Cell observation device and imaging method for cell observation device

Abstract

Provided is a cell observation device with which it is possible to acquire a captured image in which defocus is suppressed even in a divided region in which the influence of a meniscus occurs. This cell observation device is provided with: an arrangement means for arranging a cell culture vessel (D) containing cells as an object to be observed; an imaging optical system for imaging an observation image of the object to be observed; a moving unit that moves at least one of the arrangement unit and the imaging optical system from a movement start position to a movement end position along an optical axis direction of the imaging optical system; an imaging unit that captures a plurality of observation images by an imaging element during movement in the optical axis direction; an evaluation unit 513 that evaluates the degree of distribution of luminance values in the plurality of captured images; a selection unit (514) that selects, as a focus image, the captured image having the highest degree of distribution of luminance values by the evaluation unit (513); a selection unit (514) selects the focus image only in accordance with a degree of distribution of luminance values equal to or less than a given luminance value.

Description

Technical Field

[0001] The invention relates to a cell observation device and a photographing method used for the cell observation device. Background Art

[0002] When an optical cell observation device such as a bright field microscope or a phase contrast microscope is used to capture an entire image of a cell culture container (e.g., a culture dish), the culture container is divided into a plurality of partitions (segmented regions), and each segmented region is photographed to obtain a segmented image. Then, by integrating the obtained images, an integrated image is created that integrates the observed image of the entire surface of the culture container.

[0003] When imaging each segmented area of ​​the culture container, the optical axis deviates around the side wall of the culture container due to the lens effect of the meniscus. Therefore, there is a problem that among the segmented images captured using the cell observation device, there are segmented images that are not focused on the observed object, that is, out-of-focus images are generated (Patent Document 1).

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. 2016 / 084551 Summary of the invention

[0007] Problems to be solved by the invention

[0008] In order to avoid the influence of the meniscus, the optical observation device of Patent Document 1 includes an adjustment optical system for adjusting the optical axis deviation, and the adjustment optical system adjusts the optical axis deviation. However, in the optical observation device of Patent Document 1, a new optical system needs to be added to the optical observation device.

[0009] Therefore, an object of the present invention is to provide a cell observation apparatus capable of acquiring a captured image with suppressed defocus even in a divided region where the influence of a meniscus appears.

[0010] Solution to the problem

[0011] In order to achieve the above-mentioned object, the cell observation device of the present invention (hereinafter also referred to as "observation device") comprises:

[0012] a configuration unit for configuring a cell culture container containing cells as an object to be observed;

[0013] An imaging optical system for imaging an observed image of the observed object;

[0014] a moving unit, which moves at least one of the configuration unit and the imaging optical system from a moving start position to a moving end position along an optical axis direction of the imaging optical system;

[0015] A photographing unit that photographs a plurality of observation images through a photographing element during movement along the optical axis direction;

[0016] An evaluation unit, which evaluates the distribution of brightness values ​​of the plurality of captured images;

[0017] a selection unit, which selects the captured image having the highest brightness value distribution degree as the focused image through the evaluation unit;

[0018] The selection unit selects the focused image only according to the distribution degree of brightness values ​​below a given brightness value.

[0019] The photographing method of the present invention is a photographing method for an observation and photographing device having a configuration unit, an imaging optical system, a moving unit, and a photographing unit, and comprises:

[0020] an imaging step of imaging the object to be observed by using an imaging optical system capable of imaging an observation image of the object to be observed, with respect to a cell culture container containing cells arranged in the arrangement unit as the object to be observed;

[0021] a moving step of moving at least one of the configuration unit and the imaging optical system from a moving start position to a moving end position along the optical axis direction of the imaging optical system by the moving unit;

[0022] A photographing step of photographing a plurality of the observation images by a photographing element of the photographing unit during movement in the optical axis direction;

[0023] An evaluation step of evaluating the distribution of brightness values ​​of the plurality of captured images;

[0024] A selection step is performed to select, by the evaluation unit, a captured image having the highest brightness value distribution as the focused image.

[0025] In the selection step, the focused image is selected only according to the distribution degree of brightness values ​​below a given brightness value.

[0026] Effects of the Invention

[0027] According to the present invention, even in a divided area where the influence of the meniscus appears, a captured image with suppressed defocus can be acquired. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] [ Figure 1 ] Figure 1This is a schematic cross-sectional view showing an example of the observation device according to Embodiment 1.

[0029] [ Figure 2 ] Figure 2 This is a block diagram showing an example of a computing device in the observation device according to the first embodiment.

[0030] [ Figure 3 ] Figure 3 This is a diagram showing the relationship between the phase information of the drive signal received by the pulse counting device of Embodiment 1, the count of the pulse counting device, the imaging trigger signal, and the imaging of the camera.

[0031] [ Figure 4 ] Figure 4 This is a flowchart showing the photographing method according to the first embodiment.

[0032] [ Figure 4 ] Figure 5 An example of a frequency distribution diagram (histogram) of the brightness value of an image captured by using a camera in Embodiment 1 is shown.

[0033] [ Figure 6 ] Figure 6 1 is a schematic diagram showing an example of a frequency distribution diagram of brightness values ​​of a plurality of captured images captured at different positions in the Z-axis direction in the first embodiment.

[0034] [ Figure 7 ] Figure 7 This is a photograph showing the result of selecting an image closer to the focus position from a plurality of captured images in the Z-axis direction of the region affected by the meniscus based on the variance value of the luminance value or the degree of distribution of the luminance value in Embodiment 1.

[0035] [ Figure 8 ] Figure 8 1 is a diagram showing focused images captured for a region not affected by the meniscus and a region differently affected by the meniscus in Embodiment 1, and a frequency distribution diagram of brightness values ​​of the focused images.

[0036] [ Fig. 9 ] Fig. 9 1 is a diagram showing a focused image captured under conditions where different illumination light intensities are generated by illumination in Embodiment 1 and a frequency distribution diagram of the brightness value of the focused image.

[0037] [ Fig.10 ] Fig.10 The diagram shows focused images when the cell culture container is contaminated and when it is not contaminated in Embodiment 1, and a frequency distribution diagram of the brightness values ​​of the focused images.

[0038] [ Fig.11 ] Fig.111 is a schematic diagram showing an example of a frequency distribution diagram of brightness values ​​of a plurality of captured images captured at different positions in the Z-axis direction in the second embodiment.

[0039] [ Fig.12 ] Fig.12 This is a block diagram showing an example of the structure of an observation device 300 according to the third embodiment.

[0040] [ Fig.13 ] Fig.13 This is a flowchart showing the photographing method according to the third embodiment.

[0041] [ Fig.14 ] Fig.14 An example of a frequency distribution diagram of the brightness value of an image captured by using a camera in the third embodiment is shown. DETAILED DESCRIPTION

[0042] In this specification, the "optical axis direction" refers to the direction of the optical axis (symmetry axis) in the imaging optical system, also referred to as the "Z-axis direction". The optical axis direction can also be said to be a direction orthogonal (perpendicular) to the mounting surface of the observed object, for example. In addition, in the present invention, the "X-axis direction" refers to a direction in a plane (XY plane) orthogonal to the optical axis direction, and the "Y-axis direction" refers to a direction orthogonal (perpendicular) to the X-axis direction in the XY plane.

[0043] In this specification, "observation" means observing an observed object, and for example, the observation may be accompanied by photographing or may not be accompanied by photographing.

[0044] In this specification, "cell" means a cell or a structure containing a cell. The cell may be, for example, a cell, a cell mass composed of cells, a tissue, an organ, etc. The cell may be, for example, a cultured cell or a cell isolated from a living body.

[0045] The following is a detailed description of the observation device of the present invention with reference to the accompanying drawings. However, the present invention is not limited to the following description. Figures 1 to 7 In the drawings, the same parts are given the same symbols and their descriptions are omitted. In addition, in the drawings, the structures of the various parts are appropriately simplified for the convenience of description, and the size ratios of the various parts are schematically shown, which are different from the actual ones. In addition, the descriptions of the various embodiments can be mutually referenced unless otherwise specified.

[0046] (Implementation Method 1)

[0047] Embodiment 1 relates to the observation device and imaging method of the present invention.

[0048] This embodiment is an example of an observation device. Figure 1Schematic diagram of the observation device 100 according to Embodiment 1. Figure 1 As shown, the observation device 100 includes a stage 1 as a configuration unit, an objective lens 2 as an imaging optical system, a camera 3 as a photographing unit, a moving unit 4, a control unit 5, and an illumination 6 as main structures. The moving unit 4 includes a Z-axis direction moving unit 41 as a first moving unit, an X-axis direction moving unit 42 and a Y-axis direction moving unit 43 as a second moving unit. The Z-axis direction moving unit 41 includes a Z-axis motor 41a and a Z-axis round head screw 41b. The X-axis direction moving unit 42 includes an X-axis motor 42a and an X-axis round head screw 42b. The Y-axis direction moving unit 43 includes a Y-axis motor 43a and a Y-axis round head screw 43b. The control unit 5 includes a computing device 51, a program device (PLC) 52, a motor driver 53, and a pulse counting device 54. The structure of the computing device 51 will be described later. The motor driver 53 includes a motor driver 53x in the X-axis direction, a motor driver 53y in the Y-axis direction, and a motor driver 53z in the Z-axis direction. The lighting 6 includes a light source 61 and a support member 62 that supports the light source 61 .

[0049] A cell culture container D containing cells, which is a structure outside the observation device 100, is placed on the stage 1. In addition, as the stage 1, any structure capable of configuring the observed object may be adopted. As a specific example, the configuration unit may utilize the structure of a configuration unit in an optical observation device. The optical observation device may include, for example, a bright field microscope, a stereo microscope, a phase contrast microscope, a differential interference microscope, a polarizing microscope, a fluorescence microscope, a confocal laser microscope, a total reflection illumination fluorescence microscope, a Raman microscope, etc., preferably a phase contrast microscope. In the stage 1, the configuration area of ​​the cell culture container D may be configured so that the cell culture container D can be observed from the objective lens 2 arranged below the stage 1. The configuration area of ​​the cell culture container D may be formed of a light-transmitting material such as glass, quartz, plastic or resin, and a through hole may be formed in a part thereof.

