Observation device and method for visualizing phase objects

Abstract

To secure the high observation performance in observation of a phase object stored in a multi-well plate.SOLUTION: An observation device comprises: an illumination optical system 15 which is provided on the lower side relative to an installation position of a multi-well plate C; a reflector 70 which is provided on the upper side relative to the installation position and reflects light emitted from the illumination optical system 15; and an observation optical system 16 which is provided on the lower side relative to the installation position and collects light reflected by the reflector 70. The reflector 70 is installed so as to pass through a peripheral well W2 different from an on-axis well W1 located on an optical axis of the observation optical system 16 before a marginal light beam incident on the observation optical system 16 is reflected by the reflector 70.SELECTED DRAWING: Figure 5

Description

[Technical Field]

[0001] The present disclosure relates to an observation device and a method for visualizing a phase object. [Background technology]

[0002] The cultivation of biological samples such as cells is carried out in an incubator to maintain a suitable cultivation environment. The condition of the biological samples is checked periodically during cultivation, but removing the biological samples from the incubator each time a check is made may adversely affect the growth of the biological samples.

[0003] A technology related to this problem is described, for example, in Patent Document 1. By using the observation device employing the oblique illumination described in Patent Document 1, cells and the like can be observed without increasing the size of the device. This makes it possible to continuously observe the biological sample being cultured in the limited space inside the incubator without removing the biological sample from the incubator. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] US Patent Application Publication No. 2019 / 0187450 Summary of the Invention [Problem to be solved by the invention]

[0005] However, when a multiwell plate, especially a multiwell plate with a small well diameter such as a 96-well plate, is used as the culture vessel, it is difficult to achieve high observation performance with oblique illumination such as that of the observation device described above.

[0006] In view of the above circumstances, an object of one aspect of the present invention is to provide a technique that ensures high observation performance when observing a phase object contained in a multiwell plate. [Means for solving the problem]

[0007] An observation device according to one aspect of the present invention includes an illumination optical system provided below a position where a multiwell plate is installed, a reflector provided above the installation position and reflecting light emitted from the illumination optical system, and an observation optical system provided below the installation position and collecting light reflected by the reflector, wherein the reflector It is a plane mirror, Marginal rays incident on the observation optical system At least the axial marginal rays is installed so that the light passes through a peripheral well different from an axial well located on the optical axis of the observation optical system before being reflected by the reflector. , satisfying the following condition .

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[0008] A method according to one aspect of the present invention is a method for visualizing a phase object contained in a multiwell plate, the method comprising the steps of: the multiwell plate is a 384-well plate;The method includes emitting light to the multiwell plate from an illumination optical system provided below the installation position of the multiwell plate, reflecting the light emitted from the illumination optical system with a reflector provided above the installation position, and condensing the light reflected by the reflector with an observation optical system provided below the installation position, wherein reflecting the light emitted from the illumination optical system includes condensing the light that has passed through peripheral wells different from an axial well located on the optical axis of the observation optical system before being reflected by the reflector. At least on axis Including making the light beam incident on the observation optical system as a marginal light beam. The reflector is a plane mirror and satisfies the following conditional expression:

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[0009] According to the above aspect, it is possible to provide a technique for ensuring high observation performance in observing a phase object contained in a multi-well plate. [Brief explanation of the drawings]

[0010] [Figure 1] FIG. 1 is a diagram illustrating an example of the configuration of a system 1. [Figure 2] FIG. 1 is a perspective view of an observation device 10. [Figure 3] FIG. 2 is a diagram showing the internal configuration of the observation device 10. [Figure 4] FIG. 4 is a cross-sectional view taken along the line AA shown in FIG. [Figure 5] This is to show the optical path of the light rays in the observation device 10. [Figure 6] 10 is a diagram for explaining the arrangement of an illumination light emission area IL. FIG. [Figure 7] FIG. 10 is a diagram for explaining the arrangement of wells W in a multiwell plate C. [Figure 8] FIG. 10 is a diagram for explaining parameters related to the placement of a reflector 70. [Figure 9] FIG. 10 is another diagram for explaining parameters related to the placement of the reflector 70. [Figure 10] 10 is a diagram showing the relationship between marginal rays passing through the main positions of well W and parameters. FIG. [Figure 11] 10 is a graph showing conditions under which oblique illumination is realized. [Figure 12] FIG. 2 is a diagram for explaining settings in the observation device 100 according to the first embodiment. [Figure 13] FIG. 2 is a diagram illustrating the specifications of a multiwell plate C1 used in the observation device 100 according to the first embodiment. [Figure 14] 1 is a diagram showing the position of an imaging region IM relative to a well W. FIG. [Figure 15] 10 is a diagram showing the distribution of contrast formed in the imaging area IM by the observation device 100. FIG. [Figure 16] FIG. 10 is a ray diagram for light incident from an on-axis well. [Figure 17] 17 is a diagram showing the distribution of contrast formed in the imaging area IM by the light shown in FIG. 16. FIG. [Figure 18] This is a ray diagram of light that has entered from an adjacent well and passed through a region relatively distant from the emission region in the axial well. [Figure 19] 19 is a diagram showing the distribution of contrast formed in the imaging region IM by the light shown in FIG. 18. FIG. [Figure 20] This is a ray diagram of light that has entered from an adjacent well and passed through a region relatively close to the emission region in the axial well. [Figure 21] 21 is a diagram showing the distribution of contrast formed in an imaging area IM by the light shown in FIG. 20. FIG. [Figure 22] 4 is a graph showing the conditions under which oblique illumination is achieved in the observation device 100 according to the first embodiment. [Figure 23] This is a ray diagram of light incident from an adjacent well and passing through a region relatively far from the emission region in the axial well when illuminated from the opposite direction to the illumination direction shown in Figure 18. [Figure 24] 23 is a diagram showing the distribution of contrast formed in the imaging area IM by the light shown in FIG. 22. FIG. [Figure 25] FIG. 25 is a diagram showing a contrast distribution obtained by combining the contrast distribution shown in FIG. 19 and the contrast distribution shown in FIG. 24. [Figure 26] 10 is a diagram showing the positions of multiple imaging regions relative to a well W. FIG. [Figure 27] 10 is a diagram showing the illumination direction when imaging the imaging region IM1 and the distribution of contrast formed in the imaging region IM1. FIG. [Figure 28] 10 is a diagram showing the illumination direction when imaging the imaging region IM2 and the distribution of contrast formed in the imaging region IM2. FIG. [Figure 29] 10 is a diagram showing the illumination direction when imaging the imaging region IM3 and the distribution of contrast formed in the imaging region IM3. FIG. [Figure 30] 10 is a diagram showing the illumination direction when imaging the imaging region IM4 and the distribution of contrast formed in the imaging region IM4. FIG. [Figure 31] FIG. 31 is a diagram showing a contrast distribution obtained by combining the contrast distributions shown in FIGS. 27 to 30. [Figure 32] FIG. 10 is a diagram for explaining settings in an observation device 200 according to a second embodiment. [Figure 33] FIG. 10 is a diagram illustrating the specifications of a multiwell plate C2 used in an observation device 200 according to a second embodiment. [Figure 34]FIG. 10 is a ray diagram for light incident from an adjacent well. [Figure 35] 35 is a diagram showing the distribution of contrast formed in the imaging area IM by the light shown in FIG. 34. FIG. [Figure 36] 35 is a diagram showing the distribution of contrast formed in the imaging area IM when illuminated from the opposite direction to the illumination direction shown in FIG. 34. FIG. [Figure 37] FIG. 37 is a diagram showing a contrast distribution obtained by combining the contrast distribution shown in FIG. 35 and the contrast distribution shown in FIG. 36. [Figure 38] 10 is a graph showing the conditions under which oblique illumination is achieved in the observation device 200 according to the second embodiment. [Figure 39] FIG. 10 is a diagram for explaining settings in an observation device 300 according to a third embodiment. [Figure 40] 40 is a diagram showing the distribution of contrast formed in the imaging area IM by the light shown in FIG. 39. FIG. [Figure 41] 40 is a diagram showing the distribution of contrast formed in the imaging area IM when illuminated from the opposite direction to the illumination direction shown in FIG. 39. FIG. [Figure 42] 42 is a diagram showing a contrast distribution obtained by combining the contrast distribution shown in FIG. 40 and the contrast distribution shown in FIG. 41. FIG. [Figure 43] 10 is a graph showing the conditions under which oblique illumination is achieved in the observation device 300 according to the third embodiment. [Figure 44] FIG. 10 is a diagram showing an example of the positional relationship between a multiwell plate and the curved surface of a reflector. [Figure 45] FIG. 45 is a diagram for explaining the region of oblique illumination achieved by an observation device equipped with reflectors arranged in the positional relationship shown in FIG. 44. [Figure 46] FIG. 10 is a diagram showing another example of the positional relationship between the multiwell plate and the curved surface of the reflector. [Figure 47] FIG. 47 is a diagram for explaining the region of oblique illumination achieved in an observation device equipped with reflectors arranged in the positional relationship shown in FIG. 46. [Figure 48] FIG. 10 is a diagram showing another example of the positional relationship between the multiwell plate and the curved surface of the reflector. [Figure 49] FIG. 49 is a diagram for explaining the region of oblique illumination achieved by an observation device equipped with reflectors arranged in the positional relationship shown in FIG. 48. [Figure 50] FIG. 10 is a diagram for explaining parameters related to the placement of a reflector 470. [Figure 51] This figure shows the relationship between the marginal ray passing through the upper right end (ORU) of the axial well and the parameters. [Figure 52] FIG. 10 is a diagram showing the relationship between marginal rays passing through the right and left ends (OLU) of an on-axis well and parameters. [Figure 53] FIG. 10 is a diagram showing the relationship between the marginal ray passing through the right end (M+) of the cylindrical surface array and the parameters. [Figure 54] This is a diagram showing the relationship between the marginal ray passing through the right end (M-) of the cylindrical surface array and the parameters. [Figure 55] 10 is a graph showing the conditions under which there is no vignetting by the on-axis well and the light falls within the effective range of the reflecting surface of the reflector 470. [Figure 56] FIG. 10 is a diagram illustrating marginal rays passing through the upper left (LU) and lower right (RB) edges of adjacent wells. [Figure 57] 56 is a graph showing the relationship between the height of the reflector 470 and the coordinates at which the marginal ray shown in FIG. 55 passes through the object surface. [Figure 58] FIG. 10 illustrates a marginal ray passing through the upper left edge (LU) of an adjacent well. [Figure 59] FIG. 10 is a diagram illustrating a marginal ray passing through the bottom right edge (RB) of an adjacent well. [Figure 60] 59 is a graph showing the relationship between the height of the reflector 470 and the coordinates at which the marginal ray shown in FIG. 58 passes through the object surface. [Figure 61] 60 is a graph showing the relationship between the height of the reflector 470 and the coordinates at which the marginal ray shown in FIG. 59 passes through the object surface. [Figure 62]10 is a graph showing conditions under which oblique illumination is realized by a reflector 470 having an array of cylindrical surfaces. [Figure 63] FIG. 10 is a diagram for explaining settings in an observation device 400 according to a fourth embodiment. [Figure 64] FIG. 10 is a diagram for explaining the positional relationship between the multiwell plate C1 and the reflector 471. [Figure 65] FIG. 10 is a ray diagram for light incident from an adjacent well. [Figure 66] 10 is a diagram showing the illumination direction when imaging the imaging region IM1 and the distribution of contrast formed in the imaging region IM1. FIG. [Figure 67] 10 is a diagram showing the illumination direction when imaging the imaging region IM2 and the distribution of contrast formed in the imaging region IM2. FIG. [Figure 68] 10 is a diagram showing the illumination direction when imaging the imaging region IM3 and the distribution of contrast formed in the imaging region IM3. FIG. [Figure 69] 10 is a diagram showing the illumination direction when imaging the imaging region IM4 and the distribution of contrast formed in the imaging region IM4. FIG. [Figure 70] FIG. 70 is a diagram showing a contrast distribution obtained by combining the contrast distributions shown in FIGS. 66 to 69. [Figure 71] FIG. 10 is a diagram for explaining settings in an observation device 500 according to a fifth embodiment. [Figure 72] FIG. 10 is a diagram for explaining the specifications of the multiwell plate C3. [Figure 73] FIG. 10 is a ray diagram for light incident from an adjacent well. [Figure 74] FIG. 74 is a diagram showing the distribution of contrast formed in imaging area IM5 by the light shown in FIG. 73. [Figure 75] This is a ray diagram for light incident from an adjacent well in the opposite direction to the illumination direction shown in Figure 73. [Figure 76] FIG. 76 is a diagram showing the distribution of contrast formed in the imaging area IM6 by the light shown in FIG. 75. [Figure 77] FIG. 77 is a diagram showing a contrast distribution obtained by combining the contrast distribution shown in FIG. 74 and the contrast distribution shown in FIG. 76. [Figure 78] FIG. 10 is a diagram for explaining settings in an observation device 600 according to a sixth embodiment. [Figure 79] FIG. 10 is a diagram for explaining the positional relationship between the multiwell plate C1 and the reflector 571. [Figure 80] FIG. 10 is a diagram showing the contrast distribution obtained by the observation device 600. [Figure 81] FIG. 10 is a ray diagram for light incident from an adjacent well. [Figure 82] 82 is a diagram showing the distribution of contrast formed in imaging area IM10 by the light shown in FIG. 81. FIG. [Figure 83] This is a ray diagram when the imaging area is slightly shifted along the illumination direction from the state shown in Figure 81. [Figure 84] FIG. 84 is a diagram showing the distribution of contrast formed in the imaging area IM11 by the light shown in FIG. 83. [Figure 85] 10 is a diagram for explaining the imaging order in the observation device 600. FIG. [Figure 86] 10 is a diagram for explaining the contrast obtained in wells in even-numbered columns and odd-numbered columns in the observation device 600. FIG. [Figure 87] FIG. 10 is a diagram for explaining the configuration of an observation device 700 according to a seventh embodiment. [Figure 88] FIG. 10 is a ray diagram for light incident from an adjacent well. [Figure 89] FIG. 89 is a diagram showing the distribution of contrast formed in the imaging area IM10 by the light shown in FIG. 88. [Figure 90] This is a ray diagram when the imaging area is slightly shifted along the illumination direction from the state shown in Figure 88. [Figure 91] 91 is a diagram showing the distribution of contrast formed in the imaging area IM11 by the light shown in FIG. 90. FIG. [Figure 92]This is a ray diagram for light incident from an adjacent well in the opposite direction to that in Figure 88. [Figure 93] 93 is a diagram showing the distribution of contrast formed in the imaging area IM10 by the light shown in FIG. 92. FIG. [Figure 94] This is a ray diagram when the imaging area is slightly shifted along the illumination direction from the state shown in Figure 93. [Figure 95] 95 is a diagram showing the distribution of contrast formed in the imaging area IM12 by the light shown in FIG. 94. FIG. [Figure 96] 10 is a diagram for explaining the function of two reflecting surfaces of a reflector 572. FIG. [Figure 97] FIG. 10 is a diagram showing the contrast distribution obtained by the observation device 700. [Figure 98] FIG. 13 is a diagram for explaining the configuration of an observation device 800 according to an eighth embodiment. [Figure 99] FIG. 10 is a ray diagram for light incident from an adjacent well. [Figure 100] 99. FIG. 99 shows the distribution of contrast formed in the imaging area IM10. [Figure 101] This is a ray diagram when the imaging area is slightly shifted along the illumination direction from the state shown in Figure 99. [Figure 102] 102 is a diagram showing the distribution of contrast formed in the imaging area IM11 by the light shown in FIG. 101. FIG. [Figure 103] This is a ray diagram for light incident from an adjacent well in the opposite direction to that in Figure 99. [Figure 104] 104 is a diagram showing the distribution of contrast formed in the imaging area IM10 by the light shown in FIG. 103. FIG. [Figure 105] This is a ray diagram when the imaging area is slightly shifted along the illumination direction from the state shown in Figure 103. [Figure 106] 106 is a diagram showing the distribution of contrast formed in the imaging area IM12 by the light shown in FIG. 105. FIG. [Figure 107] FIG. 13 is a diagram for explaining the configuration of an observation device 900 according to a ninth embodiment. [Figure 108] 10A and 10B are diagrams for explaining adjacent wells through which illumination light incident on an axial well passes. [Figure 109] FIG. 10 is a ray diagram for light incident from an adjacent well. [Figure 110] 110 is a diagram showing the distribution of contrast formed in imaging area IM5 by the light shown in FIG. 109. FIG. [Figure 111] This is a ray diagram for light incident from an adjacent well in the opposite direction to that of Figure 109. [Figure 112] 112 is a diagram showing the distribution of contrast formed in the imaging area IM6 by the light shown in FIG. 111. FIG. [Figure 113] FIG. 10 is a diagram showing the contrast distribution obtained by the observation device 900. [Figure 114] FIG. 20 is a diagram for explaining settings in the observation device 1000 according to the tenth embodiment. [Figure 115] FIG. 10 is a diagram for explaining the positional relationship between the multiwell plate C3 and the reflector 671. [Figure 116] FIG. 10 is a ray diagram for light incident from an adjacent well. [Figure 117] 117 is a diagram showing the distribution of contrast formed in the imaging area IM by the light shown in FIG. 116. FIG. [Figure 118] 117 is a diagram showing the distribution of contrast formed in the imaging area IM by light incident from an adjacent well in the opposite direction to that in FIG. 116. [Figure 119] FIG. 1 is a diagram showing the distribution of contrast obtained by the observation device 1000. [Figure 120] FIG. 10 is a diagram for explaining the contrast of each well in multiwell plate C3. [Figure 121] FIG. 20 is a diagram for explaining the configuration of an observation device 1100 according to an eleventh embodiment. [Figure 122] 10A and 10B are diagrams for explaining the relationship between a reflective surface and a well row onto which reflected light is incident. [Figure 123]FIG. 10 is a diagram for explaining the contrast of each well in multiwell plate C3. DETAILED DESCRIPTION OF THE INVENTION

