WO2020203700A1

WO2020203700A1 - Device, method, and program for controlling observation

Info

Publication number: WO2020203700A1
Application number: PCT/JP2020/013845
Authority: WO
Inventors: 兼太松原
Original assignee: 富士フイルム株式会社
Priority date: 2019-04-02
Filing date: 2020-03-26
Publication date: 2020-10-08

Abstract

In the present invention, a condition setting unit sets an acquirement condition at which an imaging unit images an observation object contained in a container to acquire an observation image thereof. A determination unit determines, on the basis of an image for determination including the observation object, whether a container corresponding to the set acquirement condition is being used.

Description

Observation controllers, methods and programs

The present disclosure relates to an observation control device, a method, and a program for imaging and observing a container containing an observation object.

Conventionally, pluripotent stem cells such as ES (Embryonic Stem) cells and iPS (Induced Pluripotent Stem) cells and cells induced to differentiate are imaged with an observation device such as a microscope, and cell differentiation is captured by capturing the characteristics of the images. A method for determining a state or the like has been proposed.

Pluripotent stem cells such as ES cells and iPS cells have the ability to differentiate into cells of various tissues, and are attracting attention as being applicable in regenerative medicine, drug development, elucidation of diseases, and the like. There is.

As described above, when a cell is imaged with an observation device, in order to obtain a high-magnification wide-field image, the range of a container such as a culture container such as a well plate is scanned by an imaging optical system, and each observation position is scanned. It has been proposed to perform so-called tyling photography in which images are taken and then the images for each observation position are combined.

On the other hand, many types of containers such as well plates, flasks, petri dishes and dishes are used. As the well plate, well plates having different numbers of wells such as 6 wells, 12 wells, 24 wells, 48 wells and 96 wells are used. Further, regarding the well plate, the center position of the well, the distance between the wells, and the arrangement of the wells on the plate differ depending on the manufacturer of the well plate. In the observation device, the type of container is set in the photographing menu when performing imaging. If the container is a well plate, the number of wells, the manufacturer name, and the like are also set in the shooting menu. By setting the imaging menu in this way, when performing the above-mentioned tiling imaging, imaging is performed at the well position according to the well plate to be used, so that the imaging position and the well position should be matched. Can be done. Further, when the entire well plate is imaged and then only the image of the well position is extracted to generate the observation image, the image of the well position is specified according to the container to be used and the observation image is generated. be able to.

Various methods have been proposed for matching the field of view of the imaging optical system with the position to be imaged on the well plate when imaging the container in this way. For example, in Japanese Patent Application Laid-Open No. 2011-47695, an observation image of the entire container is acquired, and the position of a well in the container is specified by aligning the observation image with a template image created in advance, and the observation in the observation image is performed. It is designed to determine if the area to be located is at the well position in the container. Then, in the method described in Japanese Patent Application Laid-Open No. 2011-47695, if this determination is denied, a warning is issued and the observation image is not recorded.

However, in the method described in Japanese Patent Application Laid-Open No. 2011-47695, when a container different from the container corresponding to the template image is used, the template image and the image obtained by imaging the container do not match. In such a case, it becomes impossible to determine whether or not the area to be observed in the observation image is at the position of the well in the container. Further, in this case, the acquired observation image may be an image that does not include an image of cells in the wells of the container. Since cells change from moment to moment due to culture, a failure in imaging means that an image of the cells cannot be obtained at the required time. Therefore, if the imaging fails, the necessary opportunity to capture the observation image is lost.

The present disclosure has been made in view of the above circumstances, and an object of the present disclosure is to prevent a loss of necessary observation image imaging opportunities.

The observation control device according to the present disclosure includes a condition setting unit for setting acquisition conditions for acquiring an observation image by imaging an observation object housed in a container with an imaging unit.
It is provided with a determination unit that determines whether or not a container corresponding to the set acquisition condition is used based on the determination image including the observation target.

In the observation control device according to the present disclosure, the determination unit may give a warning when the determination is denied.

Further, the observation control device according to the present disclosure further includes an imaging control unit that acquires an observation image by imaging the observation target with the imaging unit based on the set acquisition conditions.
If the judgment is denied, the condition setting unit sets new acquisition conditions corresponding to the container being used, and sets new acquisition conditions.
The image pickup control unit may acquire a new observation image based on the new acquisition condition.