[0050] The object to be observed is an object observed by the observation device 100. In the present embodiment, the object to be observed is a cell culture container D containing cells, but the object to be observed may be any sample that can be observed by an optical observation device. Examples of the object to be observed include cell culture containers such as a culture dish, a culture plate, and a flask (cell culture flask) containing cells, and a specimen in which a sample is arranged.

[0051] The objective lens 2 forms an observation image of the cell culture container D as the object to be observed on the camera 3 as the shooting element. More specifically, the objective lens 2 forms an observation image of the cells in the cell culture container D on the camera 3. Thus, the observation device 100 can observe and photograph the cells in the cell culture container D. In the observation device 100, although the imaging optical system is configured as the objective lens 2, it is sufficient as long as it can form an observation image of the object to be observed. The imaging optical system can, for example, adopt the structure of the imaging optical system in the optical observation device. In the observation device 100, the number of objective lenses 2 is one, but it can also be multiple. At this time, the magnifications of the objective lenses 2 can be the same or different.

[0052] In the observation device 100 , the objective lens 2 is disposed below the cell culture container D, but may be disposed above the cell culture container D. The arrangement position of the objective lens 2 as the imaging optical system may be appropriately set according to the type of the optical observation device, for example.

[0053] The camera 3 is configured to be able to capture an observation image of the cell culture container D as the object to be observed, and more specifically, to be able to capture an observation image of the cells in the cell culture container D. In the observation device 100, the camera 3 having an imaging element is used as the imaging unit, but a structure capable of capturing an observation image of the object to be observed may also be used. The imaging element may be, for example, a known imaging element, and as specific examples, a charge coupled device (CCD) or a complementary metal oxide semiconductor (CMOS) may be used. Therefore, the imaging unit may be, for example, an imaging device such as a camera having these imaging elements.

[0054] The camera 3 is configured to capture an observation image of the cell culture container D as the object to be observed upon receiving a capture trigger signal transmitted from a pulse counting device 54 described later. One capture time (exposure time) of the camera 3 can be appropriately set according to the brightness of the object to be observed, for example.

[0055] In the observation device 100, after the observation image is formed on the camera 3, the observation image is photographed by the camera 3, and the obtained image is displayed on the display device 56 outside the device. In addition to displaying the image on the display device 56, the observation device 100 can also relay the image (primary image) obtained by the objective lens 2 to the eyepiece, and the user of the observation device 100 observes through the eyepiece. In this case, the observation device 100 includes, for example, a relay optical system and an eyepiece that relay the primary image to the eyepiece. The relay optical system and the eyepiece can adopt the structure of the relay optical system and the eyepiece in the above-mentioned optical observation device. A specific example of the display device 56 will be described later.

[0056] The camera 3 as the imaging optical system sends the captured image to the computing device 51. At this time, the camera 3 preferably associates the captured position of the image (for example, coordinates such as XYZ coordinates) with the captured image and sends it to the computing device 51.

[0057] The moving unit 4 is capable of moving the objective lens 2 as the imaging optical system and the camera 3 as the imaging element. In the observation device 100, although the moving unit 4 is capable of moving the objective lens 2 and the camera 3, it may be configured to be capable of moving only the objective lens 2. In addition, in addition to being capable of moving the objective lens 2 or in place of being capable of moving the objective lens 2, the moving unit 4 may be configured to be capable of moving the stage 1 as the configuration unit. In this case, the stage 1 may also be a structure in which the stage 1 and the moving unit 4 are integrated, such as an electric stage or a mechanical stage. By being configured to be capable of moving the objective lens 2 as the imaging optical system, the moving unit 4 can reduce the shaking of the liquid surface of the culture solution in the cell culture container D as the observed object, as compared with the case in which the stage 1 is configured to be capable of moving. As a result, the observation device 100 can suppress the occurrence of changes in the intensity of the illumination light, changes in the focus position, etc. caused by the shaking of the liquid surface, and can obtain a plurality of captured images including a more accurately focused image (focused image) through the camera 3.

[0058] The moving unit 4 is composed of a first moving unit that can move in the Z-axis direction (i.e., the optical axis direction) and a second moving unit that can move in the XY plane direction. In the moving unit 4, the first moving unit includes a Z-axis motor 41a and a Z-axis round head screw 41b. In addition, the second moving unit includes an X-axis motor 42a and an X-axis round head screw 42b, and a Y-axis motor 43a and a Y-axis round head screw 43b. Figure 1As shown, the Z-axis round head screw 41b, the X-axis round head screw 42b and the Y-axis round head screw 43b are respectively installed in the Z-axis, X-axis and Y-axis directions. In addition, the Z-axis round head screw 41b is connected to the nut on the X-axis round head screw 42b in a manner that the X-axis round head screw 42b can be moved in the X-axis direction. The X-axis round head screw 42b is connected to the nut on the Y-axis round head screw 43b in a manner that the Y-axis round head screw 43b can be moved in the Y-axis direction. The objective lens 2 and the camera 3 are connected to the nut on the Z-axis round head screw 41b in a manner that the Z-axis round head screw 41b can be moved in the Z-axis (optical axis) direction. Then, the Z-axis motor 41a, the X-axis motor 42a, and the Y-axis motor 43a are respectively connected to the motor driver 53z in the Z-axis direction, the motor driver 53x in the X-axis direction, and the motor driver 53y in the Y-axis direction. Therefore, in the observation device 100, the drive signal described later is sent by the motor driver 53 described later. And, based on the driving signal, the Z-axis motor 41a, the X-axis motor 42a and the Y-axis motor 43a are driven, so that the objective lens 2 and the camera 3 are moved to the XYZ coordinates specified by the driving signal. The Z-axis motor 41a, the X-axis motor 42a and the Y-axis motor 43a can be, for example, motors capable of controlling positions, and as a specific example, a stepping motor can be used.

[0059] In the observation device 100, the moving unit 4 can be any one that can move at least one of the configuration unit and the imaging optical system, and can adopt a structure corresponding to the above-mentioned optical observation device. The moving unit 4 is composed of a round head screw and a motor, but can also be composed of a linear motor, or can be combined with a bracket. When the objective lens 2 has a focus adjustment mechanism such as a lens extension mechanism, the focus adjustment mechanism can be the first moving unit in the moving unit 4.

[0060] The moving unit 4 may be configured to be able to move at least one of the configuration unit and the imaging optical system in the optical axis (Z axis) direction, and may also be further configured to be able to move in at least one of the X axis direction and the Y axis direction. For example, the movement in the X axis direction and the Y axis direction may also be referred to as movement in a plane (XY plane) in a direction perpendicular (orthogonal) to the optical axis direction.

[0061] As described above, the control unit 5 includes the operation device 51, the PLC 52, the motor driver 53, and the pulse counting device 54. In the observation device 100, the operation device 51 in the control unit 5 cooperates with the PLC 52, the motor driver 53, and the pulse counting device 54 to function as each unit including the movement control unit including the movement instruction unit 511, the shooting control unit including the shooting instruction unit 512, the evaluation unit 513, and the selection unit 514. The present invention is not limited to this, and the operation device 51 may function as the movement control unit, the shooting control unit, the evaluation unit 513, and the selection unit 514 alone, or the operation device 51 may cooperate with the motor driver 53 and the pulse counting device 54 to function as the movement control unit, the shooting control unit, the evaluation unit 513, and the selection unit 514.

[0062] The computing device 51 has a structure similar to that of a personal computer, a server computer, a workstation, or the like. Figure 2 1 is a block diagram showing an example of the computing device 51 of the observation device 100. Figure 2 As shown, the computing device 51 includes a central processing unit (CPU) 51a, a main memory (main storage device) 51b, an auxiliary storage device 51c, a video codec 51d, an I / O (input-output) interface 51e, etc., which are controlled by a controller (system controller, I / O controller, etc.) 51f and operate in coordination. The auxiliary storage device 51c can be a storage device such as a flash memory and a hard disk drive. The computing device 51 has a CPU 51a as a computing device, but can also have a GPU described later. The video codec 51d includes: a graphics processing unit (GPU) that generates a screen to be displayed based on a drawing instruction received from the CPU 51a and sends the screen signal to a display device 56 outside the observation device 100, etc., a video memory that temporarily stores screens and image data, etc. The I / O interface 51e is a device that is connected to the PLC 52, the pulse counting device 54, and the camera 3 in a communicable manner and is used to control these or obtain information such as images. The I / O interface 51e may also include a servo driver (servo controller). Furthermore, the I / O interface 51e can be connected to an input mechanism (input device) outside the observation device 100. The display device 56 can be exemplified by a monitor that outputs images (e.g., various image display devices such as a liquid crystal display (LCD) and a cathode ray tube (CRT) display). The input device can be exemplified by a touch pad, a touch panel, a pointing device such as a mouse, a keyboard, buttons, etc. that can be operated by a user with a finger.

[0063] The program and various information implemented by the computing device 51 are stored in the auxiliary storage device 51c. When the program is executed, it is read by the main memory 51b and decoded by the CPU 51a. Then, the computing device 51 functions as each part such as the movement instruction unit 511, the shooting instruction unit 512, the evaluation unit 513, and the selection unit 514 according to the program. Therefore, in the observation device 100, the CPU 51a functions as the movement instruction unit 511, the shooting instruction unit 512, the evaluation unit 513, and the selection unit 514. The functions of each part will be described later.

[0064] like Figure 1 As shown, PLC52 is connected with motor driver 53, more specifically, is connected with motor driver 53z on Z-axis direction, motor driver 53x on X-axis direction, and motor driver 53y on Y-axis direction with motor driver 53.PLC52 for example can use programmable logic controller or sequencer.PLC52 will transform the information of the moving position of the specified object lens 2 sent by computing device 51 into the motor instruction to the motor driver 53 of controlling Z-axis motor 41a, X-axis motor 42a and Y-axis motor 43a.Then, PLC52 drives Z-axis motor 41a, X-axis motor 42a and Y-axis motor 43a via motor driver 53, and when object lens 2 arrives the moving position of appointment, the information that object lens 2 has finished moving is sent to computing device 51.

[0065] In the observation device 100, the operation device 51 controls the motor driver 53 via the PLC 52. That is, the movement instruction unit 511 of the operation device 51, the PLC 52, and the motor driver 53 cooperate to function as a movement control unit that controls the movement performed by the movement unit 4. However, the present invention is not limited to this, and the operation device 51 may directly control the motor driver 53. In this case, for example, the operation device 51 includes a motor controller, a microcontroller having a motor control function, a field programmable gate array (FPGA, Field-Programmable Gate Array), etc.