[0011] FIG. 1 is a diagram illustrating the configuration of the system 1. FIG. 2 is a perspective view of the observation device 10. FIG. 3 is a diagram illustrating the internal configuration of the observation device 10. FIG. 4 is a cross-sectional view taken along the line AA in FIG. 3. FIG. 5 is a diagram illustrating the optical path of light rays in the observation device 10. FIG. 6 is a diagram illustrating the arrangement of the illumination light emission region IL. The configuration of the system 1 will be described below with reference to FIGS. 1 to 6.

[0012] The system 1 shown in Figure 1 is an observation system that observes a sample contained in a multiwell plate while culturing it. The sample to be observed can be any cultured cell, such as a colorless and transparent phase object. The number of wells in the multiwell plate is not particularly limited, but it is particularly suitable when a multiwell plate with a large number of wells, such as 96 or more, is used.

[0013] The system 1 includes one or more observation devices 10 that acquire images of samples cultured in multiwell plates, and a control device 30 that controls the observation devices 10. Each of the observation devices 10 and the control device 30 only need to be able to exchange data with each other. Therefore, each of the observation devices 10 and the control device 30 may be connected to each other so as to be able to communicate with each other via a wire, as shown in FIG. 1, or may be connected to each other so as to be able to communicate with each other wirelessly.

[0014] The observation device 10 is an imaging device that captures images of samples contained in a multiwell plate from below the multiwell plate. To capture images of the samples without removing them from the incubator 20, the observation device 10 is used while placed inside the incubator 20, for example, as shown in FIG. 1. More specifically, as shown in FIG. 2, the observation device 10 is placed inside the incubator 20 with a multiwell plate C placed on the transmission window 11 of the observation device 10, and acquires images of the samples contained in the wells of the multiwell plate C according to instructions from the control device 30.

[0015] As shown in Figure 2, the observation device 10 comprises a box-shaped housing 12 with a transparent transmission window 11 on the upper surface through which the multiwell plate C is placed, a positioning member 60 that positions the multiwell plate C at a predetermined position relative to the observation device 10 on the transmission window 11 (mounting surface), and a reflector 70 that is attached to the multiwell plate C.

[0016] The transmission window 11 provided in the housing 12 is a transparent top plate that forms the upper surface of the housing 12 of the observation device 10, and forms the mounting surface on which the multiwell plate C is placed. That is, in the observation device 10, the position above the transmission window 11 is the installation position of the multiwell plate C. The transmission window 11 is made of, for example, glass or transparent resin.

[0017] The positioning member 60 is fixed to the housing 12. However, the positioning member 60 can be removed as needed.

[0018] The reflector 70 is a flat mirror having a flat reflective surface. The reflector 70 is attached to the multiwell plate C, and is positioned above the installation position of the multiwell plate C, as shown in FIG. 2. The reflector 70 is attached, for example, by placing it on the upper surface of the multiwell plate C via a support member 80, as shown in FIG. 3. However, the support member 80 may be attached to the positioning member 60, as long as it can position the reflector 70 at a predetermined height. The reflector 70 reflects light emitted from the illumination optical system 15, which will be described later, when the observation device 10 images the sample from below the multiwell plate C.

[0019] The multiwell plate C is, for example, a 96-well plate having 96 (=12 × 8) wells W, as shown in Figure 4. The vertical and horizontal widths of the multiwell plate C are predetermined, for example, by the ANSI / SBS standard.

[0020] 3, the observation device 10 further includes, inside the housing 12, a stage 13 that moves within the housing 12, and an imaging unit 14 that is provided on the stage 13. As shown in FIG. 5, the imaging unit 14 includes an illumination optical system 15 and an observation optical system 16.

[0021] The stage 13 is an example of a moving device that moves within the observation device 10, and moves the illumination optical system 15 and the observation optical system 16 relative to the multiwell plate C in a direction perpendicular to the optical axis of the observation optical system 16. In other words, the stage 13 changes the relative position of the imaging unit 14 with respect to the multiwell plate C. The stage 13 can move in a direction perpendicular to the optical axis of the observation optical system 16, more specifically, in the X and Y directions that are parallel to the transmission window 11 (mounting surface) and perpendicular to each other, but it may also move in the Z direction that is perpendicular to both the X and Y directions.

[0022] As shown in FIG. 5, the illumination optical system 15 is provided below the installation position of the multiwell plate C (i.e., the upper surface of the transmission window 11) and includes a light source 15a and a diffuser 15b. The light source 15a includes, for example, a light-emitting diode (LED). The light source 15a may include a white LED, or may include multiple LEDs that emit light of multiple different wavelengths, such as R (red), G (green), and B (blue). The light emitted from the light source 15a is incident on the diffuser 15b.

[0023] The diffuser plate 15b diffuses the light emitted from the light source 15a. The diffuser plate 15b is not particularly limited, but may be, for example, a frosted diffuser plate with an uneven surface. However, the diffuser plate 15ba may also be an opal-type diffuser plate with a coated surface, or may be another type of diffuser plate. The light emitted from the diffuser plate 15b travels in various directions and enters the well W at various angles.

[0024] 5, the illumination optical system 15 is provided with two light sources 15a at positions symmetrical with respect to the observation optical system 16. Therefore, as shown in Fig. 6, two emission regions IL of illumination light emitted from the illumination optical system 15 are also formed at positions symmetrical with respect to the observation optical system 16. That is, the illumination optical system 15 can switch the illumination direction between two directions symmetrical with respect to the optical axis of the observation optical system 16 by selectively emitting light from the light sources 15a. Hereinafter, of the two directions orthogonal to the optical axis of the observation optical system 16, the direction in which the emission regions IL are aligned, that is, the direction in which the light sources 15a are aligned, is defined as the Y direction, and the direction orthogonal to that is defined as the X direction.

[0025] As shown in Figure 5, the observation optical system 16 includes an image sensor 16a, an aperture 16b, and a lens system 16c. The observation optical system 16 is located below the installation position of the multiwell plate C and focuses light that is reflected by the reflector 70 and transmitted through the transmission window 11 and enters the housing 12. More specifically, the lens system 16c focuses light from the bottom of the well W containing the sample onto the image sensor 16a, thereby forming an optical image of the sample on the image sensor 16a. Furthermore, the observation optical system 16 is a telecentric optical system on the sample side so that the magnification does not change when the focus is changed.

[0026] The image sensor 16a is an optical sensor that converts detected light into an electrical signal, and captures images of samples in wells located on the optical axis of the observation optical system 16. The image sensor 16a captures images of the samples at multiple different positions as the illumination optical system 15 and the observation optical system 16 are moved relative to the multi-well plate C by the stage 13. The image sensor 16a is an image sensor, and is not particularly limited to, for example, a CCD (Charge-Coupled Device) image sensor or a CMOS (Complementary MOS) image sensor.

[0027] 3 shows an example in which the illumination optical system 15 and the observation optical system 16 in the imaging unit 14 are installed on the stage 13 and move together within the housing 12, but the illumination optical system 15 and the observation optical system 16 may move independently within the housing 12. Also, while FIG. 5 shows an example in which a pair of light sources 15a are arranged on the left and right sides of the observation optical system 16, the arrangement and number of light sources 15a are not limited to this example. For example, three or more light sources 15a may be provided within the imaging unit 14, or only one light source 15a may be provided.

[0028] Observation device 10 configured as described above employs oblique illumination to visualize a sample, which is a phase object. Specifically, as shown in Fig. 5, light emitted by light source 15a is diffused by diffuser plate 15b, passes through transmission window 11, and exits housing 12. Some of the light exiting housing 12 is then reflected by, for example, the liquid interface in well W, the lid placed on the container body of multiwell plate C, or reflector 70 attached to multiwell plate C, and is thereby deflected above the sample.

[0029] Furthermore, a portion of the light deflected above the sample is irradiated onto the sample and passes through the sample and the transmission window 11 before entering the housing 12. A portion of the light entering the housing 12 is then collected by the observation optical system 16, forming an image of the sample on the image sensor 16a within the observation optical system 16. Light reflected by the reflector 70 is primarily used for image formation. In particular, to accommodate observations using multiwell plates with a narrow pitch P (see FIG. 7 ) between wells W, such as a 96-well plate, light that enters the reflector 70 via peripheral wells (particularly well W2 adjacent to the axial well) different from the axial well (well W1) located on the optical axis of the observation optical system 16 is used. This point will be described in more detail later. Finally, the observation device 10 generates an image of the sample based on the electrical signal output from the image sensor 16a and outputs the image to the control device 30.

[0030] The control device 30 is a device that controls the observation device 10. The control device 30 may include one or more processors and one or more non-transitory computer-readable media, and may be, for example, a general-purpose computer. Each of the one or more processors is an electrical circuit made up of hardware including, for example, a central processing unit (CPU), a graphics processing unit (GPU), a digital signal processor (DSP), etc., and performs programmed processing by executing programs stored in one or more non-transitory computer-readable media. The one or more processors may also include an application-specific integrated circuit (ASIC), a field-programmable gate array (FPGA), etc.

[0031] The control device 30 configured as described above transmits an image acquisition instruction to the observation device 10 placed in the incubator 20 and receives images acquired by the observation device 10. The control device 30 may display images acquired by the observation device 10 on a display device provided in the control device 30, and the system 1 may thereby function as an observation system for a user to observe a sample being cultured. The control device 30 may communicate with client terminals (client terminal 40, client terminal 50) shown in FIG. 1, and may display images acquired by the observation device 10 on a display device provided in the client terminal. The client terminal may be any device that has a display unit, and may be, for example, a desktop or notebook computer, a tablet, a smartphone, or the like.

[0032] In order to visualize a phase object sample with high contrast and to enable the recognition of cells, etc., it is important to form an image of the sample by converging light incident on the sample at an appropriate angle, i.e., to achieve oblique illumination. Specifically, it is desirable that the light incident on the sample contains light with an incident angle greater than the angle corresponding to the numerical aperture of the observation optical system 16 and light with an incident angle less than the angle.

[0033] The reason for this can be explained as follows: The angle of incidence on the object plane is converted by the observation optical system 16 into a distance from the optical axis on the pupil plane of the observation optical system 16. Therefore, a bundle of rays whose angle of incidence on the object plane is widely distributed over a relatively large angle and satisfies the above-mentioned condition will be distributed across the outer edge of the pupil of the observation optical system 16 on the pupil plane. Of this bundle of rays, those that are incident outside the pupil (i.e., light whose angle of incidence on the object plane exceeds the angle corresponding to the numerical aperture) are vignetted within the observation optical system 16 and do not reach the image sensor 16a. Therefore, the bundle of rays that reaches the image sensor 16a will produce a discontinuous intensity distribution in the angular direction on the image sensor, i.e., a steep change in the intensity distribution. As a result, shadows appear in the image of the sample, allowing for a three-dimensional image with high contrast to be obtained.

[0034] On the other hand, when light emitted from the illumination optical system 15 and incident on the multiwell plate C from the bottom of the axial well (well W1) to be observed is guided from the bottom of the same well W1 to the observation optical system 16 to observe the sample in the well W1, obtaining a high-contrast image is not necessarily easy. In particular, when using a multiwell plate with a narrow pitch P, such as a 96-well plate, it is not easy to direct light at the appropriate angle to the object surface (bottom) and then guide that light to the observation optical system 16. Even if oblique illumination could be achieved, the area where high contrast can be achieved is extremely limited within the well. Furthermore, since the well diameter becomes narrower as the number of wells increases, this becomes even more difficult when using, for example, a 384-well plate.

[0035] Therefore, in order to accommodate observations using multiwell plates with a narrow pitch P, the observation device 10 actively utilizes light from the peripheral wells. Specifically, the reflector 70 is installed so that marginal rays incident on the observation optical system 16 pass through a peripheral well different from the on-axis well (well W1) located on the optical axis of the observation optical system 16 before being reflected by the reflector 70. More specifically, by appropriately setting the height of the reflector 70 relative to the top surface of the multiwell plate C, the marginal rays are configured to enter the observation optical system 16 via the peripheral well. This achieves oblique illumination within the on-axis well, allowing samples in the on-axis well to be observed with high contrast.

[0036] Furthermore, it is usually desirable to observe at least the central portion of the field of view with good contrast. For this reason, it is desirable to install the reflector 70 so that the axial marginal rays incident on the observation optical system 16 pass through a peripheral well different from the axial well (well W1) located on the optical axis of the observation optical system 16 before being reflected by the reflector 70. This achieves oblique illumination at least in the central portion of the field of view, allowing the central portion of the field of view to be observed with high contrast.