Further, the observation control device according to the present disclosure further includes an imaging control unit that acquires an observation image by imaging the observation target with the imaging unit based on the set acquisition conditions.
If the judgment is denied, the condition setting unit sets new acquisition conditions corresponding to the container being used, and sets new acquisition conditions.
The imaging control unit may acquire both an observation image based on the acquisition conditions and a new observation image based on the new acquisition conditions.

Further, the observation control device according to the present disclosure further includes an imaging control unit that acquires an observation image by imaging the observation target with the imaging unit based on the set acquisition conditions.
The determination image may be an observation image acquired based on the set acquisition conditions.

In this case, the imaging control unit acquires an observation image including only the region where the observation target exists in the container based on the acquisition conditions.
The determination unit determines whether or not the container corresponding to the set acquisition condition is used by determining whether or not the observation target is included in the area where the observation target should exist in the observation image. It may be a thing.

Further, the observation control device according to the present disclosure may further include a determination image acquisition unit that captures an observation target and acquires a determination image.

Further, in the observation control device according to the present disclosure, the determination unit may further determine whether or not the orientation of the container included in the image to be observed is correct.

Further, in the observation control device according to the present disclosure, the culture container may be a well plate.

The observation control method according to the present disclosure includes a step of setting acquisition conditions for acquiring an observation image by imaging an observation target housed in a container with an imaging unit.
It has a step of determining whether or not a container corresponding to the set acquisition condition is used based on the determination image including the observation target.

Note that the observation control method according to the present disclosure may be provided as a program to be executed by a computer.

Other observational control devices according to the present disclosure include a memory for storing instructions to be executed by a computer and a memory.
The processor comprises a processor configured to execute a stored instruction.
The acquisition conditions for acquiring the observation image by imaging the observation target housed in the container with the imaging unit are set.
Based on the determination image including the observation target, the process of determining whether or not the container corresponding to the set acquisition condition is used is executed.

According to the present disclosure, it is possible to prevent the loss of the necessary observation image imaging opportunity.

The figure which shows the schematic structure of the microscope apparatus in the microscope observation system using the observation control apparatus by 1st Embodiment of this disclosure. A block diagram showing the configuration of the observation control device of the first embodiment. The figure which shows the scanning position of the observation position in a culture vessel Diagram showing the condition setting screen The figure for demonstrating the acquisition position of the phase difference image in the well plate of 6 wells. The figure which shows the composite retardation image The figure for demonstrating the acquisition position of the phase difference image when the culture vessel corresponding to the acquisition condition is not used. The figure which shows the synthetic phase difference image when the culture vessel corresponding to the acquisition condition is not used. The figure for demonstrating the acquisition position of the phase difference image when the culture vessel corresponding to the acquisition condition is not used. The figure which shows the synthetic phase difference image when the culture vessel corresponding to the acquisition condition is not used. Diagram showing examples of multiple templates Diagram showing the warning screen A flowchart showing the processing performed in the first embodiment The figure which shows the schematic structure of the microscope apparatus in the microscope observation system using the observation control apparatus by 1st Embodiment of this disclosure. A flowchart showing the processing performed in the second embodiment Diagram showing an example of a well plate with a fixed top and bottom The figure which shows the composite retardation image which the top and bottom are reversed

Hereinafter, a microscope observation system using the embodiment of the observation control device of the present disclosure will be described with reference to the drawings. FIG. 1 is a diagram showing a schematic configuration of a microscope device in a microscope observation system using the observation control device according to the first embodiment of the present disclosure.

The microscope device 10 is described in, for example, Japanese Patent Application Laid-Open No. 2018-054817, and obtains a phase difference image of cultured cells to be observed and combines the phase difference images to generate a composite phase difference image. To do. Specifically, as shown in FIG. 1, the microscope device 10 includes a white light source 11 that emits white light, a condenser lens 12, a slit plate 13, an imaging optical system 14, an imaging optical system driving unit 15, and an imaging element. A 16 and a detection unit 18 are provided. The retardation image and the composite retardation image correspond to the observation image.

The slit plate 13 is provided with a ring-shaped slit that transmits white light to a light-shielding plate that blocks white light emitted from the white light source 11, and is ring-shaped as the white light passes through the slit. Illumination light L is formed.