[0066] like Figure 1As shown, the motor driver 53 includes a motor driver 53z in the Z-axis direction, a motor driver 53x in the X-axis direction, and a motor driver 53y in the Y-axis direction. The motor driver 53z in the Z-axis direction, the motor driver 53x in the X-axis direction, and the motor driver 53y in the Y-axis direction are connected to the Z-axis motor 41a, the X-axis motor 42a, and the Y-axis motor 43a, respectively. In addition, the motor driver 53z in the Z-axis direction, the motor driver 53x in the X-axis direction, and the motor driver 53y in the Y-axis direction send drive signals based on the motor instructions sent from the PLC 52, and drive the Z-axis motor 41a, the X-axis motor 42a, and the Y-axis motor 43a, respectively. The drive signal is, for example, a two-phase pulse signal. In addition, the motor driver 53z in the Z-axis direction is connected to the pulse counting device 54. Therefore, in addition to sending the drive signal to the Z-axis motor 43a, the motor driver 53z in the Z-axis direction also sends the drive signal to the pulse counting device 54.

[0067] like Figure 1 As shown, the pulse counting device 54 is connected to the camera 3. The pulse counting device 54 can use a microcontroller or the like. When the pulse counting device 54 receives an on signal from the shooting instruction unit 512 of the computing device 51, it starts counting the number of times the drive signal sent from the motor driver 53z in the Z-axis direction is sent. Then, when the count reaches a given number of times, the pulse counting device 54 sends a shooting trigger signal to the camera 3 to instruct it to shoot. The given number of times can be appropriately set based on the moving distance (the given distance) of the camera 3 in the optical axis direction when the Z-axis motor 41a receives the given number of driving signals. The moving distance in the optical axis direction is, for example, 1 μm to 50 μm. By shortening the moving distance in the optical axis direction, the observation device 100 can more accurately acquire multiple captured images including an image focused on the observed object. In addition, by increasing the moving distance in the optical axis direction, the observation device 100 can acquire multiple captured images including an image focused on the observed object in a shorter time. When the pulse counting device 54 receives a shutdown signal from the shooting instruction unit 512 of the computing device 51, it stops counting the number of times the drive signal is sent. In addition, the pulse counting device 54 can also be configured to reset the count (for example, reset to 0) when receiving a signal for resetting the count of the number of times the drive signal is sent from the computing device 51.

[0068] In the observation device 100, the operation device 51 controls the pulse counting device 54 via the PLC 52 and the motor driver 53z in the Z-axis direction. That is, the shooting instruction unit 512 of the operation device 51, the PLC 52, the motor driver 53z in the Z-axis direction, and the pulse counting device 54 cooperate to function as a shooting control unit that controls the shooting by the camera 3. However, the present invention is not limited to this, and the operation device 51 may directly control the shooting by the camera 3. In this case, the operation device 51 includes, for example, a microcontroller, a field-programmable gate array (FPGA), etc., counts the number of times the drive signal sent from the motor driver 53z in the Z-axis direction is sent, and when the count reaches a given number of times, sends a shooting trigger signal to the camera 3 to instruct it to shoot.

[0069] The pulse counting device 54 is connected to the motor driver 53z in the Z-axis direction, but can also be connected to the Z-axis motor 41a. In this case, the Z-axis motor 41a has an encoder such as a linear encoder (linear scale). Then, the pulse counting device 54 can receive a signal such as a two-phase pulse output from the Z-axis motor 41a as the driving signal and count the number of times.

[0070] Will use Figure 3 The driving method of the pulse counting device 54 is described in more detail. Figure 3 : is a diagram showing the relationship between the phase information of the driving signal received by the pulse counting device 54, the count of the pulse counting device 54, the shooting trigger signal and the shooting of the camera 3. In addition, although the driving signal is a two-phase pulse signal including the A phase and the B phase and the given number of times is 100 times, the type of the driving signal and the number of given times are not limited to this. In addition, in the following description, the focus image is selected by using multiple images taken by the camera 3 having a negative contrast optical system as an example, but the present invention is not limited to this, and the focus image can also be selected by using multiple images taken by the camera 3 having a positive contrast optical system.

[0071] like Figure 3 As shown, the drive signal includes phase A and phase B with a phase difference. The pulse counting device 54 counts using the combination of the rising edge and the falling edge of these phases. Figure 3In the embodiment, the pulse counting device 54 counts once when the B phase is in the low pulse (L pulse) state and the A phase rises, and counts once when the A phase is in the high pulse (H pulse) state and the B phase rises. Furthermore, the pulse counting device 54 counts once when the B phase is in the H pulse state and the A phase falls, and counts once when the A phase is in the L pulse state and the B phase falls. And, when the cumulative count is 99 times, since one more count reaches the given number, the pulse counting device 54 sends the shooting trigger signal to the camera 3. In addition, the pulse counting device 54 also resets the count value to 0. Figure 3 Although the case of using phase A and phase B to accumulate counts is described as an example, if the drive signal is in the same Figure 3 When the phases A and B are opposite, counting is performed in the following manner. The pulse counting device 54 counts once when the B phase is in the H pulse state and the A phase rises, and counts once when the A phase is in the H pulse state and the B phase falls. Further, the pulse counting device 54 counts once when the B phase is in the L pulse state and the A phase falls, and counts once when the A phase is in the L pulse state and the B phase rises. Then, the pulse counting device 54 decreases the count from 0 to 99, 98, etc., and when the count reaches 0, the shooting trigger signal is sent to the camera 3. Thus, the pulse counting device 54 detects the movement in two directions in the direction of the optical axis, and can shoot through the camera 3.

[0072] Next, the illumination 6 illuminates the cell culture container D as the object to be observed, which is arranged on the stage 1. The illumination 6 is an arbitrary structure and may or may not be present. The illumination 6 has a light source capable of illuminating the object to be observed. In addition to the light source 61, the illumination 6 may also have an illumination optical system that guides the illumination light from the light source to the object to be observed. The light source 61 may be, for example, a halogen lamp, a tungsten lamp, and an LED (light emitting diode). The illumination optical system may, for example, adopt the structure of the illumination optical system in the above-mentioned optical observation device.

[0073] In the observation device 100, the illumination 6 is arranged opposite to the objective lens 2 and the camera 3 across the stage 1. That is, the illumination 6 is arranged above the stage 1, but the arrangement position of the illumination 6 is not limited thereto and can be appropriately set according to the type of the optical observation device. For example, it can also be arranged below the stage 1. In addition, the illumination 6 is connected to the camera 3 via the support member 62, so that it moves in conjunction with the movement of the camera 3, but the present invention is not limited thereto. The illumination 6 can be configured to be able to move independently of the camera 3 or not. When the illumination 6 is configured to be able to move independently of the camera 3, the illumination 6 preferably moves in a manner coaxial with the camera 3 in the Z-axis direction. In the observation device 100, the illumination 6 moves in conjunction with the camera 3, so that the shooting target area of ​​the cell culture container D observed and photographed by the objective lens 2 and the camera 3 can be appropriately illuminated. The shooting target area refers to the area photographed by the observation device 100.

[0074] Next, the imaging method according to the first embodiment using the observation device 100 according to the first embodiment will be described.

[0075] Figure 4 is a flowchart showing the photographing method of embodiment 1. Figure 4 As shown, the photographing method of the first embodiment includes step S1 (movement), step S2 (photographing), step S3 (evaluation) and step S4 (selection). In the photographing method of the first embodiment, step S1 and step S2 are performed in parallel.

[0076] In step S1, the objective lens 2 for forming an observation image of the cell culture container D as the observed object is moved from the movement start position to the movement end position along the optical axis direction of the objective lens 2. Specifically, in step S1, the movement instruction unit 511 of the computing device 51 sends information (e.g., XYZ coordinates) specifying the movement position of the objective lens 2 to the PLC 52. Then, the PLC 52 and the motor driver 53 cooperate based on the information specifying the movement position of the objective lens 2 to control the movement by the moving unit 4. Then, in step S1, the objective lens 2 is moved from the movement start position to the movement end position in the Z-axis direction by the moving unit 4.

[0077] The movement start position refers to the position where the movement in the optical axis direction starts, specifically, the position where the movement in the optical axis direction starts and the control of starting to shoot the observation image is implemented by the shooting instruction unit 512 described later. The movement end position refers to the position where the movement in the optical axis direction ends, specifically, the position where the movement in the optical axis direction ends and the control of ending to shoot the observation image is implemented by the shooting instruction unit 512 described later. The movement start position and the movement end position can be set in advance by the user of the observation device 100, or can be set based on the coordinates of the bottom surface in the container of the cell culture container D or the stage 1. The movement start position and the movement end position are expressed as XYZ coordinates, for example. The distance between the movement start position and the movement end position in the optical axis (Z axis) direction can be set, for example, according to the thickness of the observed object, that is, the length in the optical axis direction. When the observed object is a cell, the distance between the movement start position and the movement end position in the optical axis direction is, for example, 0.2 to 0.5 mm. The movement start position and the movement end position may be set, for example, to positions in the Z-axis direction at which a focused image is obtained in a region not affected by the meniscus.

[0078] In the photographing method of embodiment 1, in step S1, the objective lens 2 and the camera 3 as the imaging optical system are moved, but the present invention is not limited thereto. In step S1, the stage 1 as the configuration unit may be moved, or both the stage 1 and the objective lens 2 may be moved.

[0079] Next, in step S2, during the movement along the optical axis direction, a plurality of observation images are captured by the camera 3. Specifically, in step S2, the capture instruction unit 512 of the computing device 51 sends an ON signal to the PLC 52, the motor driver 53z in the Z-axis direction, and the pulse counting device 54. Then, the PLC 52, the motor driver 53z in the Z-axis direction, and the pulse counting device 54 work together based on the ON signal, and when the objective lens 2 moves along the optical axis direction in step S1, a plurality of observation images of the cells in the cell culture container D are captured by the camera 3. Then, in step S2, the camera 3 associates each captured image with the XYZ coordinates of the objective lens 2 at the time of capturing, and then sends the captured plurality of captured images to the computing device 51, and stores them in the auxiliary storage device 51c of the computing device 51. In addition, in step S2, the plurality of captured images are stored in the auxiliary storage device 51c of the computing device 51, but may also be stored in an external storage device outside the observation device 100.