[0037] 8 and 9 are diagrams for explaining parameters related to the placement of the reflector 70. FIG. 10 is a diagram showing the relationship between marginal rays passing through key positions in the well W and the parameters. FIG. 11 is a graph showing the conditions under which oblique illumination is achieved. Below, with reference to FIGS. 8 to 11, we will specifically explain how to determine the conditions under which oblique illumination is achieved, in particular the appropriate height of the reflector 70.

[0038] First, the main parameters will be described using an example in which illumination light is incident via a peripheral well on the right side of the axial well, with reference to Figures 8 and 9. In the following, the upper right end of the axial well will be abbreviated as ORU, the upper left end of the adjacent well to the right of the axial well as LU, and the lower right end of the adjacent well to the right of the axial well as RB.

[0039] The parameters shown in Figure 8 are: N a is the numerical aperture of the observation optical system 16 on the sample side. P is the distance from the center of a well W of the multiwell plate C to the center of an adjacent well W, i.e., the well pitch. D u is the inner diameter of the upper end of the well W. D b is the inner diameter of the bottom (lower end) of the well W. w is the distance from the liquid surface of the solution (culture medium) contained in the well W at the center of the well W to the bottom of the well W. b is the distance from the top to the bottom of the well W, that is, the depth of the well W.

[0040] H m is the height of the reflector 70 based on the top end of the well W. More precisely, it is the air-equivalent distance from the top end of the well W of the multiwell plate C to the reflective surface of the reflector 70. Therefore, H m When the reflector 70 is a back-coated mirror, it can be calculated by the following formula:

number

[0041] Here, the parameters shown in FIG. 9 used in the above equation are as follows: H WU-Lid is the distance (physical length) from the top edge of the well W to the bottom surface of the lid of the multiwell plate C. Lid is the thickness (physical length) of the lid of multiwell plate C. n Lid is the refractive index of the lid of multiwell plate C. H O is the distance (physical length) from the top surface of the lid of the multiwell plate C to the bottom surface of the reflector 70. Mrp is the thickness (physical length) from the bottom surface of the reflector 70 to the reflecting surface. Mrp is the refractive index of the medium (glass) from the lower surface of the reflector 70 to the reflecting surface.

[0042] Other, n wis the refractive index of the solution (culture solution) contained in the well W. Also, A is the refractive index of the liquid surface of the solution (culture solution) contained in the well W, and A c2 If we use the coefficient when approximating the concave shape of the liquid surface with a quadratic curve, then (n w -1)A c2 It is calculated as follows.

[0043] Next, various constraints that arise when realizing oblique illumination will be considered with reference to Figures 10 and 11. First, constraints associated with ORU will be considered with reference to Figure 10(a).

[0044] When the numerical aperture is derived from the light rays passing through an arbitrary observation position on the object plane and the ORU, the numerical aperture N aORU is expressed by the following formula. s is the Y coordinate of the observation position (Y coordinate).

number

[0045] The observation optical system 16 has a numerical aperture N aORU If the numerical aperture is greater than the coordinate Y, s Since the light beam from the observation optical system 16 enters the pupil diameter, it cannot be distributed across the outer edge of the pupil of the observation optical system 16 on the pupil plane. s cannot be observed with high contrast. aORU The equation (2) that defines the upper limit of the numerical aperture of the observation optical system 16 indicates the upper limit of the numerical aperture of the observation optical system 16. s are the coordinates of the minimum position to be observed, it is desirable to satisfy the following conditional expression.

number

[0046] Next, the numerical aperture N of the ORU and the observation optical system 16 a In this case, the coordinates (Y coordinate) Y of the marginal ray passing through the ORU, as shown in Fig. 10(a), aresORU is expressed by the following formula:

number

[0047] The marginal ray has coordinate Y on the object surface. sORU If you try to pass through the area on the lighting side (positive side in this example) of the coordinate Y, it will be blocked by the ORU. sORU The equation (4) that defines the maximum coordinate in the Y direction that a marginal ray can pass through on the object surface. Therefore, the area where oblique illumination is established in the observation device 10 and high contrast can be obtained is at least within the coordinate Y sORU The observation position coordinate Y is the area on the negative side of the s will satisfy the following conditional expression:

number

[0048] The observation device 10 calculates the coordinates of the position to be observed at least as a minimum by using the coordinate Y s When the condition (3) and the condition (5) are satisfied, it is desirable that both the condition (3) and the condition (5) are satisfied.

[0049] Next, we will consider the constraints associated with LU and RB with reference to Figures 10(b) and 10(c). When tracing a marginal ray from the pupil position toward the light source, LU and RB become constraints in the peripheral wells it passes through. If it attempts to pass through a position closer to the optical axis than LU, it will be vignetted by the edge of the peripheral well. Also, if it attempts to pass through a position farther from the optical axis than RB, it will be vignetted by the side of the peripheral well. This is true not only when it passes through an adjacent well next to an on-axis well, but also when it passes through any peripheral well (for example, the well two next to it).

[0050] That is, all marginal rays incident on the observation optical system 16 pass between the marginal rays passing through LU and the marginal rays passing through RB. For this reason, we will consider the marginal rays passing through LU and the marginal rays passing through RB.

[0051] Numerical aperture N of the observation optical system 16 a When is known, the passing position of the marginal ray at the pupil position (the outer edge of the pupil) is determined, and as a result, two points through which the ray passes are determined together with LU or RB. However, the path of the ray changes depending on the height at which the reflector 70 is installed. Therefore, first, the coordinates on the object plane of the marginal ray passing through LU or RB are considered by fixing the height at which the reflector 70 is installed (i.e., by making it a variable).

[0052] In this case, as shown in Fig. 10(b), the coordinates of the marginal ray passing through LU on the object plane are defined as Y sFLU As shown in Fig. 10(c), the coordinates of the marginal ray passing through RB on the object plane are defined as Y sFRB Then, the coordinate Y of the observation position where oblique illumination is established is s will satisfy the following conditional expression:

number

[0053] Here, the coordinate Y sFLU and the coordinate Y sFRB is the height H of the reflector 70 m When defined using the formula, it is expressed by the following formula: k is an integer of 1 or more, and indicates how many neighboring wells the marginal ray passes through are located next to the on-axis well.

number

[0054] The observation device 10 calculates the coordinates of the position to be observed at least as a minimum by using the coordinate Y s When the condition (6) is satisfied, it is desirable to satisfy the condition (6) in addition to the condition (3) and the condition (5).

[0055] Next, the coordinates on the object plane through which the marginal ray passes are fixed (that is, defined as variables), and the height of the reflector 70 at which oblique illumination is established is considered. In this case, the coordinates Y s The height of the reflector 70 when the marginal ray passing through passes through LU is the height H mFLU and the coordinate Y on the object surface s The height of the reflector 70 when the marginal ray passing through passes through RB is the height H mFRB Then, the height H of the reflector 70 where oblique illumination is established is m will satisfy the following conditional expression:

number

[0056] Here, the height H mFLU and height H mFRB is the Y coordinate s When defined using the formula, it is expressed by the following formula: k is an integer of 1 or more, and indicates how many neighboring wells the marginal ray passes through are located next to the on-axis well.

number

[0057] The observation device 10 calculates the coordinates of the position to be observed at least as a minimum by using the coordinate Y s When the condition (9) is satisfied, it is desirable to satisfy the condition (9) in addition to the condition (3) and the condition (5).

[0058] The above-described relationship is shown in Fig. 11. The graph shown in Fig. 11 shows the relationship between the numerical aperture N a is a fixed value, and this is an example where k is 1. Line L1 shows the relationship between the object plane coordinates of the marginal ray passing through LU and the height of the reflector 70. Line L2 shows the relationship between the object plane coordinates of the marginal ray passing through RB and the height of the reflector 70.

[0059] The area between lines L1 and L2 satisfies the condition for preventing marginal rays from being vignetted in adjacent wells, and the area to the left of line L3 satisfies the condition for preventing marginal rays from being vignetted in on-axis wells. Therefore, the area to the left of line L3 and between lines L1 and L2 is the area where oblique illumination is established.

[0060] Generally, when observing the inside of a well, it is desirable to have oblique illumination at the center of the well. s It is desirable that oblique illumination be established when θ = 0. In this case, it is desirable that the observation device 10 satisfy the following conditional expressions instead of conditional expressions (3) and (5).

number

[0061] where N aCORU is N aORU Y in the definition of s is substituted with 0 and is defined as follows:

number

[0062] Furthermore, it is desirable that the observation device 10 satisfy the following condition, which is indicated by line L4 in FIG.

number

[0063] Here, the height H mFCLU and height H mFCRB are the height H mFLU and height H mFRB Y in the definition of s is substituted with 0 and is defined as follows:

number

[0064] When observing the center of a well, it is desirable that the observation device 10 satisfy conditional formula (9-1) in addition to conditional formulas (3-1) and (5-1).

[0065] Based on the above, a standard 96-well plate was used, and the central part of the well (Y s = 0) with good contrast using oblique illumination, the observation device 10 preferably satisfies the following conditions: Hm is expressed in mm and N a The unit of is a dimensionless quantity.

number

[0066] Here, the various parameters when using a standard 96-well plate are as follows: P=9mm, D u = 6.9 mm, D b = 6.4 mm, n W =1.332, A=0.0763 / mm, H b =10.7mm, H w =1.8mm, k=1

[0067] In addition, using a standard 384-well plate, the central part of the well (Y s = 0) with good contrast using oblique illumination, the observation device 10 preferably satisfies the following conditions: Hm is expressed in mm and N a The unit of is a dimensionless quantity.

number

[0068] That is, when light from an adjacent well is used (k=1), it is desirable that the observation device 10 satisfy the following conditional expression.

number

[0069] Furthermore, when light from the next adjacent well is used (k=2), it is desirable that the observation device 10 satisfy the following conditional expression.

number

[0070] Here, the various parameters when using a standard 384-well plate are as follows: P=4.5mm, D u =3.8mm, D b =3.2mm, n W =1.332, A=0.153 / mm, H b =11mm, H w =1.8mm, k=1 or 2

[0071] A specific example of the observation device 10 using a plane mirror as a reflector will be described below in the following embodiment. [First embodiment] Fig. 12 is a diagram for explaining the settings in the observation device 100 according to this embodiment. Fig. 13 is a diagram for explaining the specifications of the multiwell plate C1 used in the observation device 100 according to this embodiment. Fig. 14 is a diagram showing the position of the imaging region IM relative to the well W. The configuration and settings of the observation device 100 will be explained below with reference to Figs. 12 to 14.

[0072] Observation device 100 is an apparatus for observing samples in multiwell plate C1, and achieves oblique illumination mainly using light incident from an adjacent well (k=1). Observation device 100 has a configuration similar to observation device 10, and as shown in Fig. 12, it includes illumination optical system 115, reflector 170 that reflects light emitted from illumination optical system 115, and observation optical system 116 that collects the light reflected by reflector 170. Illumination optical system 115 includes light source 115a and diffuser 115b.

[0073] The illumination optical system 115 and the observation optical system 116 are installed below the installation position of the multiwell plate C1. In contrast, the reflector 170 is installed above the installation position of the multiwell plate C1. The reflector 170 is a plane mirror and is installed so that on-axis marginal rays incident on the observation optical system 116 pass through adjacent wells before being reflected by the reflector 170.

[0074] In the observation device 100, the specifications of the observation optical system 116, the specifications of the multiwell plate C1, the state of the culture solution in the wells, and the parameters related to the installation conditions of the reflector 170 are as follows, as shown in Figures 12 and 13. The multiwell plate C1 is a 96-well plate. The thickness of the lid of the multiwell plate C1 is 1.5 mm, which is equivalent to 1.0 mm in air. N a =0.25, P=9mm, D u = 6.9 mm, D b = 6.4 mm, n W =1.332, A c2 =0.11497 / mm, A=0.0763 / mm, H b =10.7mm, H w =1.8mm, H m =10mm

[0075] At this time, in the observation device 100, N aCORU , Y sORU , H mFCLU , H mFCRB are as follows: N aCORU =0.31, Y sORU =0.39mm, H mFCLU = 6.0 mm, H mFCRB =11.5mm

[0076] Therefore, the observation device 100 satisfies the conditional formulas (3-1), (5-1), and (9-1), and can observe at least the central portion of the well well with oblique illumination.

[0077] The observation device 100 configured as above performs observation with the optical axis of the observation optical system 116 aligned with the center of the axial well W1, as shown in FIG.

[0078] FIG. 15 is a diagram showing the distribution of contrast formed in the imaging region IM by the observation device 100. FIG. 16 is a ray diagram for light incident from an axial well. FIG. 17 is a diagram showing the distribution of contrast formed in the imaging region IM by the light shown in FIG. 16. FIG. 18 is a ray diagram for light incident from an adjacent well that has passed through a region relatively far from the emission region in the axial well. FIG. 19 is a diagram showing the distribution of contrast formed in the imaging region IM by the light shown in FIG. 18. FIG. 20 is a ray diagram for light incident from an adjacent well that has passed through a region relatively close to the emission region in the axial well. FIG. 21 is a diagram showing the distribution of contrast formed in the imaging region IM by the light shown in FIG. 20. FIG. 22 is a graph showing the conditions under which oblique illumination is achieved in the observation device 100. 15, 17, 19, and 21, the region R1 indicates the region where oblique illumination is established, and the region R2 indicates the region where bright field illumination is established.

[0079] In the observation device 100, as shown in FIG. 15, oblique illumination is realized over a wide range (region R1) within the imaging region IM, enabling good observation. More specifically, as shown in FIG. 16, with light incident from the axial well W1, bright-field illumination is realized over a wide range (region R2) as shown in FIG. 17, but oblique illumination is not realized. However, as shown in FIG. 18, oblique illumination is realized in region R1 shown in FIG. 19 by light incident from the adjacent well W2 and passing through a region relatively far from the emission region within the axial well W1. Note that, as shown in FIG. 20, bright-field illumination is realized in region R2 shown in FIG. 21 by light incident from the adjacent well W2 and passing through a region relatively close to the emission region within the axial well W1. As a result, oblique illumination is realized over a wide range (region R1) of the imaging region IM as shown in FIG. 15.

[0080] Specifically, H shown by line L5 in FIG. m = Y at 10 mm sFLU From Y sFRB Oblique illumination is established within the range up to the center of the field of view (Y s In order to achieve oblique illumination with the reflector 70 at the angle L6 (=0), the height of the reflector 70 must be set to H mFCLU From H mFCRB You can set it to a height up to

[0081] Fig. 23 is a ray diagram of light incident from an adjacent well and passing through a region relatively distant from the emission region in the axial well when illuminated from the opposite direction to the illumination direction shown in Fig. 18. Fig. 24 is a diagram showing the distribution of contrast formed in the imaging region IM by the light shown in Fig. 23. Fig. 25 is a diagram showing the distribution of contrast obtained by combining the contrast distribution shown in Fig. 19 and the contrast distribution shown in Fig. 24.

[0082] Although an example in which an axial well is illuminated from one direction has been shown, images obtained by sequentially illuminating and capturing images from two or more directions may also be synthesized. For example, as shown in Fig. 23, by illuminating from the opposite direction to that shown in Fig. 18, a region R1 is obtained that is approximately symmetrical about the optical axis to that shown in Fig. 19, as shown in Fig. 24. Taking advantage of this fact, by using illumination from two symmetric directions, oblique illumination can be established over a wider range within the field of view, as shown in Fig. 25, allowing for high-contrast observation.

