (i.e., there is no continuous increase relative to 1.5 times the average standard deviation), the rightmost value readable from the direct-reading scale DS will be used as the PR reading for depth of field. If neither PL nor 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.

(Stage transformation)
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 in the horizontal direction.

The transparent member 51 transmits light that enters 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. Even more preferably, 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, plastic (polystyrene resin, polyester resin, polyethylene resin, polypropylene resin, acrylic resin, polycarbonate resin, fluororesin, methylpentene resin, vinyl chloride resin, etc.).

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. Therefore, when the container 20 is slid on the transparent member 51, the container 20 does not fall off the transparent member 51. Preferably, the four corners 56a of the second opening 56 are positioned so that, when one corner 20a1 of the container 20 contacts each corner, the well 21 closest to the corner 20a2 that is vertex-opposite to the corner 20a1 can be imaged by the imaging unit 40.

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.

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.

Furthermore, as shown in Figure 19, a ruler 57 with markings may be fixed along the X and Y axes of the stage 50. A pole 58 may also be connected to each ruler 57. Each pole 58 is movable along the X and Y axes. This allows for easy positioning of the well 21 to be imaged. 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 spacing of each well 21. This allows for confirmation of which well 21 is being imaged using the markings on the ruler 57.

The multiple components disclosed in each of the above embodiments and variations can be combined as needed. Alternatively, some components may be removed from all the components shown in each of the above embodiments and variations.

10 Imaging device 20 Container 21 Well 22 Opening 22a Outer edge 23 Side wall 23a First side wall portion 23b Second side wall portion 24 Bottom 24a Outer edge 25 Top plate 30 Illumination device 31 Light source 40 Imaging unit 41 Camera 42 Lens 50 Stage

Claims (5)

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