[0080] The shooting control unit controls the shooting by the camera 3 so that the camera 3 shoots a plurality of the observation images during the movement along the optical axis direction. The number of observation images shot can be determined by the movement distance in the optical axis direction and the distance between the movement start position and the movement end position. The number of observation images shot can be multiple, and there is no particular upper limit. Preferably, the shooting instruction unit 512 controls the camera 3 so that the observation image is shot by the shooting element every time a given distance is moved during the movement in the optical axis direction, thereby shooting a plurality of observation images. Thus, the observation device 100 can select a focused image that is more focused on the observed object in the selection of the selection unit 514 described later.

[0081] Next, in steps S3 and S4, a focused image is selected from a plurality of captured images stored in the auxiliary storage device 51c. Figure 5 and Figure 6 Steps S3 and S4 which are characteristic processing of the first embodiment will be described. Figure 5 FIG. 2 shows an example of a frequency distribution diagram of brightness values ​​of an image captured by the camera 3 of the same cell culture container D. Figure 5 In FIG. 1 , (A) shows a frequency distribution diagram of brightness values ​​of a captured image in an area not affected by the meniscus, and (B) shows a frequency distribution diagram of brightness values ​​of a captured image in an area affected by the meniscus. Figure 5 (A) and Figure 5 In (B), each frequency distribution diagram shows, from the top, a frequency distribution diagram of the brightness value of a captured image captured at a position offset from the focus position toward the positive side in the coordinates of the Z-axis direction (optical axis direction), a frequency distribution diagram of the brightness value of a captured image captured at the focus position, and a frequency distribution diagram of the brightness value of a captured image captured at a position offset from the focus position toward the negative side in the coordinates of the Z-axis direction (optical axis direction). Figure 5 In the figure, the horizontal axis is the brightness value and the vertical axis is the frequency. In addition, in other frequency distribution diagrams below, the parameters on the horizontal axis and the vertical axis are also the same.

[0082] In the area not affected by the meniscus, e.g. Figure 5 As shown in (A), a brightness peak caused by cells is observed, but no high brightness peak caused by the refracted light generated by the meniscus appears. On the other hand, in the area affected by the meniscus, Figure 5As shown in (B), in addition to the brightness peak caused by the cell, there is also a high brightness peak caused by the refracted light. Generally speaking, when selecting a focused image from a plurality of captured images acquired at different coordinates in the Z-axis direction, the focused image is selected by evaluating the variance value of the brightness value. Thus, a focused image can be selected in an area that is not affected by the meniscus. On the other hand, in an area affected by the meniscus, the focused image may not be selected using the evaluation of the variance value. This is because, in an area affected by the meniscus, due to the influence of the high brightness peak caused by the refracted light, the variance value is a variance value that includes the brightness value caused by the refracted light. Therefore, in an area affected by the meniscus, the focused image among the plurality of captured images is selected based on the variance value that includes the brightness value caused by the refracted light, which may result in a situation where an accurate evaluation cannot be performed.

[0083] The inventors found that: Figure 5 As shown by arrows X in (A) and 5(B), in the focused image, regardless of the presence or absence of the influence of the meniscus, its tail will expand toward the low brightness side of the brightness peak caused by the cells. On the other hand, the inventors have found that: Figure 5 (A) and Figure 5 As shown by arrow Y in (B), in the captured image obtained when the image is far from the focus position, the tail end on the low brightness side is relatively narrowed compared to the focused image, and when it is farther from the focus position, it is almost or not observed at all. This is because, in the focused image, the resolution of the image is high, the difference in brightness (contrast) of the brightness value is large, and the captured image can be clearly extracted even in the area with low brightness value. On the other hand, it is inferred that in the captured image far from the focus position, the resolution of the image is reduced, the difference in brightness of the brightness value is reduced, and it becomes difficult to extract the captured image in the area with low brightness value. Therefore, in the observation device 100 of embodiment 1, the evaluation unit 513 evaluates the degree of expansion of the tail end on the low brightness side of the brightness peak caused by cells as the distribution degree of brightness value for multiple captured images. Then, in the observation device 100 of embodiment 1, the selection unit 514 selects the captured image with the highest distribution degree of brightness value as the focused image using the distribution degree of brightness value obtained by the evaluation unit 513. Thus, in the observation device 100 according to the first embodiment, regardless of the presence or absence of the influence of the meniscus, it is possible to more accurately select a captured image closer to the focus position.

[0084] Next, use Figure 6 The details of step S3 will be described. Figure 6 1 is a schematic diagram showing an example of a frequency distribution diagram of brightness values ​​of a plurality of captured images captured at different positions in the Z-axis direction. Figure 6In FIG. 1 , (A) shows the positional relationship of each captured image in the Z-axis direction, (B) shows the frequency distribution diagram of each captured image, and (B) is a schematic diagram showing the number of brightness values ​​below the threshold value ν. Figure 6 As shown in (A), images 1 to 4 are a portion of the images among the plurality of captured images, wherein image 1 is a captured image with the maximum Z coordinate, image 2 is a captured image between image 1 and image 3, image 3 is a focused image, and image 4 shows a captured image with the minimum Z coordinate. As described above, in the captured image closer to the focus position, since the tail end of the low brightness side of the brightness peak caused by the cell is relatively increased, the minimum brightness value I is reduced. Therefore, in step S3, as Figure 6 As shown in (C), a threshold value ν of the brightness value is set, and the distribution degree of the brightness value is evaluated by calculating the area of ​​the brightness value below the threshold value ν or less than ν in the captured image, that is, the number of pixels having the brightness value below the threshold value ν or less than ν in the captured image. In addition, in this embodiment, the lower limit of the calculation range of the number of pixels is not set, which is substantially greater than 0 and less than ν (or less than ν), but another threshold value μ can be arbitrarily set to be greater than μ (or greater than μ) and less than ν (or less than ν).

[0085] Specifically, in step S3, the evaluation unit 513 generates a frequency distribution diagram showing the captured images ( Figure 4(B) (Image 1 and Image 4) contain the distribution of brightness values ​​of all pixels in the captured images, and extract the minimum brightness values ​​I1 and I4 from the frequency distribution diagram. Next, in step S3, the two minimum brightness values ​​I1 and I4 are compared by the evaluation unit 513, and the minimum brightness value I1 with a larger brightness value is set as the threshold value ν. Then, in step S3, the number of pixels with brightness values ​​below the threshold value ν of each of the multiple captured images is counted by the evaluation unit 513. In addition, when calculating the minimum brightness values ​​I1 and I4, it is preferred to remove the noise below the noise threshold in advance, or to take the part of the frequency distribution diagram that is greater than the noise threshold as the object. The noise threshold is, for example, the frequency of the brightness value, and can be set by the user. In addition, in the present embodiment, the minimum brightness value I in the two captured images at both ends of the Z-axis direction is set as the threshold value ν, but the present invention is not limited thereto, and the minimum brightness value I of any one of the multiple captured images may be set as the threshold value ν, or the minimum brightness values ​​I of any two or more captured images may be compared, and the maximum minimum brightness value I may be set as the threshold value ν, or the minimum brightness values ​​I of each captured image may be compared for all the multiple captured images, and the maximum minimum brightness value I may be set as the threshold value ν. In addition, in step S3, the evaluation unit 513 removes noise, but it may not remove noise. In this case, in step S3, the evaluation unit 513 extracts the minimum brightness value I from the brightness values ​​of all pixels included in each captured image for the multiple captured images. Then, in step S3, the minimum brightness values ​​I of each captured image are compared, and the maximum minimum brightness value I is set as the threshold value ν.

[0086] Next, in step S4, the selection unit 514 selects a captured image (focused image) closer to the focus position using the number of pixels of brightness values ​​below the threshold value v of each captured image calculated in step S3. Figure 6 As shown in (C), in the captured image closer to the focus position, the tail end of the low brightness side of the brightness peak caused by the cells becomes relatively large, so that the number of pixels with brightness values ​​below the threshold value ν becomes relatively large. Therefore, in step S4, the captured image with the largest number of pixels with brightness values ​​below the threshold value ν is selected as the captured image with the highest brightness value distribution. When the observed object is captured for each segmented area, in step S4, the selection unit 514 selects the captured image with the largest number of pixels with brightness values ​​below the threshold value ν as the captured image with the highest brightness value distribution for each segmented area.

[0087] Next, when the observed object is divided into a plurality of regions (divided regions) that can be photographed within one field of view of the camera 3, and the observed object is photographed for each divided region, in step S1, the movement instruction unit 511 of the computing device 51 sends information (e.g., XYZ coordinates) specifying the moving position of the objective lens 2 for photographing the next divided region to the PLC 52. Then, the PLC 52 and the motor driver 53 cooperate to control the movement by the moving unit 4 based on the information specifying the moving position of the objective lens 2, and the objective lens 2 moves to the new divided region. Preferably, the movement to the new divided region is a movement on the XY plane, and more preferably, it is a movement to the adjacent new divided region on the XY plane. Then, after moving to the new divided region, the observation device 100 of embodiment 1 similarly implements steps S1 to S4. Then, the observation device 1 of embodiment 1 similarly moves to the new divided region, and repeats the processing of steps S1 to S4 in the new divided region until there is no divided region that has not been photographed.

[0088] After capturing, evaluating, and selecting all the segmented areas, the observation device 100 of Embodiment 1 may integrate the focused images selected from the plurality of captured images of the segmented areas to generate a focused image of the entire cell culture container D. The integration may be performed by arranging the focused images based on the XYZ coordinates associated with the focused image of each segmented area. Thus, the observation device 100 of Embodiment 1 can obtain a focused image of the entire cell culture container D.

[0089] In addition, although the observation device 100 of the first embodiment is described by using a plurality of captured images captured by a negative contrast optical system as an example, the observation device 100 of the first embodiment may select a focused image using a plurality of captured images captured by a positive contrast optical system. In this case, in the description of the processing of step S3 and step S4, the observation device 100 of the first embodiment may replace "below" with "above", replace "the largest minimum brightness value" with "the smallest maximum brightness value", replace "the minimum brightness value" with "the maximum brightness value", replace "the larger brightness value" with "the smaller brightness value", replace "greater than" with "less than", and replace "the low brightness side" with "the high brightness side", and can select the focused image by performing the same processing. In addition, the same implementation can be carried out in other embodiments.