The imaging optical system 14 includes a phase difference lens and an imaging lens including an objective lens and a phase plate. The imaging optical system 14 is moved in the optical axis direction by the imaging optical system driving unit 15 shown in FIG. In the present embodiment, the optical axis direction and the Z direction (vertical direction) of the imaging optical system 14 are the same directions. Autofocus control is performed by moving the imaging optical system 14 in the Z direction, and the contrast of the phase difference image captured by the image sensor 16 is adjusted.

The imaging optical system drive unit 15 includes an actuator such as a piezoelectric element, and drives the image based on a control signal output from an autofocus control unit 31 (see FIG. 2) described later. The imaging optical system driving unit 15 is configured to pass the phase difference image that has passed through the imaging optical system 14 as it is. Further, the configuration of the imaging optical system driving unit 15 is not limited to the piezoelectric element, as long as the imaging optical system 14 can be moved in the Z direction, and other known configurations can be used.

The image sensor 16 captures a phase difference image imaged by the imaging optical system 14. As the image sensor 16, a CCD (Charge-Coupled Device) image sensor, a CMOS (Complementary Metal-Oxide Semiconductor) image sensor, or the like is used. As the image sensor 16, an image sensor provided with an RGB (Red Green Blue) color filter may be used, or a monochrome image sensor may be used. The image sensor 16 constitutes the image pickup unit.

The detection unit 18 detects the position of the culture vessel 50 installed on the stage 51 in the Z direction (vertical direction). Specifically, the detection unit 18 includes a first displacement sensor 18a and a second displacement sensor 18b. The first displacement sensor 18a and the second displacement sensor 18b are provided side by side in the X direction shown in FIG. 1 with the imaging optical system 14 interposed therebetween. The first displacement sensor 18a and the second displacement sensor 18b in the present embodiment are laser displacement meters, and by irradiating the culture vessel 50 with laser light and detecting the reflected light, the Z on the bottom surface of the culture vessel 50 is detected. Detect the position of the direction. The bottom surface of the culture vessel 50 is a boundary surface between the bottom portion of the culture vessel 50 and the cells to be observed, that is, an observation target installation surface.

The position information indicating the position of the culture vessel 50 in the Z direction detected by the detection unit 18 is output to the autofocus control unit 31. The autofocus control unit 31 controls the imaging optical system drive unit 15 based on the input position information, and performs autofocus control.

A stage 51 is provided between the slit plate 13, the imaging optical system 14, and the detection unit 18. A culture vessel 50 containing cells to be observed is installed on the stage 51. The culture vessel 50 is aligned with a predetermined position on the stage 51 and installed on the stage 51.

As the culture container 50, a petri dish, a dish, a well plate, or the like can be used. The cells contained in the culture vessel 50 include pluripotent stem cells such as iPS cells and ES cells, nerves, skin, myocardial and liver cells induced to differentiate from stem cells, and skin and retina extracted from the human body. There are cells of myocardium, blood cells, nerves and organs.

The stage 51 is moved in the X and Y directions orthogonal to each other by the horizontal drive unit 17 (see FIG. 2) described later. The X and Y directions are orthogonal to the Z direction and are orthogonal to each other in the horizontal plane. In the present embodiment, the X direction is the main scanning direction and the Y direction is the sub scanning direction. At the center of the stage 51, a rectangular opening 51a shown by a line in FIG. 1 is formed. The culture vessel 50 is installed on the member forming the opening 51a, and the illumination light L transmitted through the cells in the culture vessel 50 is configured to pass through the opening 51a.

Next, the configuration of the observation control device 20 that controls the microscope device 10 will be described. FIG. 2 is a block diagram showing the configuration of the observation control device of the first embodiment. Regarding the microscope device 10, a block diagram of a part of the configuration controlled by each part of the observation control device 20 is shown.

The observation control device 20 controls the entire microscope device 10 in the present embodiment, and is realized by installing the observation control program according to the first embodiment on the computer. As shown in FIG. 2, the observation control device 20 includes a CPU (Central Processing Unit) 21, a memory 22, and a storage 23 as a standard workstation configuration. Further, the observation control device 20 is connected to a display unit 24 such as a liquid crystal display and an input unit 25 such as a keyboard and a mouse.