[0083] FIG. 26 is a diagram showing the positions of multiple imaging regions relative to the well W. FIG. 27 is a diagram showing the illumination direction when imaging imaging region IM1 and the distribution of contrast formed in imaging region IM1. FIG. 28 is a diagram showing the illumination direction when imaging imaging region IM2 and the distribution of contrast formed in imaging region IM2. FIG. 29 is a diagram showing the illumination direction when imaging imaging region IM3 and the distribution of contrast formed in imaging region IM3. FIG. 30 is a diagram showing the illumination direction when imaging imaging region IM4 and the distribution of contrast formed in imaging region IM4. FIG. 31 is a diagram showing the contrast distribution obtained by combining the contrast distributions shown in FIGS. 27 to 30. Of the regions R1 located inside and outside region R2, region R1 on the outside of the well is a region where oblique illumination is achieved by light that passes through the axial well and is reflected by reflector 170.

[0084] Although the above describes an example in which the imaging area IM is aligned with the center of the axial well for observation, it is also possible to image and combine multiple different imaging areas. For example, as shown in FIG. 26, the imaging unit 14 may be moved relative to the axial well W1 to sequentially image four imaging areas (imaging area IM1, imaging area IM2, imaging area IM3, and imaging area IM4). In this case, as shown in FIGS. 27 to 30, it is desirable to illuminate the imaging areas from the direction opposite to the direction in which they are offset from the center of the axial well W1.

[0085] By shifting the imaging area from the center of the on-axis well, the region R1 where oblique illumination is achieved appears not only in the center of the well but also in an area slightly away from the center, as shown in Figures 27 to 30. Therefore, by combining the obtained images, it is possible to observe with high contrast the area near the center of the well and an annular area slightly away from it, as shown in Figure 31, allowing for observation of a wider range within the well.

[0086] [Second embodiment] Fig. 32 is a diagram for explaining the settings in the observation device 200 according to this embodiment. Fig. 33 is a diagram for explaining the specifications of the multiwell plate C2 used in the observation device 200 according to this embodiment. The configuration and settings of the observation device 200 will be explained below with reference to Figs. 32 and 33.

[0087] Observation device 200 is an apparatus for observing samples in multiwell plate C2, and mainly realizes oblique illumination using light incident from an adjacent well (k=1). Observation device 200 has a configuration similar to observation device 10, and as shown in Fig. 32, it includes illumination optical system 215, reflector 270 that reflects light emitted from illumination optical system 215, and observation optical system 216 that collects the light reflected by reflector 270. Illumination optical system 215 includes light source 215a and diffuser 215b.

[0088] The illumination optical system 215 and the observation optical system 216 are installed below the installation position of the multiwell plate C2. In contrast, the reflector 270 is installed above the installation position of the multiwell plate C2. The reflector 270 is a plane mirror and is installed so that on-axis marginal rays incident on the observation optical system 216 pass through adjacent wells before being reflected by the reflector 270.

[0089] In the observation device 200, the specifications of the observation optical system 216, the specifications of the multiwell plate C2, the state of the culture solution in the wells, and the parameters related to the installation conditions of the reflector 270 are as follows, as shown in Figures 32 and 33. The multiwell plate C2 is a 384-well plate. The thickness of the lid of the multiwell plate C2 is 1.5 mm, which is equivalent to 1.0 mm in air. N a =0.12, P=4.5mm, D u =3.8mm, D b = 3.24 mm, n W =1.332, A c2 =0.2306 / mm, A=0.153 / mm, H b =12.6mm, H w=1.8mm, H m =7mm

[0090] At this time, in the observation device 200, N aCORU , Y sORU , H mFCLU , H mFCRB are as follows: N aCORU =0.13, Y sORU =0.066mm, H mFCLU = 4.8 mm, H mFCRB =8.3mm

[0091] Therefore, the observation device 200 satisfies the conditional formulas (3-1), (5-1), and (9-1), and at least the central portion of the well can be observed well with oblique illumination.

[0092] Similarly to the observation device 100, the observation device 200 configured as described above also performs observation with the optical axis of the observation optical system 216 aligned with the center of the axial well W1.

[0093] FIG. 34 is a ray diagram for light incident from an adjacent well. FIG. 35 is a diagram showing the distribution of contrast formed in the imaging region IM by the light shown in FIG. 34. FIG. 36 is a diagram showing the distribution of contrast formed in the imaging region IM when illuminated from the direction opposite to the illumination direction shown in FIG. 34. FIG. 37 is a diagram showing the contrast distribution obtained by combining the contrast distribution shown in FIG. 35 and the contrast distribution shown in FIG. 36. FIG. 38 is a graph showing the conditions under which oblique illumination is achieved in the observation device 200 according to this embodiment. Note that region R1 in FIGS. 35 to 37 indicates the region where oblique illumination is achieved, and region R2 indicates the region where bright-field illumination is achieved.

[0094] In the observation device 200, as shown in Fig. 34, light incident from an adjacent well W2 establishes oblique illumination in a crescent-shaped range (region R1) as shown in Fig. 35. Furthermore, by switching the illumination direction, oblique illumination is established in a crescent-shaped range (region R1) facing in the opposite direction to that in Fig. 35, as shown in Fig. 36. Therefore, by combining images obtained by switching the illumination direction, oblique illumination can be achieved over a relatively wide range (region R1), as shown in Fig. 37, making it possible to observe the inside of wells of a 384-well plate, which was previously almost impossible to observe.

[0095] Specifically, H shown by line L7 in FIG. m = Y at 7mm sFLU From Y sFRB Oblique illumination is established within the range up to the center of the field of view (Y s In order to achieve oblique illumination with the reflector 270 at the center of the reflector 270, the height of the reflector 270 must be set to H mFCLU From H mFCRB It must be set to a height of

[0096] [Third embodiment] 39 is a diagram for explaining the settings in the observation device 300 according to this embodiment. Hereinafter, the configuration and settings of the observation device 300 will be explained with reference to FIG.

[0097] Observation device 300 is an apparatus for observing samples in multiwell plate C3, and achieves oblique illumination mainly using light incident from an adjacent well (k=1) and light incident from the well two wells away (k=2). Observation device 300 has a configuration similar to observation device 10, and as shown in Fig. 39, it includes an illumination optical system, a reflector 370 that reflects light emitted from the illumination optical system, and an observation optical system 316 that collects the light reflected by reflector 370. The illumination optical system includes a light source (not shown) and a diffuser 315b.

[0098] The illumination optical system and observation optical system 316 are installed below the installation position of the multiwell plate C3. In contrast, the reflector 370 is installed above the installation position of the multiwell plate C3. The reflector 370 is a plane mirror and is installed so that the on-axis marginal light beam incident on the observation optical system 316 passes through the peripheral wells (the adjacent well and the well two adjacent thereto) before being reflected by the reflector 370.

[0099] In observation device 300, the specifications of observation optical system 316, specifications of multiwell plate C3, state of culture solution in the wells, and parameters related to installation conditions of reflector 370 are as follows, as shown in Figure 39. Note that multiwell plate C3 is a 384-well plate. The thickness of the lid of multiwell plate C3 is 1.5 mm, which is equivalent to 1.0 mm in air. N a =0.08, P=4.5mm, D u =3.8mm, D b =3.2mm, n W =1.332, A c2 =0.2306 / mm, A=0.153 / mm, H b =11mm, H w =1.8mm, H m =39.2mm

[0100] At this time, in the observation device 300, N aCORU , Y sORU , H mFCLU , H mFCRB are as follows: N aCORU =0.13, Y sORU =0.375mm (When k=2)H mFCLU =39.1mm, H mFCRB =43.2mm

[0101] Therefore, the observation device 300 satisfies the conditional formula (3-1), the conditional formula (5-1), and the conditional formula (9-1) where k=2, and at least the central portion of the well can be observed well with oblique illumination.

[0102] Similarly to the observation device 100, the observation device 300 configured as above also performs observation with the optical axis of the observation optical system 316 aligned with the center of the axial well W1.

[0103] FIG. 40 is a diagram showing the contrast distribution formed in the imaging region IM by the light shown in FIG. 39. FIG. 41 is a diagram showing the contrast distribution formed in the imaging region IM when illuminated from the direction opposite to the illumination direction shown in FIG. 39. FIG. 42 is a diagram showing the contrast distribution obtained by combining the contrast distribution shown in FIG. 40 and the contrast distribution shown in FIG. 41. FIG. 43 is a graph showing the conditions under which oblique illumination is achieved in the observation device 300 according to this embodiment. Note that regions R1, R12, and R13 in FIGS. 40 to 42 indicate regions where oblique illumination is achieved, and region R2 indicates a region where bright-field illumination is achieved.

[0104] In the observation device 300, as shown in Fig. 40, an oblique illumination region (R12) formed by light incident from the adjacent well W2 and an oblique illumination region (R13) formed by light incident from the well W3 next to it are simultaneously generated. Furthermore, by switching the illumination direction, oblique illumination regions (regions R12 and R13) symmetrical to those in Fig. 40 are generated, as shown in Fig. 41. Therefore, by combining images obtained by switching the illumination direction, oblique illumination can be achieved over a relatively wide range (region R1), as shown in Fig. 42, making it possible to observe the inside of wells of a 384-well plate, which was previously almost impossible to observe.

[0105] Specifically, the light from the adjacent well W2 causes the H m = 39.2 mm sFLU From Y sFRB In addition, the light from the well W3 next to the well H is illuminated by the line L10 in FIG. m = 39.2 mm sFLU From Y sFRB Oblique lighting is achieved within the range.

[0106] The observation device using a plane mirror as a reflector has been described above, but hereinafter, an observation device using a curved mirror as a reflector will be described.

[0107] In multi-well plates with 96 or more wells, the well diameter is small, so the refractive power of the liquid surface (concave surface) of the culture medium contained in the well acts relatively strongly. If the reflector is a flat mirror, light reflected by the reflector after passing through the surrounding wells will be incident on the axial well at a relatively large angle. Therefore, when refracted by the negative refractive power of the liquid surface, an increased amount of light will miss the pupil. As explained below, by configuring the reflector as a curved mirror with positive power, such as a concave mirror, the angle of incidence on the liquid surface can be reduced, thereby reducing the amount of light that misses the pupil. This further expands the area in which oblique illumination is possible.

[0108] Figures 44, 46, and 48 are diagrams illustrating the positional relationship between a multiwell plate and the curved surface of a reflector. Figures 45, 47, and 49 are diagrams for explaining the oblique illumination area achieved by an observation device equipped with reflectors positioned in the positional relationships shown in Figures 44, 46, and 48, respectively. Three patterns of observation devices using curved mirrors will be explained below with reference to Figures 44 to 49.

[0109] First, we will explain the common features of the three patterns. The curved mirror used as a reflector has multiple cylindrical surface rows aligned with their concave surfaces facing the multiwell plate, as shown in Figures 44, 46, and 48. Furthermore, the cylindrical surface rows are aligned without any gaps, and the axial direction of the cylindrical surface rows is oriented in the direction (X direction) perpendicular to the alignment direction of the light source (Y direction).

[0110] Next, the features of each pattern will be described. Reflector 470 of the first pattern shown in Figure 44 has the following two features: (1) The center of the cylindrical surface array is located between wells. That is, reflector 470 is installed so that each center of curvature of the multiple cylindrical surface arrays is located on a plane that passes between adjacent wells in the multiwell plate and is parallel to the optical axis of the observation optical system. (2) The pitch of the cylindrical surface array is equal to the well pitch P.

[0111] When the center of the axial well W1 is used as the reference, the center coordinate Y of the cylindrical surface row on the axial well is mc , the coordinates Y of both the left and right ends m- , Y m+ are as follows, respectively: Y mc =P / 2 Y m- =0 Y m+ =P

[0112] The reflector 470 has a shape symmetrical with respect to the center of the axial well. Furthermore, this relationship is the same regardless of which well in the multiwell plate C1 is designated as the axial well W1. Therefore, as shown in FIG. 45, the orientation of the oblique illumination region R1 is reversed depending on the illumination direction. Furthermore, regardless of which well is observed, oblique illumination is established in the same region within the well, allowing for observation with good contrast. Note that FIG. 45 shows an example in which four wells arranged in the Y direction are imaged using two illumination directions, and six imaging regions (imaging regions IM1 to IM6) are imaged within each well.

[0113] Reflector 570 of the second pattern shown in Figure 46 has the following two features: (1) The center of the cylindrical surface array is located between wells. That is, reflector 570 is installed so that each center of curvature of the multiple cylindrical surface arrays is located on a plane that passes between adjacent wells in the multiwell plate and is parallel to the optical axis of the observation optical system. (2) The pitch of the cylindrical surface array is equal to twice the well pitch P. That is, reflector 570 is the same as reflector 470 in terms of (1) but differs in terms of (2).

[0114] When the center of the axial well W1 is used as the reference, the center coordinate Y of the cylindrical surface row on the axial well is mc , the coordinates Y of both the left and right ends m- , Y m+ are as follows: There are two patterns for when the axial well W1 is located to the left or right of the center of the cylindrical surface row. (When there is an axial well to the left of the center of the cylindrical surface row) Y mc =P / 2 Y m- =-P / 2 Y m+ =3P / 2 (When there is an axial well to the right of the center of the cylindrical surface row) Y mc =-P / 2 Y m- =-3P / 2 Y m+ =P / 2

[0115] Unlike the reflector 470, the reflector 570 has an asymmetric shape with respect to the center of the axial well (i.e., the optical axis of the observation optical system). Furthermore, this asymmetric relationship is not the same regardless of which well in the multiwell plate C1 is designated as the axial well W1, but occurs for every other well. Therefore, unlike the reflector 470, if the oblique illumination area simply faces in the opposite direction depending on the illumination direction, as shown in FIG. 47, an oblique illumination area R1 is generated for every other illumination direction. Note that the reflector 570 can obtain a wider area R1 than the reflector 470 in a single image capture. On the other hand, because oblique illumination is only possible in one illumination direction, the reflector 470 can ultimately provide oblique illumination for a wider area when switching illumination directions for image capture.

[0116] Reflector 670 of the third pattern shown in Figure 48 has the following two features: (1) The center of the cylindrical surface array is located at the center of the well. That is, reflector 670 is installed so that each center of curvature of the multiple cylindrical surfaces is located on a plane passing through the centers of the wells contained in the multiwell plate and parallel to the optical axis of the observation optical system. (2) The pitch of the cylindrical surface array is equal to twice the well pitch P. That is, reflector 670 is the same as reflector 570 in terms of (2) but differs in terms of (1).

[0117] When the center of the axial well W1 is used as the reference, the center coordinate Y of the cylindrical surface row on the axial well is mc , the coordinates Y of both the left and right ends m- , Y m+ are as follows: There are two patterns for each case: when the center of the cylindrical surface array is located on the axial well W1, and when the center of the cylindrical surface array is located on the adjacent well. (When the center of the cylindrical surface array is on the axial well W1) Y mc =0 Y m- =-P Y m+ =P (When the center of the cylindrical surface row is on the adjacent well W2) Y mc =P Y m- =0 Y m+ =2P

[0118] Like reflector 470, reflector 670 has a shape symmetrical with respect to the center of the axial well (i.e., the optical axis of the observation optical system). However, not all wells have the same symmetrical shape when observed; every other well has a different symmetrical shape. Therefore, as shown in FIG. 49, like reflector 470, the oblique illumination region R1 simply faces in the opposite direction depending on the illumination direction, but like reflector 570, every other illumination direction produces an oblique illumination region R1. That is, regardless of the illumination direction, wells in which oblique illumination is effective and wells in which it is not effective occur alternately. Therefore, for wells in which oblique illumination is effective, a wide range can be observed with good contrast by switching the illumination direction and observing them.