[0090] (Effects of Embodiment 1)

[0091] In the observation device 100 of the first embodiment, the evaluation unit 513 evaluates the degree of expansion of the tail end of the low brightness side of the brightness peak caused by the cells in the plurality of captured images that is not affected by the meniscus and is related to the focus position as the number of pixels with brightness values ​​below the threshold value v. In the observation device 100 of the first embodiment, the selection unit 514 selects the captured image (focused image) that is closer to the focus position using the number of pixels with brightness values ​​below the threshold value v. Therefore, the observation device 100 of the first embodiment is not affected by the meniscus, so the captured image that is closer to the focus position can be selected in both the area affected by the meniscus and the area not affected by the meniscus. In addition, according to the observation device 100 of the first embodiment, it is possible to select the captured image that is closer to the focus position, so the observation device 100 of the first embodiment can be appropriately used in combination with a device that uses the selected captured image to distinguish the type of cell, identify the treatment object, or perform cell treatment. As an example, by combining the observation device 100 of embodiment 1 with a laser processing device (laser processing machine), cells to be removed by laser can be more accurately identified, thereby suppressing the reduction in yield caused by laser processing of cells that do not need to be removed.

[0092] Figure 7 This is a photograph showing the result of selecting an image closer to the focus position from a plurality of images captured in the Z-axis direction of the region affected by the meniscus, based on the variance value of the brightness value or the degree of distribution of the brightness value. Figure 7 In FIG. 1 , (A) shows the shooting position in the cell culture container D, (B) shows the result of selection using the variance value, and (C) shows the result of selection based on the distribution degree of the brightness value. Figure 7 As shown in (A), the central portion of the cell culture container D is not affected by the meniscus, while the peripheral portion of the cell culture container D is affected by the meniscus. Figure 7 As shown in (B), when the selection is made based on the variance value, the out-of-focus captured image is selected. Figure 7 As shown in (C), when selection is performed based on the distribution degree of the brightness value, a captured image closer to the focus position can be selected compared to when selection is performed based on the variance value.

[0093] Figure 8 is a diagram showing focused images captured for an area not affected by a meniscus and an area differently affected by a meniscus, and a frequency distribution diagram of brightness values ​​of the focused images. Figure 8 In the figure, (A) shows the result of focusing the image taken of the area not affected by the meniscus, (B) shows the result of focusing the image taken of the area affected by the meniscus and having a medium brightness, and (C) shows the result of focusing the image taken of the area affected by the meniscus and having a high brightness. Figure 8 (A) to Figure 8 In (C), the left column shows the focused images, and the right column shows the frequency distribution diagram of each focused image. Figure 8 (A)~ Figure 8 As shown in (C), regardless of the presence or absence of the influence of the meniscus, the tail end of the low brightness side of the brightness peak caused by the cell is observed to expand as shown by the arrow X. Figure 8 (A)~ Figure 8 As shown in (C), by setting the threshold value ν to the low brightness side of the brightness peak caused by cells, it is possible to select a captured image closer to the focus position even when affected by different curved liquid surfaces by selecting based on the distribution degree of the brightness value.

[0094] Fig. 9 : is a diagram showing a focused image captured under different conditions of the intensity of the illumination light from the illumination 6 and a frequency distribution diagram of the brightness value of the focused image. Fig. 9 In FIG. 1 , the intensity of the illumination light from the illumination 6 increases in the order of (A) to (D). Fig. 9 (A) to Fig. 9 In (D), the left column shows the focused images, and the right column shows the frequency distribution diagram of each focused image. Fig. 9 (A) to Fig. 9 As shown in (D), regardless of the intensity of the illumination light from illumination 6, the tail end of the low brightness side of the brightness peak caused by the cells was observed to expand. Fig. 9 (A) to Fig. 9 As shown in (D), by setting the threshold value ν to the low brightness side of the brightness peak caused by the cells, which is lower than the peak value, even when the intensity of the illumination light of the illumination 6 is different, it is possible to select a captured image closer to the focus position by selecting based on the distribution degree of the brightness value.

[0095] When selecting a focused image, a brightness peak caused by factors other than the meniscus can also affect the selection of a focused image. According to the observation device 100 of this embodiment, when selecting a focused image, it is also possible to suppress the optical influence caused in the captured image by factors other than the meniscus. As an example, even when the image is affected by dirt attached to the cell culture container, an image closer to the focus position can be selected by evaluating the tail end expansion degree on the low brightness side of the brightness peak caused by the cells. Fig.10 : is a diagram showing a focused image when the cell culture container D is contaminated and when it is not contaminated, and a frequency distribution diagram of the brightness value of the focused image. Fig.10 In the figure, (A) shows the result of the focused image when there is no dirt, and (B) shows the result of the focused image when there is dirt in the same area as (A). Fig.10 (A) and Fig.10(B), the left column shows the focused images, and the right column shows the frequency distribution graphs of the respective focused images. As Fig.10 (A) and Fig.10 (B) show, regardless of the presence or absence of dirt, as indicated by arrow X, a tail end expansion is observed on the low-brightness side of the brightness peak caused by the cells. Therefore, as Fig.10 (A) to Fig.10 (B) show, by setting the threshold ν on the low-brightness side lower than the peak value of the brightness peak caused by the cells, regardless of the presence or absence of dirt, it is possible to select, based on the degree of distribution of the brightness values, a captured image closer to the focus position.

[0096] (Embodiment 2)

[0097] In Embodiment 1, the number of pixels with a brightness value below the threshold ν is evaluated as the degree of distribution of the brightness values. As Figure 5 (A) and Figure 5 (B) and Figure 6 (A) and Figure 6 (B) show, in a captured image closer to the focus position, due to a relatively larger expansion at the tail end on the low-brightness side of the brightness peak caused by the cells, the lowest brightness value I relatively decreases, that is, the degree of distribution of the brightness values relatively increases. Thus, in Embodiment 2, an observation device is described that evaluates the lowest brightness value I of a plurality of captured images as the degree of distribution of the brightness values and selects a captured image closer to the focus position.

[0098] In the observation device 200 of Embodiment 2, in step S3, the evaluation unit 513 evaluates the lowest brightness value I of each captured image, and in step S4, the selection unit 514 selects the captured image with the lowest lowest brightness value I as the focused image. Except for this, the observation device 200 of Embodiment 2 has the same structure as the observation device 100 of Embodiment 1 and its description can be cited.

[0099] A more detailed description of steps S3 and S4 will be given using Fig.11 . Fig.11 is a schematic diagram showing an example of a frequency distribution graph of the brightness values of a plurality of captured images (Z1, Z k , Z l , Z m , Z n , k < l < m < n, where k, l, m, and n are integers greater than or equal to 2) captured at different positions in the Z-axis direction. In Fig.11 , (A) shows the positional relationship of each captured image (Z1 to Z n ) in the Z-axis direction, and (B) shows the frequency distribution graphs of each captured image (Z1 to Z n ). As Fig.11As shown in (A), each captured image (Z1 to Z n ) is a part of a plurality of captured images, image Z1 shows the captured image with the maximum Z coordinate, and image Z k Showing image Z1 and image Z l The captured images between, image Z l shows the focused image, image Z m Shown is image Z l and image Z n The captured images between, image Z n As described above, in the captured image closer to the focus position, the lowest brightness value I becomes relatively smaller because the tail end of the brightness peak caused by the cell becomes relatively larger on the low brightness side. Therefore, in step S3, as shown in FIG. Fig.11 As shown in (B), by extracting each captured image (Z1 to Z n ) in the lowest brightness value I(I1,I k ,I l ,I m ,I n ), the distribution degree of the brightness value is evaluated.

[0100] Specifically, in step S3, the evaluation unit 513 evaluates each captured image (Z1 to Z n ) generates a frequency distribution diagram showing the distribution of brightness values ​​of all pixels contained in the captured image. Next, in step S3, for the frequency distribution diagram of each captured image, the lowest brightness value among the brightness values ​​having a frequency greater than the noise threshold is extracted as the lowest brightness value I (I1 to I2) of the lowest brightness information. n ).

[0101] Next, in step S4, the selection unit 514 uses the extracted plurality of lowest brightness values ​​I (I1 to I n ) to select a captured image (focused image) that is closer to the focus position. Specifically, Fig.11 As shown in (B), the minimum luminance value I becomes relatively low in the captured image closer to the focus position. Therefore, in step S4, the minimum luminance values ​​I (I1 to I2) evaluated in step S3 are compared. n ), select the one with the lowest minimum brightness value I l The captured image is used as the focused image.

[0102] In the observation device 200 of the second embodiment, the evaluation unit 513 evaluates the minimum brightness value I among the plurality of captured images that are not affected by the meniscus and are related to the focus position as the distribution degree of the brightness value. Then, in the observation device 200 of the second embodiment, the selection unit 514 selects a captured image (focused image) that is closer to the focus position using the minimum brightness value I. Therefore, in the observation device 200 of the second embodiment, since it is not affected by the meniscus, a captured image that is closer to the focus position can be selected in both the area affected by the meniscus and the area not affected by the meniscus. In addition, in the observation device 200 of the second embodiment, a captured image that is closer to the focus position can be selected without setting a threshold value v.

[0103] (Implementation 3)

[0104] In Embodiment 1, for the threshold value ν, by comparing the minimum brightness values ​​I of a captured image having the maximum Z coordinate and a captured image having the minimum Z coordinate among a plurality of captured images, the larger minimum brightness value I is set as the threshold value ν. As described above, in the present invention, by evaluating the degree of tail expansion on the low brightness side of the brightness peak caused by cells, a captured image (focused image) closer to the focus position can be selected. Therefore, the threshold value ν can also be set using the peak value (cell peak) in the brightness peak caused by cells as a reference. Therefore, in Embodiment 3, an observation device is described that is capable of selecting a captured image (focused image) closer to the focus position by using the threshold value ν set by the cell peak after detecting the brightness peak caused by the cell.