The storage 23 is composed of a hard disk drive or the like, and stores various information including information necessary for processing performed by the observation control device 20 according to the present embodiment.

Further, the memory 22 stores the observation control program according to the present embodiment. The observation control program defines autofocus control processing, scanning control processing, imaging control processing, condition setting processing, determination processing, and display control processing as processing to be executed by the CPU 21. Then, when the CPU 21 executes these processes according to the program, the computer functions as an autofocus control unit 31, a scanning control unit 32, an imaging control unit 33, a condition setting unit 34, a determination unit 35, and a display control unit 36. The observation control device 20 may be configured only by the condition setting unit 34 and the determination unit 35. Further, the observation control device 20 may be configured only by the image pickup control unit 33, the condition setting unit 34, and the determination unit 35.

As will be described later, the display unit 24 displays a setting screen for setting acquisition conditions for the phase difference image, a generated composite phase difference image, and the like, and includes, for example, a liquid crystal display. Further, the display unit 24 may be configured by a touch panel and may also be used as the input unit 25.

The input unit 25 is provided with a mouse, a keyboard, and the like, and accepts various setting inputs by the operator. The input unit 25 of the present embodiment accepts setting inputs such as image acquisition conditions.

The autofocus control unit 31 controls the imaging optical system drive unit 15 based on the position information in the Z direction of the culture vessel 50 detected by the detection unit 18 as described above. Then, the imaging optical system 14 moves in the optical axis direction by driving the imaging optical system driving unit 15, and autofocus control is performed.

The scanning control unit 32 drives and controls the horizontal drive unit 17, thereby moving the stage 51 in the X direction and the Y direction. The horizontal drive unit 17 is composed of an actuator having a piezoelectric element or the like.

In the present embodiment, for example, by the method described in Japanese Patent Application Laid-Open No. 2018-054817, the stage 51 is moved in the X and Y directions under the control of the scanning control unit 32, and the imaging optical system 14 is placed in the culture vessel 50. Scans in a two-dimensional manner and captures a phase difference image of each observation position by the imaging optical system 14. FIG. 3 is a diagram showing the scanning position of the observation position in the culture vessel 50 by the solid line M. In this embodiment, a well plate having 6 wells W is used as the culture container 50.

As shown in FIG. 3, the imaging optical system 14 moves along the solid line M from the scanning start point S to the scanning end point E. That is, the observation position on the culture vessel 50 by the imaging optical system 14 is scanned in the positive direction (right direction in FIG. 3) in the X direction, then moves in the Y direction (downward direction in FIG. 3), and vice versa. It is scanned in the negative direction (leftward in FIG. 3). The observation position then moves again in the Y direction and is scanned again in the positive direction. In this way, the imaging optical system 14 scans the inside of the culture vessel 50 in a two-dimensional manner by repeatedly moving back and forth in the X direction and moving in the Y direction. At this time, the observation position R of the culture vessel 50 is preceded by the imaging optical system 14 in the moving direction (that is, the main scanning direction) by the first displacement sensor 18a and the second displacement sensor 18b according to the moving direction. The position in the Z direction is detected. Then, when the observation position R moves to the position of the imaging optical system 14, the autofocus control unit 31 performs autofocus control using the position information of the culture vessel 50 detected in advance in the Z direction. , The culture vessel 50 is imaged and a phase difference image is acquired.

Returning to FIG. 2, the image pickup control unit 33 controls to take an image of the culture vessel 50 by the image pickup element 16 and acquire a phase difference image at each observation position by the imaging optical system 14 in the movement of the stage 51. The image sensor 16 is controlled by the image pickup control unit 33 based on the phase difference image acquisition conditions set by the condition setting unit 34.

The condition setting unit 34 sets the acquisition conditions when acquiring the phase difference image. Specifically, an input of the type of the culture container 50 to be used by the operator is received from the input unit 25, and the acquisition condition is set based on the input type of the culture container 50. Therefore, the display control unit 36 displays a condition setting screen for setting the acquisition condition on the display unit 24. FIG. 4 is a diagram showing a condition setting screen. As shown in FIG. 4, the condition setting screen 60 displays a plurality of commands for instructing the type of the culture vessel 50 to be used. For example, commands 61A-61C for selecting well plates, flasks and petri dishes as the type of culture vessel 50 are displayed. Also, for well plates in particular, commands 62A-62C for selecting well plate manufacturers (Company A, Company B and Company C) are displayed. FIG. 4 shows a state in which Company A is selected as the manufacturer. Therefore, in FIG. 4, for Company A, commands 63A-63D for selecting 6-well, 12-well, 24-well, 48-well, and 96-well are displayed.