[0119] FIG. 50 is a diagram for explaining parameters related to the placement of the reflector 470. FIG. 51 is a diagram showing the relationship between parameters and marginal rays passing through the upper right end (ORU) of the axial well. FIG. 52 is a diagram showing the relationship between parameters and marginal rays passing through the right and left ends (OLU) of the axial well. FIG. 53 is a diagram showing the relationship between parameters and marginal rays passing through the right end (M+) of the cylindrical surface array. FIG. 54 is a diagram showing the relationship between parameters and marginal rays passing through the right end (M-) of the cylindrical surface array. FIG. 55 is a graph showing conditions under which a ray is not vignetted by the axial well and falls within the effective range of the reflecting surface of the reflector 470. FIG. 56 is a diagram showing an example of marginal rays passing through the upper left end (LU) and lower right end (RB) of an adjacent well. FIG. 57 is a graph showing the relationship between the height of the reflector 470 and the coordinates at which the marginal rays shown in FIG. 56 pass through the object plane. FIG. 58 is a diagram illustrating a marginal ray passing through the upper left end (LU) of an adjacent well. FIG. 59 is a diagram illustrating a marginal ray passing through the lower right end (RB) of an adjacent well. FIG. 60 is a graph showing the relationship between the height of the reflector 470 and the coordinates at which the marginal ray shown in FIG. 58 passes through the object plane. FIG. 61 is a graph showing the relationship between the height of the reflector 470 and the coordinates at which the marginal ray shown in FIG. 59 passes through the object plane. FIG. 62 is a graph showing the conditions under which oblique illumination is achieved with a reflector 470 having an array of cylindrical surfaces. Hereinafter, with reference to FIGS. 50 to 62, the conditions for achieving oblique illumination in an observation device equipped with a curved mirror will be described using the reflector 470 as an example, particularly a method for deriving the height of the reflector 470 and the radius of curvature of the reflecting surface of the reflector 470.

[0120] As shown in FIG. 50, the coordinates of the marginal ray incident on the axial well on the reflecting surface of the reflector 470 are expressed as coordinates Y m and the radius of curvature of the reflecting surface is denoted by the radius of curvature R. The other parameters shown in FIG. 50 are as described above. Also, as shown in FIG. 51, the coordinates on the object plane of the marginal ray passing through the upper right end (ORU) of the axial well are denoted by the coordinate Y sORUAs shown in Figure 52, the coordinates on the object plane of the marginal ray passing through the upper left end (LRU) of the axial well are denoted by the coordinate Y sOLU Furthermore, as shown in FIG. 53, the object plane Y s The marginal ray passing through the right end of the cylindrical surface array (coordinate Y m+ ) the height of the reflector 470 when reflected at height H mYm+ and, as shown in Figure 54, the object plane Y s The marginal ray passing through the left end of the cylindrical surface array (coordinate Y m- ) the height of the reflector 470 when reflected at height H mYm- The height of a reflector with a curved reflective surface is defined as the distance from the top edge of the multiwell plate to the top of the reflective surface.

[0121] First, let us consider the constraints imposed by the ORU with reference to Fig. 51. The constraints imposed by the ORU are the same as those described above for the reflector made of a flat mirror. That is, the conditional expressions (3) and (5) must be satisfied.

[0122] In the case of a curved mirror such as reflector 470, it is possible that a marginal ray may be incident from the direction opposite to the illumination direction. For this reason, we will also consider the constraints associated with the OLU as shown in FIG.

[0123] Numerical aperture N of the observation optical system a Using the above, the coordinate (Y coordinate) of the marginal ray passing through the OLU on the object plane is sORU is expressed by the following formula:

number

[0124] The marginal ray has coordinate Y on the object surface. sOLU If you try to pass through the negative side of the coordinate Y, it will be blocked by OLU. sОRU Equation (12) defining the equation (12) indicates the minimum coordinate in the Y direction that the marginal ray can pass through on the object surface.

[0125] Therefore, in order to avoid being blocked by ORU and OLU, it is desirable that the following conditional expression be satisfied.

number

[0126] However, the numerical aperture N of the observation optical system a is the threshold N aOLU If it exceeds the Y coordinate sOLU reaches the left end of the well. In other words, the above equation is a <N aOLU This is an equation that holds when N a >N aOLU When , it is desirable that the following formula be satisfied:

number

[0127] In addition, the threshold N aOLU is expressed by the following formula:

number

[0128] Next, we will consider the constraints imposed by the effective range of the reflecting surface with reference to Figures 53 and 54. s and the coordinate Y of the reflecting surface m The height H of the reflecting surface when passing through mYm is expressed by the following formula:

number

[0129] To guide the marginal ray into the well, the coordinates of the marginal ray on the reflecting surface must be within the effective range (Y m- From Y m+ ) but the object plane coordinate Y s Whether or not the marginal ray passing through falls within the effective range of the reflecting surface depends on the height of the reflector 470.

[0130] As can be seen from equation (16), the height H mYm is Y s is a hyperbola with variables, and the coordinate of its asymptote is Y sASY is expressed by the following formula:

number

[0131] Y coordinate of the object surface s is the Y coordinate sASY greater than (Y s >Y sASY ) when the coordinate Y of the reflecting surface m Y m+ (Y m =Y m+ ) when the height of the reflecting surface is H mYm+ becomes positive, and the height of the reflecting surface H mYm- On the other hand, the coordinate Y of the object plane s is the Y coordinate sASY is smaller than (Y s <Y sASY ) when the coordinate Y of the reflecting surface m Y m+ (Y m =Y m+ ) when the height of the reflecting surface is H mYm+ becomes negative, and the height of the reflecting surface H mYm- will be positive.

[0132] Therefore, Y s >Y sASY In this case, in order to keep the marginal ray within the effective range of the reflecting surface, the height of the reflecting surface H m is the height H mYm+ It is desirable that the value of the saturation voltage be smaller than 1 / 2 and that the value of the saturation voltage be larger than 1 / 2.

number

[0133] Y s <Y sASY In this case, in order to keep the marginal ray within the effective range of the reflecting surface, the height of the reflecting surface H mis the height H mYm- It is desirable that the value of the saturation voltage be smaller than 1 / 2 and that the value of the saturation voltage be larger than 1 / 2.

number

[0134] In addition, the height H mYm+ , height H mYm- are the height H mYm The coordinate Y in the definition of s coordinate Y s+ , coordinate Y s+ is substituted, as follows:

number

[0135] In other words, to prevent vignetting by the axial well W1 and to make the marginal ray incident within the effective range of the reflecting surface, the Y S The coordinate Y shown on the line L11 in Figure 55 sORU and the coordinate Y shown on line L12 sOLU While fitting the reflector 470 between the coordinates Y S It is necessary to set the height between the height indicated by the line L13 and the height indicated by the line L14.

[0136] Next, the constraints associated with adjacent wells will be considered. With reference to Figures 56 and 57, a marginal ray passing through the upper left end (LU) and lower right end (RB) of the adjacent well W2 will be considered.

[0137] As shown in FIG. 56, a marginal ray passing through both the upper left end (LU) and the lower right end (RB) of the adjacent well W2 has a coordinate Y s The height H of the reflector 470 when passing through mLU-RB is expressed by the following formula:

number

[0138] Reflector 470 is this height HmLU-RB If the height of the reflector 470 is lower than , it cannot pass through the adjacent well. m It is desirable that the height H mLU-RB It is desirable to utilize the area above the line L15 in FIG.

number

[0139] Referring to Figures 58 to 61, consider a marginal ray passing through the upper left edge (LU) or lower right edge (RB) of adjacent well W2.

[0140] As shown in FIG. 58, a marginal ray passing through the upper left corner (LU) of the adjacent well W2 has a coordinate Y s The radius of curvature R of the reflecting surface of reflector 470 when passing through LU is the height H of the reflector 470 m Then, it is expressed by the following formula:

number

[0141] radius of curvature R LU is the height H m is a hyperbola with a variable, and its asymptote height H mLU is expressed by the following formula:

number

[0142] In order for the marginal ray to pass to the right of the upper left end (LU) of the adjacent well W2 without being vignetted by LU, the height H of the reflector 470 must be m is the height H mLU lower than (H m <H mLU ) Sometimes, the radius of curvature needs to be small, and it is desirable to satisfy the following formula:

number

[0143] Also, the height H of the reflector 470 m is the height H mLU Higher than (H m >H mLU ) Sometimes, the radius of curvature needs to be large, and it is desirable to satisfy the following formula:

number

[0144] In other words, to avoid vignetting at the upper left end (LU) of the adjacent well W2, the height H mLU In the region above line L16 in FIG. 60, it is desirable that the reflecting surface satisfy conditional formula (25), and in the region below line L16, it is desirable that the reflecting surface satisfy conditional formula (24).

[0145] As shown in Figure 59, a marginal ray passing through the lower right edge (RB) of the adjacent well W2 has a coordinate Y s The radius of curvature R of the reflecting surface of reflector 470 when passing through RB is the height H of the reflector 470 m Then, it is expressed by the following formula:

number

[0146] radius of curvature R RB is the height H m is a hyperbola with a variable, and its asymptote height H mRB is expressed by the following formula:

number

[0147] In order for the marginal ray to pass to the left of the lower right edge (RB) of the adjacent well W2 without being vignetted by RB, the height H of the reflector 470 must be m is the height H mRB lower than (H m <HmRB ) Sometimes, the radius of curvature needs to be large, and it is desirable to satisfy the following formula:

number

[0148] Also, the height H of the reflector 470 m is the height H mRB Higher than (H m >H mRB ) Sometimes, the radius of curvature needs to be small, and it is desirable to satisfy the following formula:

number

[0149] In other words, to avoid vignetting at the bottom right edge (RB) of the adjacent well W2, the height H mRB In the region above line L17 in FIG. 61, it is desirable that the reflecting surface satisfy conditional formula (29), and in the region below line L17, it is desirable that the reflecting surface satisfy conditional formula (28).

[0150] Considering the constraints associated with the above-mentioned on-axis well W1, the constraints associated with the effective range of the reflecting surface, and the constraints associated with the adjacent well W2, the height of the reflector 470 and the radius of curvature of the reflecting surface may be determined by the following procedure.

[0151] First, the coordinate Y of the observation position s The coordinate Y indicated by the line L11 in Figure 62 sORU and the coordinate Y indicated by line L12 sOLU Next, the height H of the reflector 470 is determined. m The height H indicated by the line L14 mYm- and the height H indicated by line L13 mYm+ Between the height H indicated by the line L15 mLU-RB Set it to a height higher than

[0152] Finally, the radius of curvature R is set according to the set height. Specifically, the set height is the height H indicated by the line L16. mLUIf the radius of curvature R is lower than the radius of curvature R LU The radius of curvature R is smaller than RB is larger than R RB <R<R LU The height is set to be equal to the height H indicated by the line L16. mLU The height H indicated by the line L17 is higher than the mRB If the radius of curvature R is lower than the radius of curvature R LU Radius of curvature R is larger than RB is greater than R, i.e., R>R RB And R>R LU Furthermore, the set height is set to the height H indicated by the line L17. mRB If the curvature radius R is higher than the curvature radius R LU Radius of curvature R is larger than RB is smaller than R LU <R<R RB Set it so that:

[0153] This allows the axis well to have coordinate Y s Since oblique illumination is established, the observation position can be observed well with high contrast.

[0154] The various parameters when using a standard 96-well plate or 384-well plate are expressed as follows. The units of height, coordinates, and radius of curvature are mm and N. a The units of are dimensionless quantities. These units will be applied in the future unless otherwise specified. (For 96-well plates)

number

[0155] (For 384-well plates)

number

[0156] Each of the three patterns will be described in more detail below. First, in order to achieve oblique illumination using reflector 470 of the first pattern, all of the following must be satisfied.

number

[0157] The height and coordinate parameters are k=1, Y m- =0, Y m+ =P, the result is as follows:

number

[0158] The height of the reflector 470 is H m Height H at mSym- The Y coordinate of sSymHm is as follows:

number

[0159] From the above, when applied to a standard 96-well plate, the following is derived: When using a standard 96-well plate, the coordinate Y s , height H m It is desirable that the following conditions be met:

number

[0160] Also, at this time, the height H mSym- The Y coordinate of sSymHm becomes as follows:

number

[0161] Similarly, applying to a standard 384-well plate, the following is derived: If a standard 96-well plate is used, the coordinate Y s , height Hm It is desirable that the following conditions be met:

number

[0162] Also, at this time, the height H mSym- Coordinate Y sSymHm becomes as follows:

number

[0163] Next, the conditions for observing the center of the well with at least oblique illumination in the first pattern will be described. To observe the center of the well with at least oblique illumination, all of the following must be met.

number

[0164] Furthermore, the height H of the reflector 470 m Depending on the situation, the following must also be met:

number

[0165] The parameters of the numerical aperture, height, and radius of curvature are Y s = 0 and is derived as follows:

number

[0166] In the first pattern, it is desirable to further satisfy the following condition: This makes it possible to widen the polarized illumination range to the negative side.

number

[0167] The parameters of the numerical aperture, height, and radius of curvature are Ys Y s The minimum possible value of Y sSym± This is derived by substituting:

number

[0168] In addition, the coordinate Y sSym± is the height H mSym+C H mSym- The coordinates are as follows:

number

[0169] In the first pattern, it is desirable to further satisfy the following condition: sORU It is possible to expand it to.

number

[0170] where H mSymORU is as follows:

number

[0171] When the above conditions described for the first pattern are applied to a 96-well plate, the results are as follows.

[0172] The conditions for observing at least the center of the well with oblique illumination are as follows.

number

[0173] The conditions for extending the oblique illumination range to the negative side are as follows:

number

[0174] The conditions for extending the oblique illumination range to the positive side are as follows.

number

[0175] When the above conditions described for the first pattern are applied to a 384-well plate, the results are as follows.

[0176] The conditions for observing at least the center of the well with oblique illumination are as follows.

number

[0177] The conditions for extending the oblique illumination range to the negative side are as follows:

number

[0178] The conditions for extending the oblique illumination range to the positive side are as follows.

number

[0179] [Fourth embodiment] Fig. 63 is a diagram for explaining the settings in the observation device 400 according to this embodiment. Fig. 64 is a diagram for explaining the positional relationship between the multiwell plate C1 and the reflector 471 used in the observation device 400 according to this embodiment. The configuration and settings of the observation device 400 will be explained below with reference to Figs. 63 and 64.

[0180] The observation device 400 is an apparatus for observing samples in a multiwell plate C1, and mainly realizes oblique illumination using light incident from an adjacent well (k=1). The observation device 400 has a configuration similar to that of the observation device 10, and as shown in Fig. 63, includes an illumination optical system 415, a reflector 471 that reflects light emitted from the illumination optical system 415, and an observation optical system 416 that collects the light reflected by the reflector 471. The illumination optical system 415 includes a light source 415a and a diffuser 415b.