[0105] Fig.12 FIG. 2 is an example of the structure of the observation device 300 according to the third embodiment. Fig.12 As shown, the observation device 300 of the third embodiment includes a detection unit 515 in addition to the calculation device 51 of the observation device 100 of the first embodiment. Otherwise, the observation device 300 of the third embodiment has the same structure as the observation device 100 of the first embodiment, and the description thereof can be cited.

[0106] Fig.13 A flowchart of a photographing method using the observation device 300 of Embodiment 3 is shown. Fig.13 As shown, the photographing method of the third embodiment further includes step S5 (detection step) in addition to the photographing method of the first embodiment, and step S5 is performed after step S2.

[0107] The detection unit 515 detects the cell peak using one or more of the plurality of captured images. Fig.14 The processing of the detection unit 515 in step S5 is described in more detail. Fig.14 (A) and Fig.14 As shown in (B), regardless of the presence or absence of the influence of the meniscus, the cell peaks (maximum brightness values) in multiple captured images are roughly the same. Fig.14 (A) and Fig.14 As shown in (B), the cell peak is the lowest maximum brightness value among the multiple brightness value peaks of each captured image. Therefore, in step S5, first, a frequency distribution diagram is generated for one or more captured images in the multiple captured images by the detection unit 515. Then, in step S5, a maximum value is detected from the frequency distribution diagram, and then, among the one or more detected maximum values, the maximum value of the lowest brightness value is detected as the cell peak. When detecting the peak value of the maximum value, it is preferred to remove the noise below the noise threshold in advance.

[0108] Next, in step S3, the brightness value of the cell peak is used as a threshold value v to evaluate the distribution of brightness values ​​in the plurality of captured images, that is, the number of pixels having brightness values ​​below the threshold value v in the plurality of captured images is calculated. In addition, in the present embodiment, the cell peak is used as the threshold value v, but the threshold value v may be any brightness value between the cell peak and the lowest brightness value I. As a specific example, the threshold value v may also be set to a value obtained by subtracting a predetermined value (e.g., 10, 20, etc.) from the cell peak, or a value obtained by subtracting a value obtained by multiplying the brightness value difference between the cell peak and the lowest brightness value I by a given ratio (e.g., 1 / 2, 1 / 3, 1 / 4, etc.) from the brightness value of the cell peak.

[0109] Then, in step S4 , the captured image having the largest number of pixels with brightness values ​​below the threshold value ν is selected as the captured image having the highest degree of distribution of brightness values.

[0110] In the observation device 300 of the third embodiment, since the cell peak value can be detected in the processing of the computing device 51 of the observation device 300, the threshold value v is calculated based on the cell peak value, and the captured image with the highest brightness value distribution degree is selected. Therefore, in the observation device 300 of the third embodiment, it is not necessary to set the threshold value v, and even when capturing the cell to be measured for the first time, it is possible to appropriately select a focused image.

[0111] (Implementation 4)

[0112] In the first embodiment, a threshold value v is set for each segmented region, and a different threshold value v is used for each segmented region to perform evaluation and selection. Fig.14 (A) and Fig.14As shown in (B), when the same cell culture container D is photographed under the same conditions, the brightness peak value caused by the cells and the cell peak value are substantially the same. Therefore, in Embodiment 4, an observation device that sets a common threshold value v for each divided area for evaluation is described.

[0113] In the observation device 400 of the fourth embodiment, in step S3, the evaluation unit 513 sets the threshold value v using a plurality of captured images obtained in one of the plurality of divided regions. Except for this, the observation device 400 of the fourth embodiment has the same structure as the observation device 100 of the first embodiment, and the description thereof can be cited.

[0114] In step S3, the segmented area for setting the threshold value ν may be a segmented area in an area not affected by the meniscus, a segmented area in an area affected by the meniscus, or a segmented area including both, but since the threshold value ν is easy to set, it is preferable to set the segmented area in an area not affected by the meniscus. Then, in the observation device 400 of embodiment 4, the minimum brightness value I1 in the captured image of the maximum Z coordinate is compared with the minimum brightness value I4 in the captured image of the minimum Z coordinate in the same manner as the observation device 100 of embodiment 1, and the threshold value ν is set. Next, in step S3, the number of pixels having brightness values ​​less than the threshold value ν in the captured image is counted using the plurality of captured images of each segmented area and the threshold value ν.

[0115] In the observation device 400 of the fourth embodiment, since the threshold value v obtained in one segmented area is used, a focused image can be selected even without setting the threshold value v for each segmented area. Therefore, according to the observation device 400 of the fourth embodiment, the processing required for obtaining a focused image of the entire cell culture container D can be reduced, so that a focused image of the entire cell culture container D can be obtained in a shorter time.

[0116] In addition, in the observation device 400 of the fourth embodiment, the threshold value ν is set by the same processing as the observation device 100 of the first embodiment, but the threshold value ν may be set by the same processing as the observation device 300 of the third embodiment.

[0117] (Implementation 5)

[0118] In Embodiment 1, a threshold value ν is set based on a plurality of captured images, and the threshold value ν is used to perform evaluation and selection. On the other hand, if the capturing conditions of the cell culture container D are the same, the brightness peaks caused by the cells will be generated within a numerical range of approximately the same brightness value. Therefore, for each of the capturing conditions, a threshold value ν is set in advance for the brightness peak caused by the cells to be obtained, and a focused image can be selected by using the threshold value ν. Therefore, in Embodiment 5, an observation device is described in which a table of threshold values ​​ν associated with the capturing conditions of the cell culture container D is prepared in advance, and in the processing of the computing device 51, the threshold value ν is read from the capturing conditions associated with the plurality of captured images, and a focused image is selected based on the threshold value ν.

[0119] In the observation device 500 of the fifth embodiment, in step S2, in addition to the plurality of captured images, the capturing conditions of the plurality of captured images are stored, and in step S3, the threshold value v is set based on the capturing conditions and a table in which the capturing conditions are associated with the threshold value v. Except for this, the observation device 500 of the fifth embodiment has the same structure as the observation device 100 of the first embodiment, and the description thereof can be referred to.

[0120] In the observation device 500 of the fifth embodiment, a table is created before the observation device 500 performs processing. The table can be created as follows. First, a plurality of images captured under different shooting conditions are acquired using the observation device 500. Next, a threshold value v is set in the same manner as the observation device 100 of the first embodiment based on the acquired plurality of images. Then, the shooting conditions and the threshold value are associated and stored in the auxiliary storage device 51c. The shooting conditions may include, for example, the intensity of the illumination light of the illumination 6, the gain value of the camera 3, and the exposure time.

[0121] Next, in step S3, the evaluation unit 513 obtains and sets the threshold value v associated with the shooting condition from the shooting condition and the table stored in the auxiliary storage device 51c. Next, in step S3, the number of pixels having a brightness value below the threshold value v in the shot image is counted using the plurality of shot images of each segmented area and the threshold value v.

[0122] In the observation device 500 of the fifth embodiment, since the threshold value v associated with the shooting condition is used, a focused image can be selected without setting the threshold value v for each of the plurality of shot images. Therefore, according to the observation device 500 of the fifth embodiment, a focused image can be obtained at a higher speed.

[0123] Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above-described embodiments. The configuration and specific contents of the present invention can be modified in various ways that can be understood by those skilled in the art within the scope of the present invention.

[0124] This application claims priority based on Japanese patent application No. 2022-113571 filed on Jul. 15, 2022, the disclosure of which is incorporated herein in its entirety.

[0125] <Notes>

[0126] A part or all of the above-mentioned embodiments and examples are as described in the following supplementary notes, but are not limited thereto.

[0127] <Cell Observation Device>

[0128] (Note 1)

[0129] A cell observation device having:

[0130] a configuration unit for configuring a cell culture container containing cells as an object to be observed;

[0131] An imaging optical system for imaging an observed image of the observed object;

[0132] a first moving unit, which moves at least one of the configuration unit and the imaging optical system from a moving start position to a moving end position along the optical axis direction of the imaging optical system;

[0133] A photographing unit that photographs a plurality of observation images through a photographing element during movement along the optical axis direction;

[0134] The evaluation unit evaluates the plurality of captured images only according to the distribution degree of the brightness values ​​in a given brightness value range.

[0135] The selection unit selects a captured image having the highest brightness value distribution as a focused image.

[0136] (Note 2)

[0137] The cell observation device according to Supplementary Note 1, wherein the evaluation unit measures the number of pixels having brightness values ​​below a given threshold value ν in the plurality of captured images;

[0138] The selection unit selects a captured image having the largest measured number of pixels.

[0139] (Note 3)

[0140] The cell observation device according to Supplementary Note 2, wherein the evaluation unit generates the threshold value ν using at least one of the plurality of images.

[0141] (Note 4)

[0142] A cell observation device according to Note 3, wherein the evaluation unit generates a frequency distribution diagram showing the distribution of brightness values ​​of all pixels contained in at least one of the multiple captured images, and uses the generated frequency distribution diagram to determine the threshold value ν.

[0143] (Note 5)

[0144] According to the cell observation device described in Supplementary Note 4, the evaluation unit extracts the minimum brightness value for each of the plurality of captured images, and sets the largest minimum brightness value among the extracted plurality of minimum brightness values ​​as the threshold value ν.

[0145] (Note 6)

[0146] According to the cell observation device described in Appendix 4, the minimum value of the brightness value contained in the captured image at the starting position of the movement is compared with the minimum value of the brightness value contained in the captured image at the ending position of the movement, and the larger brightness value is set as the threshold value ν.

[0147] (Note 7)

[0148] The cell observation device according to Supplement 5 or 6, wherein the evaluation unit removes brightness values ​​below a given number of pixels as noise when measuring the number of pixels having brightness values ​​below the threshold value v;

[0149] The selection unit selects the focused image only according to the distribution degree of brightness values ​​below a given brightness value from which the noise has been removed.

[0150] (Note 8)

[0151] The cell observation device according to Supplement 2, wherein the cell observation device further comprises:

[0152] an illumination unit, which is arranged opposite to the imaging optical system and the photographing element via the arrangement unit;

[0153] The lighting unit is configured to illuminate the cell culture container containing the cells and the culture solution.

[0154] (Note 9)

[0155] The cell observation device according to Supplementary Note 8, wherein the imaging optical system images the observation image of the observed object corresponding to the divided area as a divided image for each of the plurality of divided divided areas of the observed object;

[0156] The selection unit selects a captured image having the largest number of pixels for each of the divided images of the divided region.