The operator inputs the type of culture vessel to be used by selecting commands for the type of culture vessel, the manufacturer, and the number of wells using the input unit 25.

The condition setting unit 34 sets the acquisition condition for acquiring the phase difference image based on the input of the operator. In the present embodiment, as shown in FIG. 3, a phase difference image is acquired while operating the culture vessel 50 two-dimensionally. When acquiring the phase difference image in this way, in the present embodiment, the image sensor 16 is driven only at the position corresponding to the well W in the culture container 50 to image the culture container 50 and acquire the phase difference image. The image pickup control unit 33 controls the image pickup so as to be performed. FIG. 5 is a diagram for explaining the acquisition position of the phase difference image in the 6-well well plate. In the present embodiment, it is assumed that 3 × 3 = 9 phase difference images are acquired as the number of fields of view in each well. The small squares in FIG. 5 indicate the imaging field of view at the observation position where the phase difference image can be acquired during the two-dimensional scanning of the culture vessel 50.

In the present embodiment, since 3 × 3 retardation images are acquired in each well W, the culture vessel 50 is imaged at the position where the diagonal line is given as shown in FIG. 5 to acquire the retardation image. As described above, the image pickup control unit 33 controls the image pickup element 16. In the present embodiment, the culture vessel 50 is positioned and installed at a predetermined position on the stage 51. Therefore, the culture vessel 50 is installed on the stage 51, and at least one of the position to be imaged after the start of scanning, the imaging timing, the size and magnification of the imaging field of view at the position to be imaged, and the like are set as acquisition conditions. By doing so, the culture vessel 50 can be imaged and a phase difference image can be obtained at the position where the diagonal line shown in FIG. 5 is provided.

Therefore, in the present embodiment, the storage 23 stores a table in which various culture containers are associated with acquisition conditions including the position to be imaged according to the culture container, the imaging timing, the size and magnification of the imaging field of view, and the like. ing. The condition setting unit 34 sets the acquisition conditions with reference to the table according to the type of the culture vessel 50 input from the input unit 25 by the operator. The set acquisition conditions are output to the imaging control unit 33. The image pickup control unit 33 acquires a phase difference image by controlling the image pickup device 16 and the like based on the input acquisition conditions to image the culture vessel 50.

Here, in the culture vessel 50, the position where the cells to be observed are present differs depending on the type. For example, when the culture vessel 50 is a well plate, the position where the cells are present differs depending on the number of wells. Further, even if the number of wells is the same, the size of the plate, the center position and size of each well differ depending on the manufacturer of the well plate, and therefore, the position where the cells exist in the well plate differs depending on the manufacturer. Therefore, when the position to be imaged, the imaging timing, and the like are set as the acquisition conditions as in the present embodiment, if the operator mistakenly inputs the type of the culture container 50 to be used, the culture container 50 to be used and the acquisition conditions are input. Will not match. If the culture vessel 50 used and the acquisition conditions do not match in this way, imaging will not be performed at the position where the cells exist in the culture vessel 50 used, and as a result, a phase difference image including the cells necessary for observation will not be performed. Will not be acquired. That is, as shown in FIG. 5, when a 6-well well plate of a certain manufacturer is used as the culture container 50, if it is input as the culture container 50 to be used, acquisition according to the type of the culture container 50 to be used. Since the conditions are set, as shown by the diagonal line in FIG. 5, a phase difference image having a number of fields of view of 3 × 3 is acquired in each well W. However, if the number of well plates is mistaken for, for example, 12 wells and 96 wells, the phase difference image will not be acquired at the position of the shaded line shown in FIG.

In the present embodiment, the determination unit 35 determines whether or not the culture vessel 50 corresponding to the acquisition conditions set by the condition setting unit 34 is used based on the determination image including the cells to be observed. Do. Therefore, in the present embodiment, the culture vessel 50 is first imaged to generate a synthetic retardation image. That is, one composite retardation image is obtained by scanning the culture vessel 50, acquiring the retardation image at the observation position where the retardation image should be acquired in the culture vessel 50, and combining the acquired retardation images. Generate. The display control unit 36 generates the composite retardation image.