[0181] The illumination optical system 415 and the observation optical system 416 are disposed below the installation position of the multiwell plate C1. In contrast, the reflector 471 is disposed above the installation position of the multiwell plate C1. The reflector 471 is a curved mirror, particularly a curved mirror having the first pattern described above, and is disposed so that on-axis marginal rays incident on the observation optical system 416 pass through adjacent wells before being reflected by the reflector 471. More specifically, as shown in FIG. 64, the edge E of the concave reflecting surface is disposed so as to coincide with the center of the well.

[0182] In the observation device 400, the specifications of the observation optical system 416, the specifications of the multiwell plate C1, the state of the culture solution in the wells, and the parameters related to the installation conditions of the reflector 471 are as follows, as shown in Figures 63 and 64. The multiwell plate C1 is a 96-well plate. The thickness of the lid of the multiwell plate C1 is 1.5 mm, which is equivalent to 1.0 mm in air. N a =0.25, P=9mm, D u = 6.9 mm, D b = 6.4 mm, n W =1.332, A c2 =0.11497 / mm, A=0.0763 / mm, H b =10.7mm, H w =1.8mm, H m =13mm, R=25mm

[0183] At this time, in the observation device 400, the parameter values ​​related to various conditions are as follows: N aCORU =0.31, H mCLU-RB =3.1mm, H mSym+C =22.5mm, H mLUC = 5mm, H mRBC =11.5mm, R LUC =11.1mm, R RBC =101.7mm, H mLU-BRSym± =10.3mm, R RBSymm± =22.1mm, R LUSymm± =25.5mm, H mSymORU =18.2mm, Y sORU =0.39mm, Y sOSym± =-2.6mm, Y sSymHm =-2.4mm,

[0184] Therefore, the observation device 400 satisfies the conditions for obliquely illuminating at least the central portion of the well, for extending the oblique illumination range to the negative side, and for extending the oblique illumination range to the positive side, and can observe the sample in the well with high contrast.

[0185] In the observation device 400 configured as above, as shown in FIG. 65, the axial well W1 is obliquely illuminated with light incident from the adjacent well W2, and images of the four imaging regions shown in FIG. 26 are captured.

[0186] FIG. 65 is a ray diagram for light incident from an adjacent well. FIG. 66 is a diagram showing the illumination direction when imaging area IM1 and the distribution of contrast formed in imaging area IM1. FIG. 67 is a diagram showing the illumination direction when imaging area IM2 and the distribution of contrast formed in imaging area IM2. FIG. 68 is a diagram showing the illumination direction when imaging area IM3 and the distribution of contrast formed in imaging area IM3. FIG. 69 is a diagram showing the illumination direction when imaging area IM4 and the distribution of contrast formed in imaging area IM4. FIG. 70 is a diagram showing the contrast distribution obtained by combining the contrast distributions shown in FIGS. 66 to 69. Note that area R1 in FIGS. 66 to 70 indicates the area where oblique illumination is established.

[0187] In the observation device 400, by illuminating with light incident from an adjacent well W2 as shown in Fig. 65 while moving the imaging region, different regions can be obliquely illuminated in each imaging region as shown in Fig. 66 to Fig. 69. Therefore, by combining images obtained while changing the illumination direction and imaging region, it is possible to observe a wide range (region R1) within well W1 of a 96-well plate with oblique illumination, as shown in Fig. 70.

[0188] [Fifth embodiment] Fig. 71 is a diagram for explaining the settings in the observation device 500 according to this embodiment. Fig. 72 is a diagram for explaining the specifications of the multiwell plate C3. The configuration and settings of the observation device 500 will be explained below with reference to Figs. 71 and 72.

[0189] Observation device 500 is an apparatus for observing samples in multiwell plate C3, and achieves oblique illumination mainly using light incident from adjacent wells (k=1). Observation device 500 has a configuration similar to observation device 10, and as shown in Fig. 71, includes an illumination optical system 515, a reflector 472 that reflects light emitted from illumination optical system 515, and an observation optical system 516 that collects the light reflected by reflector 472. Illumination optical system 515 includes a light source 515a and a diffuser 515b.

[0190] The illumination optical system 515 and the observation optical system 516 are disposed below the installation position of the multiwell plate C3. In contrast, the reflector 472 is disposed above the installation position of the multiwell plate C3. The reflector 472 is a curved mirror, particularly a curved mirror having the first pattern described above, and is disposed so that on-axis marginal rays incident on the observation optical system 516 pass through adjacent wells before being reflected by the reflector 472.

[0191] In observation device 500, the specifications of observation optical system 516, specifications of multiwell plate C3, state of culture solution in the wells, and parameters related to installation conditions of reflector 472 are as follows, as shown in Figures 71 and 72. Multiwell plate C3 is a 384-well plate. The thickness of the lid of multiwell plate C3 is 1.5 mm, which is equivalent to 1.0 mm in air. N a =0.12, P=4.5mm, D u =3.8mm, D b =3.2mm, n W =1.332, A c2 =0.2306 / mm, A=0.1530 / mm, H b =11mm, H w =1.8mm, H m =15mm, R=25mm

[0192] At this time, in the observation device 500, the parameter values ​​related to various conditions are as follows: N aCORU =0.15, H mCLU-RB =2.4mm, H mSym+C =20.8mm, H mLUC =3.8mm, H mRBC =9.8mm, R LUC =13.1mm, R RBC = 47.6 mm, H mLU-BRSym± =9.1mm, R RBSymm± =22.2mm, R LUSymm± =29.7mm, H mSymORU =15.2mm, Y sORU =0.18mm, Y sOSym± =-0.81mm, Y sSymHm =-0.78mm,

[0193] Therefore, the observation device 500 satisfies the conditions for obliquely illuminating at least the central portion of the well, for extending the oblique illumination range to the negative side, and for extending the oblique illumination range to the positive side, and can observe the sample in the well well with high contrast.

[0194] In the observation device 500 configured as above, images of the two imaging regions are captured while the imaging regions are slightly shifted in the direction opposite to the illumination direction.

[0195] FIG. 73 is a ray diagram for light incident from an adjacent well. FIG. 74 is a diagram showing the distribution of contrast formed in imaging region IM5 by the light shown in FIG. 73. FIG. 75 is a ray diagram for light incident from an adjacent well in the direction opposite to the illumination direction shown in FIG. 73. FIG. 76 is a diagram showing the distribution of contrast formed in imaging region IM6 by the light shown in FIG. 75. FIG. 77 is a diagram showing the contrast distribution obtained by combining the contrast distribution shown in FIG. 74 and the contrast distribution shown in FIG. 76. Note that region R1 in FIGS. 74, 76, and 77 indicates the region where oblique illumination is established.

[0196] In the observation device 500, the imaging region is illuminated with light incident from the adjacent well W2 while switching the illumination direction and moving the imaging region. As a result, when illuminating from the direction shown in Fig. 73, the imaging region IM5 shown in Fig. 74 is imaged, and when illuminating from the direction shown in Fig. 75, the imaging region IM6 shown in Fig. 76 is imaged. By combining the images obtained from the two different imaging regions in this way, it is possible to observe a wide range (region R1) in the center of well W1 of a 384-well plate with oblique illumination, as shown in Fig. 77.

[0197] Next, the second pattern will be described in detail. In order to achieve oblique illumination using reflector 570 of the second pattern, all of the following must be satisfied.

number

[0198] The height and coordinate parameters are as follows. The coordinate parameters are the same as the first pattern. The height parameters are the height H mLU-RB k=1 to H mYm- and H mYm+ Effective range Y m-=-P / 2, Y m+ =3P / 2.

number

[0199] When the above conditions described for the second pattern are applied to a 96-well plate, it is desirable that the height of the reflector 570 satisfy the following conditions.

number

[0200] In this case, the various coordinates are as follows:

number

[0201] When the above conditions described for the second pattern are applied to a 384-well plate, it is desirable that the height of the reflector 570 satisfy the following conditions.

number

[0202] In this case, the various coordinates are as follows:

number

[0203] In the second pattern, the conditions for observing at least the well center with oblique illumination are as follows.

number

[0204] In addition, the height H mWideC is the height H mWide Y s is substituted with 0, as follows:

number

[0205] The conditions for observing the center of a well in a 96-well plate with oblique illumination in the second pattern are as follows.

number

[0206] In addition, the conditions for observing the center of a well in a 384-well plate with oblique illumination in the second pattern are as follows.

number

[0207] Furthermore, in the second pattern, the conditions for extending the oblique illumination range to the negative side are as follows.

number

[0208] However, the above conditions are applicable if the following conditions are met:

number

[0209] The definitions of each parameter are as follows: s =-D b This is calculated by substituting / 2.

number

[0210] In the second pattern, the coordinates of the edges of the wells are -D as described above. b It is desirable to extend the range of oblique illumination to the positive limit Y / 2 and further satisfy the following condition.sORU It is possible to expand it to.

number

[0211] In addition, H mWideORU is as follows:

number

[0212] When the above-mentioned condition of expanding the polarized illumination range to the positive and negative sides is applied to a 96-well plate, a If >0.22, the height and radius of the reflector 570 must satisfy the following conditions to achieve -D b It can provide oblique lighting up to 1 / 2.

[0213]

number

[0214] Furthermore, if the height of the reflector 570 satisfies the following conditions, Y sORU Oblique illumination can be achieved up to

number

[0215] [Sixth embodiment] Fig. 78 is a diagram for explaining the settings in the observation device 600 according to this embodiment. Fig. 79 is a diagram for explaining the positional relationship between the multiwell plate C1 and the reflector 571. The configuration and settings of the observation device 600 will be explained below with reference to Figs. 78 and 79.

[0216] Observation device 600 is an apparatus for observing samples in multiwell plate C1, and mainly realizes oblique illumination using light incident from an adjacent well (k=1). Observation device 600 has a configuration similar to observation device 10, and as shown in Fig. 78, includes an illumination optical system 615, a reflector 571 that reflects light emitted from illumination optical system 615, and an observation optical system 616 that collects the light reflected by reflector 571. Illumination optical system 615 includes a light source 615a and a diffuser 615b.

[0217] The illumination optical system 615 and the observation optical system 616 are disposed below the installation position of the multiwell plate C1. In contrast, the reflector 571 is disposed above the installation position of the multiwell plate C1. The reflector 571 is a curved mirror, particularly a curved mirror having the second pattern described above, and is disposed so that on-axis marginal rays incident on the observation optical system 616 pass through an adjacent well before being reflected by the reflector 571. More specifically, as shown in FIG. 79, the reflector is disposed so that the edge E of the concave reflecting surface is located between two adjacent wells.

[0218] In the observation device 600, the specifications of the observation optical system 616, the specifications of the multiwell plate C1, the state of the culture solution in the wells, and the parameters related to the installation conditions of the reflector 571 are as follows, as shown in Figures 78 and 79. The multiwell plate C1 is a 96-well plate. The thickness of the lid of the multiwell plate C1 is 1.5 mm, which is equivalent to 1.0 mm in air. N a =0.25, P=9mm, D u = 6.9 mm, D b = 6.4 mm, n W =1.332, A c2 =0.11497 / mm, A=0.0763 / mm, H b =10.7mm, H w =1.8mm, H m =15mm, R=27mm

[0219] At this time, in the observation device 600, the parameter values ​​related to various conditions are as follows: N aCORU =0.31, H mCLU-RB =3.1mm, H mWideC =38.3mm, H mLUC = 5mm, H mRBC =11.5mm, R LUC =13.2mm, R RBC =62.4mm, H mELU-BR =12.3mm, R LUE =27.6mm, R RBE =25.1mm, Y sORU =0.39mm, Y sWide± =-3.9mm, Y sWideORU =-3.9mm

[0220] Therefore, the observation device 600 satisfies the conditions for obliquely illuminating at least the central portion of the well, for extending the oblique illumination range to the negative side, and for extending the oblique illumination range to the positive side, and can observe the sample in the well well with high contrast.

[0221] Fig. 80 is a diagram showing the contrast distribution obtained by the observation device 600. In the observation device 600 configured as described above, the axial well W1 is obliquely illuminated with light incident from the adjacent well W2, and six imaging regions are captured, making it possible to observe a wide range (region R1) within the well with oblique illumination, as shown in Fig. 80.

[0222] Figure 81 is a ray diagram for light incident from an adjacent well. Figure 82 is a diagram showing the distribution of contrast formed in imaging region IM10 by the light shown in Figure 81. Figure 83 is a ray diagram when the imaging region is slightly shifted along the illumination direction from the state shown in Figure 81. Figure 84 is a diagram showing the distribution of contrast formed in imaging region IM11 by the light shown in Figure 83. Note that region R1 in Figures 82 to 84 indicates the region where oblique illumination is established.

[0223] In the observation device 600, as shown in Figures 81 to 84, when observing an axial well, the imaging area is moved to imaging area IM10 and imaging area IM11 and images are taken twice from the same illumination direction, thereby achieving oblique illumination over a wide range as shown in Figure 80.

[0224] Fig. 85 is a diagram for explaining the imaging order in the observation device 600. Fig. 86 is a diagram for explaining the contrast obtained in wells in even-numbered rows and odd-numbered rows in the observation device 600. In the observation device 600, the contrast obtained in wells in even-numbered rows is different from that obtained in wells in odd-numbered rows.

[0225] Specifically, as shown in Figures 85(a) and 85(b), when observing axial wells in even-numbered rows, oblique illumination is achieved with illumination from the right, but not with illumination from the left. Conversely, as shown in Figures 85(c) and 85(d), when observing axial wells in odd-numbered rows, oblique illumination is achieved with illumination from the left, but not with illumination from the right. For this reason, there is no need to switch the illumination direction for imaging the same well; by illuminating the even and odd rows from predetermined directions, a wide range for each well can be observed with oblique illumination, as shown in Figure 86.

[0226] [Seventh embodiment] Fig. 87 is a diagram for explaining the configuration of an observation device 700 according to this embodiment. The reflecting surface of a reflector 572 of the observation device 700 is divided into six parts per well in the axial direction (X direction) of the cylindrical surface row. Each of the six divided reflecting surfaces has a width of 1.5 mm and includes three reflecting surfaces 572a and three reflecting surfaces 572b, the centers of curvature of which are positioned one well apart from each other. As shown in Fig. 87, the reflecting surfaces 572a and the reflecting surfaces 572b are arranged alternately in the axial direction.

[0227] The specifications of the observation optical system in the observation device 700, the specifications of the multiwell plate C1, the state of the culture solution in the wells, and other parameters related to the installation conditions of the reflector 572 are the same as those in the sixth embodiment.

[0228] FIG. 88 is a ray diagram for light incident from an adjacent well. FIG. 89 is a diagram showing the distribution of contrast formed in imaging region IM10 by the light shown in FIG. 88. FIG. 90 is a ray diagram when the imaging region is slightly shifted along the illumination direction from the state shown in FIG. 88. FIG. 91 is a diagram showing the distribution of contrast formed in imaging region IM11 by the light shown in FIG. 90. FIG. 92 is a ray diagram for light incident from an adjacent well in the opposite direction to FIG. 88. FIG. 93 is a diagram showing the distribution of contrast formed in imaging region IM10 by the light shown in FIG. 92. FIG. 94 is a ray diagram when the imaging region is slightly shifted along the illumination direction from the state shown in FIG. 93. FIG. 95 is a diagram showing the distribution of contrast formed in imaging region IM12 by the light shown in FIG. 94. FIG. 96 is a diagram for explaining the function of the two reflecting surfaces of reflector 572. FIG. 97 is a diagram showing the distribution of contrast obtained by observation device 700. It should be noted that the region R1 in FIGS. 89, 91, 93, 95 and 97 indicates the region where oblique illumination is established.