[0157] (Note 10)

[0158] According to the cell observation device described in Appendix 9, wherein the imaging optical system images the observation image of the observed object corresponding to the divided area affected by the curved liquid surface generated in the cell culture container and the divided area not affected by the curved liquid surface in the observed object as divided images.

[0159] (Note 11)

[0160] The cell observation device according to Supplementary Note 9, wherein the cell observation device further comprises:

[0161] a second moving unit capable of moving at least one of the configuration unit and the imaging optical system in a plane direction perpendicular to the optical axis direction;

[0162] The photographing unit photographs a plurality of photographed images at any position in the plane direction;

[0163] The selection unit selects the focused image.

[0164] (Note 12)

[0165] The cell observation device according to Supplementary Note 11, wherein the cell observation device further comprises:

[0166] The integration unit integrates the focused images of the divided regions to generate a focused image of the entire cell culture container.

[0167] Using the second moving unit, the photographing unit photographs a plurality of photographed images for all the divided areas;

[0168] selecting, by the selection unit, focused images in all segmented areas of the cell culture container;

[0169] The integration unit combines the selected focused images to generate a focused image of the entire cell culture container.

[0170] (Note 13)

[0171] The cell observation device according to Supplementary Note 11, wherein the first moving unit changes the moving direction along the optical axis direction in the divided region unit;

[0172] When the second moving unit moves to a new divided area, the moving direction is changed from the moving direction of the previous divided area along the optical axis direction.

[0173] (Note 14)

[0174] The cell observation device according to Supplement 1 or 8, wherein the cell observation device is a phase contrast microscope.

[0175] (Note 15)

[0176] A cell observation device having:

[0177] a configuration unit for configuring a cell culture container containing cells as an object to be observed;

[0178] An imaging optical system for imaging an observed image of the observed object;

[0179] a first moving unit, which moves at least one of the configuration unit and the imaging optical system from a moving start position to a moving end position along the optical axis direction of the imaging optical system;

[0180] A photographing unit that photographs a plurality of observation images through a photographing element during movement along the optical axis direction;

[0181] An evaluation unit, which evaluates the distribution of brightness values ​​of the plurality of captured images and extracts minimum brightness information associated with the minimum brightness value;

[0182] A selection unit selects a focused image based on the plurality of minimum brightness information.

[0183] (Note 16)

[0184] According to the cell observation device described in Note 15, the evaluation unit generates a frequency distribution diagram showing the distribution of brightness values ​​of all pixels contained in the multiple captured images respectively, evaluates the generated frequency distribution diagram, and extracts the minimum brightness information.

[0185] (Note 17)

[0186] According to the cell observation device described in Supplement 16, wherein the evaluation unit removes brightness values ​​below a given number of pixels as noise when evaluating the generated frequency distribution diagram, and extracts the minimum brightness value in the frequency distribution diagram from which the noise has been removed as minimum brightness value information.

[0187] (Note 18)

[0188] The cell observation device according to Supplement 17, wherein the cell observation device further comprises:

[0189] an illumination unit, which is arranged opposite to the imaging optical system and the photographing element via the arrangement unit;

[0190] The lighting unit is configured to illuminate the cell culture container containing the cells and the culture solution.

[0191] (Note 19)

[0192] The cell observation device according to Supplementary Note 18, wherein the imaging optical system images, for each of a plurality of divided regions of the observed object, an observed image of the observed object corresponding to the divided region as a divided image;

[0193] The selection unit selects a captured image having the largest number of pixels for each of the divided images of the divided region.

[0194] (Note 20)

[0195] According to the cell observation device described in Appendix 19, wherein the imaging optical system images the observation image of the observed object corresponding to the divided area affected by the curved liquid surface generated in the cell culture container and the divided area not affected by the curved liquid surface in the observed object as divided images.

[0196] (Note 21)

[0197] The cell observation device according to Supplementary Note 20, wherein the cell observation device further comprises:

[0198] a second moving unit capable of moving at least one of the configuration unit and the imaging optical system in a plane direction perpendicular to the optical axis direction;

[0199] The photographing unit photographs a plurality of photographed images at any position in the plane direction;

[0200] The selection unit selects the focused image.

[0201] (Note 22)

[0202] The cell observation device according to Supplementary Note 21, wherein the cell observation device further comprises:

[0203] an integration unit, integrating the focused images of the divided areas to generate a focused image of the entire cell culture container;

[0204] Using the second moving unit, the photographing unit photographs a plurality of photographed images for all the divided areas;

[0205] selecting, by the selection unit, focused images in all segmented areas of the cell culture container;

[0206] The integration unit combines the selected focused images to generate a focused image of the entire cell culture container.

[0207] (Note 23)

[0208] The cell observation device according to Supplementary Note 22, wherein the first moving unit changes the moving direction along the optical axis direction in the divided region unit;

[0209] When the second moving unit moves to a new divided area, the moving direction is changed from the moving direction of the previous divided area along the optical axis direction.

[0210] (Note 24)

[0211] The cell observation device according to Supplementary Note 15, wherein the cell observation device is a phase contrast microscope.

[0212] <Photographing method for cell observation device>

[0213] (Note 25)

[0214] A photographing method is a photographing method for an observation photographing device having a configuration unit, an imaging optical system, a first moving unit, and a photographing unit, comprising:

[0215] An imaging step of imaging a cell culture container containing cells as an object to be observed and arranged in the arrangement unit, using an imaging optical system capable of imaging an observation image of the object to be observed;

[0216] a moving step of moving at least one of the configuration unit and the imaging optical system from a moving start position to a moving end position along the optical axis direction of the imaging optical system by a first moving unit;

[0217] A photographing step of photographing a plurality of the observation images by a photographing element of the photographing unit during movement in the optical axis direction;

[0218] An evaluation step of evaluating the plurality of captured images only according to the distribution of the brightness values ​​within a given brightness value range;

[0219] In the selection step, a captured image having the highest brightness value distribution degree is selected as the focused image.

[0220] (Note 26)

[0221] The photographing method according to supplementary note 25, wherein in the evaluation step, the number of pixels having brightness values ​​below a given threshold value ν in the plurality of photographed images is measured;

[0222] The selecting step selects a captured image having the largest measured number of pixels.

[0223] (Note 27)

[0224] The photographing method according to Supplementary Note 26, wherein, in the evaluation step, the threshold value ν is generated using at least one of the plurality of images.

[0225] (Note 28)

[0226] According to the shooting method described in Note 27, wherein, in the evaluation step, a frequency distribution graph showing the distribution of brightness values ​​of all pixels contained in at least one of the multiple captured images is generated, and the threshold value ν is determined using the generated frequency distribution graph.

[0227] (Note 29)

[0228] According to the shooting method described in Supplement 28, in the evaluation step, the minimum brightness value is extracted for each of the multiple captured images, and the largest minimum brightness value among the extracted multiple minimum brightness values ​​is set as the threshold value ν.

[0229] (Note 30)

[0230] According to the shooting method described in Note 28, wherein, in the evaluation step, the minimum value of the brightness value contained in the captured image at the starting position of the movement is compared with the minimum value of the brightness value contained in the captured image at the ending position of the movement, and the larger brightness value is set as the threshold value ν.

[0231] (Note 31)

[0232] The photographing method according to Supplement 29 or 30, wherein in the evaluation step, when measuring the number of pixels having brightness values ​​below the threshold value ν, brightness values ​​below a given number of pixels are removed as noise;

[0233] In the selection step, the focused image is selected based on the distribution degree of brightness values ​​below a given brightness value from which the noise has been removed.

[0234] (Note 32)

[0235] The photographing method according to Supplementary Note 26, wherein the observation photographing device further comprises:

[0236] an illumination unit, which is arranged opposite to the imaging optical system and the photographing element via the arrangement unit;

[0237] The lighting unit illuminates the cell culture container containing the cells and the culture solution.

[0238] (Note 33)

[0239] The photographing method according to Supplementary Note 32, wherein the imaging optical system images, for each of a plurality of divided divided regions of the observed object, an observed image of the observed object corresponding to the divided region as a divided image;

[0240] In the selection step, a captured image having the largest number of pixels is selected for each of the divided images of the divided area.

[0241] (Note 34)

[0242] According to the shooting method described in Appendix 33, wherein the imaging optical system images the observation image of the observed object corresponding to the segmented area affected by the curved liquid surface generated in the cell culture container and the segmented area not affected by the curved liquid surface in the observed object as segmented images.

[0243] (Note 35)

[0244] The photographing method according to Supplement 33, wherein the observation photographing device further comprises:

[0245] a second moving unit capable of moving at least one of the configuration unit and the imaging optical system in a plane direction perpendicular to the optical axis direction;

[0246] In the photographing step, a plurality of photographed images are photographed at any position in the plane direction;

[0247] In the selecting step, the focused image is selected.

[0248] (Note 36)

[0249] The shooting method according to Supplement 35 further comprises:

[0250] an integration step of integrating the focused images of the divided areas to generate a focused image of the entire cell culture container;

[0251] In the photographing step, the second moving unit is used to photograph a plurality of images for all the divided areas through the photographing unit;

[0252] In the selecting step, focusing images in all segmented areas of the cell culture container are selected;

[0253] In the integration step, a focused image of the entire cell culture container is generated by combining the selected focused images.

[0254] (Note 37)

[0255] The photographing method according to supplementary note 35, wherein the first moving unit changes the moving direction along the optical axis direction in the divided area unit;

[0256] When the second moving unit moves to a new divided area, the moving direction is changed from the moving direction of the previous divided area along the optical axis direction.

[0257] (Note 38)

[0258] The photographing method according to Appendix 25 or 32, wherein the observation and photographing device is a phase contrast microscope.

[0259] (Note 39)

[0260] A photographing method is a photographing method for an observation photographing device having a configuration unit, an imaging optical system, a first moving unit, and a photographing unit, comprising:

[0261] An imaging step of imaging a cell culture container containing cells as an object to be observed and arranged in the arrangement unit, using an imaging optical system capable of imaging an observation image of the object to be observed;

[0262] a moving step of moving at least one of the configuration unit and the imaging optical system from a moving start position to a moving end position along the optical axis direction of the imaging optical system by the first moving unit;

[0263] A photographing step of photographing a plurality of the observation images by a photographing element of the photographing unit during movement in the optical axis direction;

[0264] An evaluation step of evaluating the brightness value distribution of the plurality of captured images and extracting minimum brightness information associated with the minimum brightness value;

[0265] A selecting step is to select a focused image based on the plurality of minimum brightness information.