The determination unit 35 uses the composite retardation image generated by the display control unit 36 as the determination image to perform the above determination. Here, when the culture vessel 50 corresponding to the acquisition condition set by the condition setting unit 34 is used, the phase difference image is acquired at the position of the shaded line shown in FIG. Therefore, as shown in FIG. 6, the synthetic retardation image 70 includes an image of cells at the position of each well W of the culture vessel 50. In the composite retardation image 70, since the image of the culture vessel 50 is not included in the region other than the region including the retardation image, the composite retardation image 70 has a predetermined density (white, black, gray, etc.). There is. Further, in FIG. 6, for the sake of explanation, the edges of the six wells W contained in the culture vessel 50 are shown by broken lines.

On the other hand, when the culture vessel 50 corresponding to the acquisition condition set by the condition setting unit 34 is not used, for example, when the culture vessel 50 used is a 6-well well plate of company A, the operator makes a mistake. When input as a culture vessel 50 using a 12-well well plate of company A, as shown in FIG. 7, the position of the well of the culture vessel 50 used and the position where the phase difference image shown by the diagonal line is acquired. Does not match. As a result, as shown in FIG. 8, the synthetic retardation image 71 does not include the image of the cells in the well W that should be originally included. Also in FIG. 8, for the sake of explanation, the edges of the six wells contained in the culture vessel 50 are shown by broken lines.

Further, when the culture vessel 50 used is a 6-well well plate of company A and the operator mistakenly inputs it as the culture vessel 50 using the 6-well well plate of company B, FIG. 9 shows. As shown, the position of the well of the culture vessel 50 used and the position where the phase difference image was acquired do not match. As a result, as shown in FIG. 10, the synthetic retardation image 72 does not include the image of the cells in the well W that should be originally included. Also in FIG. 10, for the sake of explanation, the edges of the six wells contained in the culture vessel 50 are shown by broken lines.

The determination unit 35 analyzes the generated synthetic retardation image and determines whether or not the culture vessel 50 corresponding to the acquisition conditions set by the condition setting unit 34 is used. When the culture vessel 50 corresponding to the set acquisition conditions is used as in the synthetic retardation image 70 shown in FIG. 6, the edge of the well W is shown in the image of the cells included in the synthetic retardation image 70. Not included. On the other hand, when the culture vessel 50 corresponding to the set acquisition conditions is not used as in the

synthetic retardation images

71 and 72 shown in FIGS. 8 and 10, the image of the cells contained in the

synthetic retardation images

71 and 72 Includes the edge of the well W.

Here, the characteristics of the culture vessel 50 such as the position and curvature of the edge of the well W differ depending on the manufacturer and the number of wells. For example, even in the same manufacturer, the position and curvature of the edge of the well W are different between the 6-well well plate and the 12-well well plate. Further, when the manufacturers are different, the positions of the edges of the wells W are different even in the same 6-well well plate. In the present embodiment, the type of culture vessel, the manufacturer, and the number of wells of the culture vessel 50 that are expected to be used are imaged in advance to generate synthetic retardation images corresponding to various culture vessels. The generated synthetic retardation image can recognize the characteristics of the culture vessel 50 such as the position and curvature of the edge of the well in the various culture vessels 50. In the first embodiment, a plurality of composite retardation images generated in advance are stored in the storage 23 as templates. FIG. 11 is a diagram showing an example of a plurality of templates T0.

The determination unit 35 first uses the acquired composite retardation image as the determination image, and the culture container 50 corresponding to the set acquisition conditions is used depending on whether or not the edge of the well W is included. Determine if it has been done. That is, when the composite retardation image as the determination image does not include the edge portion of the well W, the determination unit 35 determines that the culture container 50 corresponding to the set acquisition condition is used. For example, when the composite retardation image 70 shown in FIG. 6 is acquired, the composite retardation image 70 does not include the edge of the well W. In this case, the determination unit 35 determines that the culture vessel 50 that matches the acquisition conditions set by the condition setting unit 34 has been used.