[0229] In the observation device 700, as shown in Figures 88 to 91, imaging is performed twice by moving the imaging area to imaging area IM10 and imaging area IM11 while illuminating from the right side. Furthermore, as shown in Figures 92 to 95, the illumination direction is switched and the same well is illuminated from the left side, and imaging is performed twice by moving the imaging area to imaging area IM10 and imaging area IM12.

[0230] As shown in Fig. 97, the light reflected by reflector 572 can be spread in the X direction by the two staggered reflective surfaces (reflective surface 572a, reflective surface 572b) and illuminate the well. Therefore, by capturing images a total of four times using illumination from the left and right, almost the entire inside of the well can be observed with oblique illumination, as shown in Fig. 97.

[0231] In the sixth and seventh embodiments, an example in which the formula (151) is satisfied is shown, but in the second pattern, the following conditional formula may also be satisfied.

number

[0232] In this case, the oblique lighting range is set to the negative side by the coordinate Y sWide± The conditions for extending to are as follows:

number

[0233] The definitions of each parameter are as follows: s to height H mWideC H in mWide- The Y coordinate of sWide± Here, the coordinate Y sWide± is the height H mWideC H in mWide- are the coordinates of

number

[0234] In addition, the coordinate Y sWide± is as follows:

number

[0235] In the second pattern, the coordinate Y sWide± In addition to expanding the range of oblique illumination to the positive limit Y sORU It is possible to expand it to.

number

[0236] In addition, H mWideORU and H mWOLU-RB is as follows:

number

[0237] For the widest oblique illumination range, set the height of the Reflector 570 to H mLU-RB and H mWide- The height H of the intersection mWideBest Let (H m =H mWideBest ) and the radius of curvature R is R WideBest In this case, the coordinate Y where oblique illumination is established is s The minimum value of Y sWideBest is.

number

[0238] When the above-mentioned condition of expanding the polarized illumination range to the positive and negative sides is applied to a 96-well plate, a If <0.22, the height and radius of the reflector 570 must satisfy the following conditions, and for the negative side, -D b / 2 but Y sWide± Oblique illumination can be achieved up to

number

[0239] In addition, Y at this time sWide± is as follows:

number

[0240] Furthermore, if the height of the reflector 570 satisfies the following conditions, Y sORU Oblique illumination can be achieved up to

number

[0241] Furthermore, the oblique illumination range is maximized when the height and curvature radius of the reflector 570 satisfy the following conditions:

number

[0242] When the above-mentioned condition of expanding the polarized illumination range to the positive and negative sides is applied to a 384-well plate, N a If <0.2, the height and radius of the reflector 570 must satisfy the following conditions, and for the negative side, -D b / 2 but Y sWide± Oblique illumination can be achieved up to

number

[0243] In addition, Y at this time sWide± is as follows:

number

[0244] Furthermore, if the height of the reflector 570 satisfies the following conditions, Y sORU Oblique illumination can be achieved up to

number

[0245] Furthermore, the oblique illumination range is maximized when the height and curvature radius of the reflector 570 satisfy the following conditions:

number

[0246] [Eighth embodiment] 98 is a diagram for explaining the settings in the observation device 800 according to this embodiment. Hereinafter, the configuration and settings of the observation device 800 will be explained with reference to FIG.

[0247] Observation device 800 is an apparatus for observing samples in multiwell plate C1, and mainly realizes oblique illumination using light incident from an adjacent well (k=1). Observation device 800 has a configuration similar to observation device 10, and as shown in Fig. 98, it includes an illumination optical system, a reflector 573 that reflects light emitted from the illumination optical system, and an observation optical system 816 that collects the light reflected by reflector 573. The illumination optical system includes a light source and a diffuser 815b.

[0248] The illumination optical system and observation optical system 816 are provided below the installation position of the multiwell plate C1. In contrast, the reflector 573 is provided above the installation position of the multiwell plate C1. The reflector 573 is a curved mirror, particularly a curved mirror having the second pattern described above, and is provided so that on-axis marginal rays incident on the observation optical system 816 pass through adjacent wells before being reflected by the reflector 573.

[0249] The reflecting surface of reflector 573 of observation device 800 is divided into six parts per well in the axial direction (X direction) of the cylindrical surface row, similar to the reflecting surface of reflector 572 of observation device 700. Each of the six divided reflecting surfaces has a width of 1.5 mm and includes three reflecting surfaces 573a and three reflecting surfaces 573b, the centers of curvature of which are positioned one well apart from each other. Reflecting surfaces 573a and 573b are arranged alternately in the axial direction.

[0250] In observation device 800, the specifications of observation optical system 816, specifications of multiwell plate C1, state of culture solution in the wells, and parameters related to installation conditions of reflector 573 are as follows, as shown in Figure 98. Note that multiwell plate C1 is a 96-well plate. The thickness of the lid of multiwell plate C1 is 1.5 mm, which is equivalent to 1.0 mm in air. N a =0.15, P=9mm, D u = 6.9 mm, D b = 6.4 mm, n W =1.332, Ac2 =0.11497 / mm, A=0.0763 / mm, H b =10.7mm, H w =1.8mm, H m =16mm, R=25.9mm

[0251] At this time, in the observation device 800, the parameter values ​​related to various conditions are as follows: N aCORU =0.31, H mCLU-RB = 4.9 mm, H mWideC =71.5mm, H mLUC =11.7mm, H mRBC =25.8mm, R LUC =-3.99mm, R RBC =2.84mm, H mLU-BR―WideC =13.5mm, R LUWideC =26.7mm, R RB =25.1mm, H mWideORU = 40.8 mm, H mWORU-RB =14.3mm, H mWideBest =16mm, R WideBest =25.9mm, Y sORU =1.1mm, Y sWide± =-2.5mm, Y sWideORU =-2.7mm, Y sWideBest =-3mm

[0252] Therefore, the observation device 800 satisfies the conditions for obliquely illuminating at least the central portion of the well, for extending the oblique illumination range to the negative side, and for extending the oblique illumination range to the positive side, and is therefore capable of observing the sample in the well with high contrast.

[0253] FIG. 99 is a ray diagram for light incident from an adjacent well. FIG. 100 is a diagram showing the distribution of contrast formed in imaging region IM10 by the light shown in FIG. 99. FIG. 101 is a ray diagram when the imaging region is slightly shifted along the illumination direction from the state shown in FIG. 99. FIG. 102 is a diagram showing the distribution of contrast formed in imaging region IM11 by the light shown in FIG. 101. FIG. 103 is a ray diagram for light incident from an adjacent well in the opposite direction to FIG. 99. FIG. 104 is a diagram showing the distribution of contrast formed in imaging region IM10 by the light shown in FIG. 103. FIG. 105 is a ray diagram when the imaging region is slightly shifted along the illumination direction from the state shown in FIG. 103. FIG. 106 is a diagram showing the distribution of contrast formed in imaging region IM12 by the light shown in FIG. 105. Note that region R1 in FIGS. 100, 102, 104, and 106 indicates the region where oblique illumination is established.

[0254] In the observation device 800, as shown in Figures 99 to 102, imaging is performed twice by moving the imaging area to imaging area IM10 and imaging area IM11 while illuminating from the right side. Furthermore, as shown in Figures 103 to 106, the illumination direction is switched and the same well is illuminated from the left side, and imaging is performed twice by moving the imaging area to imaging area IM10 and imaging area IM12.

[0255] As a result, similar to the seventh embodiment, the light reflected by reflector 573 is spread in the X direction by the two staggered reflecting surfaces to illuminate the well, making it possible to observe almost the entire area inside the well with oblique illumination. Furthermore, compared to the seventh embodiment, the oblique illumination area can be widened by Δ in the illumination direction when the central part of the well is illuminated.

[0256] [Ninth embodiment] Fig. 107 is a diagram for explaining the settings in the observation device 900 according to this embodiment. Fig. 108 is a diagram for explaining adjacent wells through which illumination light incident on an axial well passes. The configuration and settings of the observation device 900 will be explained below with reference to Figs. 107 and 108.

[0257] Observation device 900 is an apparatus for observing samples in multiwell plate C3, and achieves oblique illumination mainly using light incident from adjacent wells (k=1). Observation device 900 has a configuration similar to observation device 10, and as shown in Fig. 107, it includes an illumination optical system 915, a reflector 574 that reflects light emitted from illumination optical system 915, and an observation optical system 916 that collects the light reflected by reflector 574. Illumination optical system 915 includes a light source 915a and a diffuser 915b.

[0258] The illumination optical system 915 and the observation optical system 916 are disposed below the installation position of the multiwell plate C3. In contrast, the reflector 574 is disposed above the installation position of the multiwell plate C3. The reflector 574 is a curved mirror, particularly a curved mirror having the second pattern described above, and is disposed so that on-axis marginal rays incident on the observation optical system 916 pass through adjacent wells before being reflected by the reflector 574. Specifically, as shown in FIG. 108, illumination light is incident not only from adjacent wells adjacent to the on-axis well along the illumination direction, but also from wells diagonally to the side (wells adjacent to the adjacent well in the X direction).

[0259] The reflecting surface of reflector 574 of observation device 900 is divided into two parts per well in the axial direction (X direction) of the cylindrical surface row. Each of the two divided reflecting surfaces has a width of 2.25 mm and includes reflecting surface 574a and three reflecting surfaces 574b, whose centers of curvature are positioned one well apart from each other. Reflecting surfaces 574a and 574b are arranged alternately in the axial direction.

[0260] In the observation device 900, the specifications of the observation optical system 916, the specifications of the multiwell plate C3, the state of the culture solution in the wells, and the parameters related to the installation conditions of the reflector 574 are as follows, as shown in Figure 107. The multiwell plate C3 is a 384-well plate. The thickness of the lid of the multiwell plate C3 is 1.5 mm, which is equivalent to 1.0 mm in air. N a =0.12, P=4.5mm, D u =3.8mm, D b =3.2mm, n W =1.332, A c2 =0.2306 / mm, A=0.1530 / mm, H b =11mm, H w =1.8mm, H m =15.5mm, R=25mm

[0261] At this time, in the observation device 900, the values ​​of the parameters related to various conditions are as follows: N aCORU =0.15, H mCLU-RB =2.4mm, H mWideC =36.3mm, H mLUC =3.8mm, H mRBC =9.8mm, R LUC =13.6mm, R RBC = 46.5 mm, H mLU-BR―WideC =13.5mm, R LUWideC =25.8mm, R RBWideC =24.6mm, H mWideORU =28.4mm, H mWORU-RB =14.04mm, H mLU-RB-WideBest =15.5mm, R WideBest =25mm, Y sORU =0.17mm, Y sWide± =-1.13mm, Y sWideORU =-1.16mm, Y sWideBest =-1.25mm

[0262] Therefore, the observation device 900 satisfies the conditions for obliquely illuminating at least the central portion of the well, for extending the oblique illumination range to the negative side, and for extending the oblique illumination range to the positive side, and can observe the sample in the well well with high contrast.

[0263] FIG. 109 is a ray diagram for light incident from an adjacent well. FIG. 110 is a diagram showing the distribution of contrast formed in imaging region IM5 by the light shown in FIG. 109. FIG. 111 is a ray diagram for light incident from an adjacent well in the opposite direction to FIG. 109. FIG. 112 is a diagram showing the distribution of contrast formed in imaging region IM6 by the light shown in FIG. 111. FIG. 113 is a diagram showing the distribution of contrast obtained by observation device 900. Note that region R1 in FIGS. 110, 112, and 113 indicates the region where oblique illumination is established.

[0264] In the observation device 900, as shown in Figures 109 to 112, the illumination direction of the imaging area is slightly different when illumination is performed from the right side or the left side. In other words, the imaging area is switched in accordance with the switching of the illumination direction. This makes it possible to observe a wide area (area R1) inside the well with oblique illumination, as shown in Figure 113.

[0265] Next, a specific description will be given of the third pattern. In order to achieve oblique illumination using the reflector 670 of the third pattern, all of the following must be satisfied.

number

[0266] The height and coordinate parameters are as follows. The coordinate parameters are the same as those of the first and second patterns. The height parameters are the height H mLU-RB k=2, H mYm- and H mYm+ Effective range Y m- =-0, Y m+ =2P = 2P.

number

[0267] When the above conditions described for the second pattern are applied to a 96-well plate, it is desirable that the height of the reflector 570 satisfy the following conditions.

number

[0268] In this case, the various coordinates are the same as those in the second pattern, as follows:

number

[0269] When the above conditions described for the second pattern are applied to a 384-well plate, it is desirable that the height of the reflector 570 satisfy the following conditions.

number

[0270] In this case, the various coordinates are the same as those in the second pattern, as follows:

number

[0271] In the third pattern, the conditions for observing at least the well center with oblique illumination are as follows.

number

[0272] Each parameter is H mStoc+ , H mLU-RB , H mLU , H mRB , R LU , R RB Y sSubstituting 0 for and 2 for k, the result is as follows.

number

[0273] Furthermore, in the third pattern, the conditions for extending the oblique illumination range to the negative side are as follows.

number

[0274] The definitions of each parameter are as follows: mLU-RB , H mLU , H mRB , R LU , R RB Y s =Y sStp It is calculated by substituting Y sStp is H mStpC H at the height mStp- This refers to the Y coordinate of the point.

number

[0275] In the third pattern, it is desirable to extend the range of oblique illumination to the negative side as described above, and further satisfy the following condition: sORU It is possible to expand it to.

number

[0276] In addition, H mStp-ORU is as follows:

number

[0277] The conditions that the third pattern must satisfy when applied to a 96-well plate are as follows: First, the conditions for obliquely illuminating the center of the well are as follows:

number

[0278] Furthermore, the conditions for extending the oblique illumination region to the negative side are as follows:

number

[0279] Furthermore, the conditions for extending the oblique illumination region to the positive side are as follows:

number

[0280] The conditions that the third pattern must satisfy when applied to a 384-well plate are as follows: First, the conditions for obliquely illuminating the center of the well are as follows:

number

[0281] Furthermore, the conditions for extending the oblique illumination region to the negative side are as follows:

number

[0282] Furthermore, the conditions for extending the oblique illumination region to the positive side are as follows:

number

[0283] [Tenth embodiment] Fig. 114 is a diagram for explaining the settings in the observation device 1000 according to this embodiment. Fig. 115 is a diagram for explaining the positional relationship between the multiwell plate C3 and the reflector 671. The configuration and settings of the observation device 1000 will be explained below with reference to Figs. 114 and 115.

[0284] Observation device 1000 is an apparatus for observing samples in multiwell plate C3, and mainly realizes oblique illumination using light incident from the well two adjacent wells (k=2). Observation device 1000 has a configuration similar to observation device 10, and as shown in FIG. 114, it includes an illumination optical system 1015, a reflector 671 that reflects light emitted from illumination optical system 1015, and an observation optical system 1016 that collects the light reflected by reflector 671. Illumination optical system 1015 includes a light source 1015a and a diffuser 1015b.

[0285] The illumination optical system 1015 and the observation optical system 1016 are disposed below the installation position of the multiwell plate C3. In contrast, the reflector 671 is disposed above the installation position of the multiwell plate C3. The reflector 671 is a curved mirror, particularly a curved mirror having the third pattern described above, and is disposed so that on-axis marginal rays incident on the observation optical system 1016 pass through an adjacent well before being reflected by the reflector 671. Specifically, as shown in FIG. 116, the reflector is disposed so that illumination light passes through the well two wells away from the on-axis well and enters the well.