[0266] (Note 40)

[0267] According to the shooting method described in Note 39, in the evaluation step, a frequency distribution diagram showing the distribution of brightness values ​​of all pixels contained in the captured images is generated for each of the multiple captured images, the generated frequency distribution diagram is evaluated, and the minimum brightness information is extracted.

[0268] (Note 41)

[0269] According to the shooting method described in Note 40, wherein, in the evaluation step, when evaluating the generated frequency distribution diagram, the brightness values ​​below a given number of pixels are removed as noise, and the lowest brightness value in the frequency distribution diagram from which the noise has been removed is extracted as the lowest brightness value information.

[0270] (Note 42)

[0271] The photographing method according to Supplement 41, wherein the observation photographing device further comprises:

[0272] an illumination unit, which is arranged opposite to the imaging optical system and the photographing element via the arrangement unit;

[0273] The lighting unit is configured to illuminate the cell culture container containing the cells and the culture solution.

[0274] (Note 43)

[0275] The photographing method according to supplementary note 42, wherein the imaging optical system images the observed image of the observed object corresponding to the divided area as a divided image for each of the plurality of divided divided areas of the observed object;

[0276] In the selection step, a captured image having the largest number of pixels is selected for each of the divided images of the divided area.

[0277] (Note 44)

[0278] According to the shooting method described in Appendix 43, wherein the imaging optical system images the observation image of the observed object corresponding to the segmented area affected by the curved liquid surface generated in the cell culture container and the segmented area not affected by the curved liquid surface in the observed object as segmented images.

[0279] (Note 45)

[0280] The photographing method according to Supplement 44, wherein the observation photographing device further comprises:

[0281] a second moving unit capable of moving at least one of the configuration unit and the imaging optical system in a plane direction perpendicular to the optical axis direction;

[0282] In the photographing step, a plurality of photographed images are photographed at any position in the plane direction;

[0283] In the selecting step, the focused image is selected.

[0284] (Note 46)

[0285] According to the photographing method described in Supplement 45, the observation and photographing device further comprises:

[0286] an integration step of integrating the focused images of the divided areas to generate a focused image of the entire cell culture container;

[0287] In the photographing step, the second moving unit is used to photograph a plurality of images for all the divided areas through the photographing unit;

[0288] In the selecting step, focusing images in all segmented areas of the cell culture container are selected;

[0289] In the integration step, a focused image of the entire cell culture container is generated by combining the selected focused images.

[0290] (Note 47)

[0291] The photographing method according to supplementary note 46, wherein the first moving unit changes the moving direction along the optical axis direction in the divided area unit;

[0292] When the second moving unit moves to a new divided area, the moving direction is changed from the moving direction of the previous divided area along the optical axis direction.

[0293] (Note 48)

[0294] The photographing method according to Supplementary Note 39, wherein the observation and photographing device is a phase contrast microscope.

[0295] Description of Reference Numerals

[0296] 1 Stage

[0297] 2 Objective lens

[0298] 3. Camera

[0299] 4 Mobile Units

[0300] 41 X-axis moving unit

[0301] 41a X-axis motor

[0302] 41b X-axis round head screw

[0303] 42 Y-axis moving unit

[0304] 42a Y-axis motor

[0305] 42b Y-axis round head screw

[0306] 43 Z-axis moving unit

[0307] 43a Z-axis motor

[0308] 43b Z-axis round head screw

[0309] 5 Control unit

[0310] 51 Computing device

[0311] 51a CPU

[0312] 511 Mobile Instruction Unit

[0313] 512 Shooting instruction unit

[0314] 513 Evaluation Units

[0315] 514 Select Unit

[0316] 515 Detection Unit

[0317] 51b main memory

[0318] 51c Auxiliary storage device

[0319] 51d video codec

[0320] 51e I / O interface

[0321] 51f Controller

[0322] 52 PLC

[0323] 53 Motor Driver

[0324] 53x X-axis motor driver

[0325] 53y Motor driver for Y axis

[0326] 53z Z-axis motor driver

[0327] 54 Pulse counting device

[0328] 56 Display device

[0329] 6 lighting

[0330] 61 Light Source

[0331] 62 Support components

[0332] 100, 200, 300, 400, 500 Observation device

Claims

1. Cell observation device, having: a configuration unit for configuring a cell culture container containing cells as an object to be observed; An imaging optical system for imaging an observed image of the observed object; a first moving unit, which moves at least one of the configuration unit and the imaging optical system from a moving start position to a moving end position along the optical axis direction of the imaging optical system; A photographing unit that photographs a plurality of observation images through a photographing element during movement along the optical axis direction; An evaluation unit, which evaluates the plurality of captured images only according to the distribution degree of the brightness values ​​in a given brightness value range; The selection unit selects a captured image having the highest brightness value distribution as a focused image.

2. The cell observation device according to claim 1, wherein The evaluation unit measures the number of pixels having brightness values ​​below a given threshold value v in the plurality of captured images; The selection unit selects a captured image having the largest measured number of pixels.

3. The cell observation device according to claim 2, wherein: The evaluation unit generates the threshold value v using at least one of the plurality of images.

4. The cell observation device according to claim 3, wherein: The evaluation unit generates a frequency distribution diagram showing the distribution of brightness values ​​of all pixels included in at least one of the plurality of captured images, and determines the threshold value v using the generated frequency distribution diagram.

5. The cell observation device according to claim 4, wherein: The evaluation unit extracts the lowest brightness values ​​for the plurality of captured images respectively; and sets the largest lowest brightness value among the extracted plurality of lowest brightness values ​​as the threshold value v.

6. The cell observation device according to claim 4, wherein: The minimum value of the brightness value in the captured image at the movement start position is compared with the minimum value of the brightness value in the captured image at the movement end position, and the larger brightness value is set as the threshold value v.

7. The cell observation device according to claim 5 or 6, wherein: The evaluation unit removes brightness values ​​below a given number of pixels as noise when measuring the number of pixels with brightness values ​​below the threshold value v; The selection unit selects the focused image only according to the distribution degree of brightness values ​​below a given brightness value from which the noise has been removed.

8. The cell observation device according to claim 2, wherein: The cell observation device also has: an illumination unit, which is arranged opposite to the imaging optical system and the photographing element via the arrangement unit; The lighting unit is configured to illuminate the cell culture container containing the cells and the culture solution.

9. The cell observation device according to claim 8, wherein: The imaging optical system images the observed image of the observed object corresponding to each of the plurality of divided divided regions of the observed object as a divided image; The selection unit selects a captured image having the largest number of pixels for each of the divided images of the divided region.

10. The cell observation device according to claim 9, wherein: The imaging optical system forms observation images of the observed object corresponding to the divided areas in the observed object, for the divided areas affected by the meniscus generated in the cell culture container and the divided areas not affected by the meniscus, as divided images.

11. The cell observation device according to claim 9, wherein: The cell observation device also has: a second moving unit capable of moving at least one of the configuration unit and the imaging optical system in a plane direction perpendicular to the optical axis direction; The photographing unit photographs a plurality of photographed images at any position in the plane direction; The selection unit selects the focused image.

12. The cell observation device according to claim 11, wherein The cell observation device also has: an integration unit, integrating the focused images of the divided areas to generate a focused image of the entire cell culture container; Using the second moving unit, the photographing unit photographs a plurality of photographed images for all the divided areas; selecting, by the selection unit, focused images in all segmented regions of the cell culture container; The integration unit combines the selected focused images to generate a focused image of the entire cell culture container.

13. The cell observation device according to claim 11, wherein The first moving unit changes the moving direction along the optical axis direction in the divided area unit; When the second moving unit moves to a new divided area, the moving direction is changed from the moving direction of the previous divided area along the optical axis direction.

14. The cell observation device according to claim 1 or 8, wherein: The cell observation device is a phase contrast microscope.

15. Cell observation device, having: a configuration unit for configuring a cell culture container containing cells as an object to be observed; An imaging optical system for imaging an observed image of the observed object; a first moving unit, which moves at least one of the configuration unit and the imaging optical system from a moving start position to a moving end position along the optical axis direction of the imaging optical system; A photographing unit that photographs a plurality of observation images through a photographing element during movement along the optical axis direction; An evaluation unit, which evaluates the distribution of brightness values ​​of the plurality of captured images and extracts minimum brightness information associated with the minimum brightness value; A selection unit selects a focused image based on the plurality of minimum brightness information.

16. A photographing method, which is a photographing method for an observation and photographing device having a configuration unit, an imaging optical system, a first moving unit, and a photographing unit, comprising: An imaging step of imaging a cell culture container containing cells as an object to be observed and arranged in the arrangement unit, using an imaging optical system capable of imaging an observation image of the object to be observed; a moving step of moving at least one of the configuration unit and the imaging optical system from a moving start position to a moving end position along the optical axis direction of the imaging optical system by the first moving unit; A photographing step of photographing a plurality of the observation images by a photographing element of the photographing unit during movement in the optical axis direction; An evaluation step of evaluating the plurality of captured images only according to the distribution of the brightness values ​​within a given brightness value range; In the selection step, a captured image having the highest brightness value distribution degree is selected as the focused image.

17. A photographing method, which is a photographing method for an observation and photographing device having a configuration unit, an imaging optical system, a first moving unit, and a photographing unit, comprising: An imaging step of imaging a cell culture container containing cells as an object to be observed and arranged in the arrangement unit, using an imaging optical system capable of imaging an observation image of the object to be observed; a moving step of moving at least one of the configuration unit and the imaging optical system from a moving start position to a moving end position along the optical axis direction of the imaging optical system by the first moving unit; A photographing step of photographing a plurality of the observation images by a photographing element of the photographing unit during movement in the optical axis direction; An evaluation step of evaluating the brightness value distribution of the plurality of captured images and extracting minimum brightness information associated with the minimum brightness value; A selecting step is to select a focused image based on the plurality of minimum brightness information.

Images