On the other hand, when the composite retardation image includes the edge of the well W, it is determined that the culture vessel 50 corresponding to the set acquisition condition is not used. In this case, the determination unit 35 compares each of the templates stored in the storage 23 with the position of the edge of the well W included in the composite retardation image, and further determines which culture container 50 was used. To do.

For example, when the composite retardation image 71 as shown in FIG. 8 is acquired, the composite retardation image 71 includes the edge of the well W. Therefore, the determination unit 35 determines that the culture vessel 50 corresponding to the set acquisition condition is not used. Further, the determination unit 35 compares the position of the edge portion of the well W in the synthetic retardation image 71 with the plurality of templates T0 stored in the storage 23, and determines the culture vessel 50 used. In this case, the determination unit 35 determines that the culture vessel 50 used is a 12-well well plate of company A.

Further, when the composite retardation image 72 as shown in FIG. 10 is acquired, the composite retardation image 72 includes the edge of the well W. Therefore, the determination unit 35 determines that the culture vessel 50 corresponding to the set acquisition condition is not used. Further, the determination unit 35 compares the position of the edge portion of the well W in the synthetic retardation image 72 with the plurality of templates T0 stored in the storage 23, and determines the culture vessel 50 used. In this case, the determination unit 35 determines that the culture vessel 50 used is a 6-well well plate of Company B.

In the storage 23, instead of the template T0, the coordinates and curvature of the position of the edge of the well on the synthetic retardation image corresponding to the various culture containers 50 are stored as the characteristics of the culture container 50. You may. In this case, when the composite retardation image includes the edge of the well, the determination unit 35 determines the position and curvature of the edge of the well and the edge of the well in the various culture vessels 50 stored in the storage 23. Which culture container 50 was used may be determined by comparing the position and curvature of the culture vessel 50.

Further, when the culture vessel 50 corresponding to the set acquisition condition is not used, the determination unit 35 outputs information to that effect to the condition setting unit 34. The condition setting unit 34 sets new acquisition conditions according to the culture vessel 50 used, and outputs the new setting conditions to the imaging control unit 33. The image pickup control unit 33 takes an image of the culture vessel 50 based on the input new setting conditions. As a result, a new phase difference image is acquired based on the new acquisition conditions according to the culture vessel 50 used, and the display control unit 36 obtains a new composite retardation image based on the new phase difference image. Generate.

Further, the determination unit 35 warns when it is determined that the culture vessel 50 corresponding to the set acquisition condition is not used. As an aspect of the warning, a text indicating that the type of the culture container 50 input by the operator is different from the type of the culture container 50 used is displayed on the display unit 24, or an alarm sound or the like is emitted. It may be a thing. Further, the composite retardation image generated based on the acquisition conditions set according to the input of the operator and the new composite retardation image generated based on the new acquisition conditions may be displayed on the display unit 24. FIG. 12 is a diagram showing a warning screen displayed on the display unit 24. As shown in FIG. 12, on the warning screen 80, the text 81 of "The input culture vessel is different", the synthetic phase difference image 82 generated based on the acquisition conditions set according to the input of the operator, and A new composite retardation image 83 generated based on the new acquisition conditions is displayed. Further, a text indicating that the culture container input by the operator and the culture container used may be different may be sent to the operator's e-mail address by e-mail or the like. In this case, the synthetic retardation image 82 based on the acquisition conditions set based on the type of the culture vessel 50 input by the operator and the new synthetic retardation image 83 based on the new acquisition conditions are also sent by e-mail or the like. It may be attached and sent to the operator.

Next, the process performed in the first embodiment will be described. FIG. 13 is a flowchart showing the processing performed in the first embodiment. First, the culture vessel 50 containing the cells to be observed is placed on the stage 51 (step ST1). Next, the input unit 25 accepts the input of the type of the culture container 50 to be used by the operator, so that the condition setting unit 34 sets the acquisition conditions (step ST2). Next, while the autofocus control unit 31 performs autofocus control and the scanning control unit 32 scans the stage 51, the image pickup control unit 33 images the culture vessel 50 with the image pickup element 16 to acquire a phase difference image. (Step ST3). Further, the display control unit 36 generates a composite retardation image (step ST4).