[0286] In observation device 1000, the specifications of observation optical system 1016, specifications of multiwell plate C3, state of culture solution in the wells, and parameters related to installation conditions of reflector 671 are as follows, as shown in Figures 114 and 115. Note that multiwell plate C3 is a 384-well plate. The thickness of the lid of multiwell plate C3 is 1.5 mm, which is equivalent to 1.0 mm in air. N a =0.12, P=4.5mm, D u =3.8mm, D b=3.2mm, n W =1.332, A c2 =0.2306 / mm, A=0.1530 / mm, H b =11mm, H w =1.8mm, H m =35mm, R=44mm

[0287] At this time, in the observation device 1000, the parameter values ​​related to various conditions are as follows: N aCORU =0.15, H mC2LU-RB =12.3mm, H mStpC =51.8mm, H mLUStpC =19.4mm, H mRBStpC =25.3mm, R LUStpC =31.9mm, R RBStpC =66.8mm, H mLU-BR―StpC =23.8mm, H mStpC =51.8mm, R LUStpC =47.7mm, R RBStpC = 42.6 mm, H mStpORU =41.6mm, Y sORU =0.17mm, Y sStp± =-0.87mm, Y sStpORU =-0.86mm

[0288] Therefore, the observation device 1000 satisfies the conditions for obliquely illuminating at least the central portion of the well, for extending the oblique illumination range to the negative side, and for extending the oblique illumination range to the positive side, and can observe the sample in the well well with high contrast.

[0289] Figure 116 is a ray diagram for light incident from an adjacent well. Figure 117 is a diagram showing the distribution of contrast formed in the imaging area IM by the light shown in Figure 116. Figure 118 is a diagram showing the distribution of contrast formed in the imaging area IM by light incident from an adjacent well in the opposite direction to that of Figure 116. Figure 119 is a diagram showing the distribution of contrast obtained by the observation device 1000. Figure 120 is a diagram for explaining the contrast of each well in the multiwell plate C3. Note that the region R1 in Figures 117 to 119 indicates the region where oblique illumination is established.

[0290] 116 to 118, the observation device 1000 can provide oblique illumination of different areas in the imaging area when illuminating from the right side or the left side by directing illumination light from the adjacent well to the on-axis well. This makes it possible to observe a wide area within a well (area R1) with oblique illumination, even in the case of a 384-well plate, as shown in FIG.

[0291] In the observation device 1000, as shown in Fig. 120, oblique illumination is only possible in wells in either the even or odd rows, but the oblique illumination area within the axial well can be expanded by switching the illumination direction. Furthermore, because the illumination light is incident from the well two adjacent wells, there is no risk of the illumination light being blocked by the observation optical system even when a relatively large observation optical system is used. Therefore, this configuration is suitable for observation devices with large observation optical systems.

[0292] [Eleventh embodiment] Fig. 121 is a diagram for explaining the configuration of the observation device 1100 according to this embodiment. Fig. 122 is a diagram for explaining the relationship between the reflective surface and the well row onto which reflected light is incident. Fig. 123 is a diagram for explaining the contrast of each well in multiwell plate C3. The configuration and settings of the observation device 1100 will be explained below with reference to Figs. 121 to 123.

[0293] Observation device 1100 is an apparatus for observing samples in multiwell plate C3, and achieves oblique illumination mainly with light incident from the well two adjacent wells (k=2). Observation device 1100 has a configuration similar to observation device 10, but differs from observation device 1000 in that it has reflector 672 instead of reflector 671.

[0294] The reflector 672 is a curved mirror, particularly a curved mirror having the third pattern described above, and is installed so that on-axis marginal rays incident on an observation optical system (not shown) pass through the well two adjacent to the reflector 672 before being reflected by the reflector 672. The reflecting surface of the reflector 672 is divided into two per well in the axial direction of the cylindrical surface array (X direction), and the divided reflecting surfaces 672a are arranged offset in the Y direction so that the cylindrical center of one of the divided reflecting surfaces 672a is located at the end of the other reflecting surface 672b. The reflecting surfaces 672a and 672b are arranged alternately in the axial direction.

[0295] Each reflecting surface functions in the same manner as the reflecting surface of the observation device 1000. In other words, the light reflected from each reflecting surface only provides oblique illumination to wells in either the even or odd rows. However, as shown in FIG. 122, reflecting surfaces 672a and 672b illuminate different rows. In other words, one of reflecting surfaces 672a and 672b forms an oblique illumination area in wells in the even rows, and the other of reflecting surfaces 672a and 672b forms an oblique illumination area in wells in the even rows. As a result, as shown in FIG. 123, an oblique illumination area (area R1) can be formed in all wells. Furthermore, by switching the illumination direction, the oblique illumination area can be expanded by excluding two rows of wells from the left and right ends.

[0296] The above-described embodiments are illustrative examples provided to facilitate understanding of the invention, and the present invention is not limited to these embodiments. Modifications and alternatives to the above-described embodiments may be included. In other words, the components of each embodiment may be modified without departing from the spirit and scope of the invention. Furthermore, new embodiments can be implemented by appropriately combining multiple components disclosed in one or more embodiments. Furthermore, some components may be deleted from the components shown in each embodiment, or some components may be added to the components shown in each embodiment. Furthermore, the order of the processing steps shown in each embodiment may be reversed as long as there is no contradiction. In other words, the observation device and the method for visualizing a phase object of the present invention can be variously modified and altered without departing from the scope of the claims.

[0297] In this specification, the expression "based on A" does not mean "based only on A," but also means "based at least on A," and further means "based at least partially on A." That is, "based on A" may be based on B in addition to A, or may be based on a part of A. [Explanation of symbols]

[0298] 1···System, 10, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100···Observation device, 13···Stage, 15, 115, 215, 415, 515, 615, 915, 1015···Illumination optical system, 16, 116, 216, 316, 416, 516, 616, 81 6, 916, 1016···Observation optical system, 16a···Image sensor, 70, 170, 270, 370, 470, 471, 472, 570, 571, 572, 573, 574, 670, 671, 672···Reflector, 80···Support member, C, C1, C2, C3···Multiwell plate, W, W1, W2···Well

Claims

1. An illumination optical system provided below the installation position of a multiwell plate; a reflector provided above the installation position, the reflector reflecting light emitted from the illumination optical system; an observation optical system provided below the installation position, the observation optical system converging light reflected by the reflector; The reflector is It is a plane mirror, The reflector is installed so that at least an axial marginal ray among marginal rays incident on the observation optical system passes through a peripheral well different from an axial well located on the optical axis of the observation optical system before being reflected by the reflector, An observation device characterized by satisfying the following conditional expressions: [Equation 1] where Hm is the height of the reflector based on the top of the well of the multiwell plate. Na is the numerical aperture of the observation optical system. k is an integer greater than or equal to 1. P is the pitch of the wells of the multiwell plate. Du is the inner diameter of the top of the well of the multiwell plate. Db is the inner diameter of the bottom of the well of the multiwell plate. nw is the refractive index of the solution contained in the well of the multiwell plate. Hw is the distance from the liquid surface of the solution at the center of the well of the multiwell plate to the bottom of the well. Hb is the depth of the well of the multiwell plate. A is the refractive index of the liquid surface.

2. An illumination optical system provided below the installation position of the multiwell plate; a reflector provided above the installation position, the reflector reflecting light emitted from the illumination optical system; an observation optical system provided below the installation position, the observation optical system converging light reflected by the reflector; the multi-well plate is a 96-well plate; The reflector is It is a plane mirror, The reflector is installed so that at least an axial marginal ray among marginal rays incident on the observation optical system passes through a peripheral well different from an axial well located on the optical axis of the observation optical system before being reflected by the reflector, An observation device characterized by satisfying the following conditional expressions: [Equation 2] where Hm is the height of the reflector relative to the top of the well of the multiwell plate, and Na is the numerical aperture of the observation optical system on the well side.

3. An illumination optical system provided below the installation position of the multiwell plate; a reflector provided above the installation position, the reflector reflecting light emitted from the illumination optical system; an observation optical system provided below the installation position, the observation optical system converging light reflected by the reflector; the multi-well plate is a 384-well plate; The reflector is It is a plane mirror, The reflector is installed so that at least an axial marginal ray among marginal rays incident on the observation optical system passes through a peripheral well different from an axial well located on the optical axis of the observation optical system before being reflected by the reflector, An observation device characterized by satisfying the following conditional expressions: [Equation 3] where Hm is the height of the reflector relative to the top of the well of the multiwell plate, Na is the numerical aperture of the observation optical system on the well side, and k is 1 or 2.

4. An illumination optical system provided below the installation position of the multiwell plate; a reflector provided above the installation position, the reflector reflecting light emitted from the illumination optical system; an observation optical system provided below the installation position, the observation optical system converging light reflected by the reflector; The reflector is a plurality of cylindrical surface rows aligned with concave surfaces facing the multiwell plate; At least an axial marginal ray among marginal rays incident on the observation optical system passes through a peripheral well different from an axial well located on the optical axis of the observation optical system before being reflected by the reflector, and the plurality of cylindrical surface arrays are arranged so that the centers of curvature of the arrays of cylindrical surfaces are each located on a plane passing between adjacent wells included in the multiwell plate and parallel to the optical axis of the observation optical system; The pitch of the plurality of cylindrical surface rows is equal to the pitch of the wells of the multiwell plate. An observation device characterized by:

5. The observation device according to claim 4, An observation device characterized by satisfying the following conditional expressions: [Equation 4] where Hm is the height of the reflector based on the top of the well of the multiwell plate. Na is the numerical aperture on the well side of the observation optical system. P is the pitch of the wells of the multiwell plate. Du is the inner diameter of the top of the well of the multiwell plate. Db is the inner diameter of the bottom of the well of the multiwell plate. nw is the refractive index of the solution contained in the well of the multiwell plate. Hw is the distance from the liquid surface of the solution at the center of the well of the multiwell plate to the bottom of the well. Hb is the depth of the well of the multiwell plate. A is the refractive index of the liquid surface.

6. An illumination optical system provided below the installation position of the multiwell plate; a reflector provided above the installation position, the reflector reflecting light emitted from the illumination optical system; an observation optical system provided below the installation position, the observation optical system converging light reflected by the reflector; The reflector is a plurality of cylindrical surface rows aligned with concave surfaces facing the multiwell plate; At least an axial marginal ray among marginal rays incident on the observation optical system passes through a peripheral well different from an axial well located on the optical axis of the observation optical system before being reflected by the reflector, and the plurality of cylindrical surface arrays are arranged so that the centers of curvature of the arrays of cylindrical surfaces are each located on a plane passing between adjacent wells included in the multiwell plate and parallel to the optical axis of the observation optical system; The pitch of the plurality of cylindrical surface rows is equal to twice the pitch of the wells of the multiwell plate. An observation device characterized by:

7. The observation device according to claim 6, An observation device characterized by satisfying the following conditional expressions: [Equation 5] where Hm is the height of the reflector based on the top of the well of the multiwell plate. Na is the numerical aperture on the well side of the observation optical system. k is an integer greater than or equal to 1. P is the pitch of the wells of the multiwell plate. Du is the inner diameter of the top of the well of the multiwell plate. Db is the inner diameter of the bottom of the well of the multiwell plate. nw is the refractive index of the solution contained in the well of the multiwell plate. Hw is the distance from the liquid surface of the solution at the center of the well of the multiwell plate to the bottom of the well. Hb is the depth of the well of the multiwell plate. A is the refractive index of the liquid surface.

8. An illumination optical system provided below the installation position of the multiwell plate; a reflector provided above the installation position, the reflector reflecting light emitted from the illumination optical system; an observation optical system provided below the installation position, the observation optical system converging light reflected by the reflector; The reflector is a plurality of cylindrical surface rows aligned with concave surfaces facing the multiwell plate; At least an axial marginal ray among marginal rays incident on the observation optical system passes through a peripheral well different from an axial well located on the optical axis of the observation optical system before being reflected by the reflector, and the plurality of cylindrical surface arrays are arranged so that the centers of curvature of the arrays of cylindrical surfaces are each located on a plane passing through the centers of the wells included in the multiwell plate and parallel to the optical axis of the observation optical system; The pitch of the plurality of cylindrical surface rows is equal to twice the pitch of the wells of the multiwell plate. An observation device characterized by:

9. The observation device according to claim 8, An observation device characterized by satisfying the following conditional expressions: [Equation 6] where Hm is the height of the reflector based on the top of the well of the multiwell plate. Na is the numerical aperture of the observation optical system. P is the pitch of the wells of the multiwell plate. Du is the inner diameter of the top of the well of the multiwell plate. Db is the inner diameter of the bottom of the well of the multiwell plate. nw is the refractive index of the solution contained in the well of the multiwell plate. Hw is the distance from the liquid surface of the solution at the center of the well of the multiwell plate to the bottom of the well. Hb is the depth of the well of the multiwell plate. A is the refractive index of the liquid surface.

10. The observation device according to any one of claims 1 to 9, The illumination optical system switches the illumination direction between two directions symmetrical with respect to the optical axis of the observation optical system. An observation device characterized by:

11. The observation device according to any one of claims 1 to 10, further comprising: a moving device that moves the illumination optical system and the observation optical system relative to the multiwell plate in a direction perpendicular to the optical axis of the observation optical system; the observation optical system includes an image pickup element that captures an image of the sample in the axial well; The imaging device images the sample at a plurality of different positions by moving the illumination optical system and the observation optical system relative to the multiwell plate using the moving device. An observation device characterized by:

12. The observation device according to any one of claims 1 to 11, further comprising: A support member is provided to position the reflector at a predetermined height. An observation device characterized by:

13. 1. A method for visualizing phase objects contained in a multiwell plate, comprising: the multi-well plate is a 96-well plate; emitting light onto the multiwell plate from an illumination optical system provided below the installation position of the multiwell plate; reflecting the light emitted from the illumination optical system with a reflector provided above the installation position; and collecting the light reflected by the reflector with an observation optical system provided below the installation position, reflecting the light emitted from the illumination optical system includes making the light, which has passed through a peripheral well different from an axial well located on the optical axis of the observation optical system before being reflected by the reflector, incident on the observation optical system as at least an axial marginal ray; the reflector is a plane mirror; A method characterized by satisfying the following conditional expression: [Equation 7] Here, H m is the height of the reflector relative to the top end of the well of the multiwell plate, and N a is the numerical aperture of the observation optical system on the well side.

14. A method for visualizing phase objects contained in a multiwell plate, comprising: the multi-well plate is a 384-well plate; emitting light onto the multiwell plate from an illumination optical system provided below the installation position of the multiwell plate; reflecting the light emitted from the illumination optical system with a reflector provided above the installation position; and collecting the light reflected by the reflector with an observation optical system provided below the installation position, reflecting the light emitted from the illumination optical system includes making the light, which has passed through a peripheral well different from an axial well located on the optical axis of the observation optical system before being reflected by the reflector, incident on the observation optical system as at least an axial marginal ray; the reflector is a plane mirror; A method characterized by satisfying the following conditional expression: [Equation 8] where H m is the height of the reflector relative to the top of the well of the multiwell plate, N a is the numerical aperture of the observation optical system on the well side, and k is 1 or 2.

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