Next, the determination unit 35 uses the composite retardation image as the determination image to determine whether or not the culture vessel 50 corresponding to the set acquisition conditions is used (step ST5). When step ST5 is affirmed, the composite retardation image generated based on the set acquisition conditions is saved in the storage 23 (step ST6), and the process ends.

When step ST5 is denied, the condition setting unit 34 sets a new acquisition condition corresponding to the culture vessel 50 used (step ST7), and the image pickup control unit 33 newly sets a new acquisition condition based on the new acquisition condition. Acquire a phase difference image (step ST8). Then, the display control unit 36 generates a new composite retardation image (step ST9). Further, the determination unit 35 issues a warning (step ST10) and ends the process.

As described above, in the present embodiment, the synthetic retardation image generated by imaging the culture vessel 50 is used as the determination image, and the culture vessel corresponding to the set acquisition conditions is used based on the determination image. It is determined whether or not 50 is used. Further, in the present embodiment, a warning is given based on the determination result, and a new acquisition condition corresponding to the culture vessel 50 used is set. As a result, the operator can reset the acquisition conditions or acquire the image to be observed based on the new acquisition conditions. Therefore, according to the present embodiment, it is possible to eliminate the failure of imaging, and as a result, it is possible to prevent the loss of the imaging opportunity of the necessary observation target cell.

Further, in the present embodiment, when the determination as to whether or not the culture container corresponding to the set acquisition condition is used is denied, a new acquisition condition corresponding to the culture container 50 used is set. , A composite phase difference image is generated based on the set acquisition conditions. Therefore, the operator can acquire a synthetic phase difference image appropriately including an image of cells without performing the work of re-inputting the correct type of the culture vessel 50. Therefore, according to the present embodiment, the work load of the operator can be reduced.

Next, the second embodiment of the present disclosure will be described. FIG. 14 is a diagram showing a schematic configuration of a microscope device in a microscope observation system using the observation control device according to the second embodiment of the present disclosure. In FIG. 14, the same reference numbers are assigned to the same configurations as those in FIG. 1, and detailed description thereof will be omitted. As shown in FIG. 14, the microscope observation system of the second embodiment is provided with a camera 90 for capturing an image of the culture vessel 50 installed on the stage 51 and acquiring a determination image. Different from. The image pickup of the culture vessel 50 by the camera 90 is performed by the image pickup control unit 33. Further, the camera 90 corresponds to the determination image acquisition unit.

In the second embodiment, the image pickup control unit 33 takes an image of the culture vessel 50 with the camera 90 and acquires the determination image 91 before taking the phase difference image. The determination unit 35 determines whether or not the culture vessel 50 corresponding to the set acquisition conditions is used based on the determination image 91. For this purpose, in the second embodiment, the culture container 50 of the manufacturer and the number of wells expected to be used is imaged in advance by the camera 90 to acquire the determination image 91 corresponding to the various culture containers. The acquired determination image 91 is capable of recognizing the characteristics of the culture vessel 50 such as the position and curvature of the edge of the well in the various culture vessels 50. In the second embodiment, a plurality of determination images 91 acquired in advance are stored in the storage 23 as templates.

In the second embodiment, the determination unit 35 acquires the template of the culture container 50 corresponding to the acquisition condition from the storage 23 as a comparison template. Then, the determination unit 35 matches the determination image 91 of the culture vessel 50 installed in the stage 51 with the comparison template to determine whether or not the culture vessel 50 corresponding to the set acquisition conditions is used. To judge. That is, when the determination image 91 and the comparison template match, the determination unit 35 determines that the culture container 50 corresponding to the set acquisition conditions is used. The determination of whether or not the determination image 91 and the comparison template match is determined by determining whether or not the correlation between the determination image 91 and the comparison template is equal to or greater than a predetermined threshold value. It may be done by.

On the other hand, if the determination image 91 and the comparison template do not match, the determination unit 35 determines that the culture vessel 50 corresponding to the set acquisition conditions is not used. In this case, the determination unit 35 matches each of the templates other than the comparison template stored in the storage 23 with the determination image 91, and determines the culture container 50 used. In this case, the culture container 50 corresponding to the template that most closely matches the determination image 91 may be determined to be the culture container 50 in use.

If it is determined that the culture vessel 50 of the type corresponding to the set acquisition condition is not used, the determination unit 35 outputs information to that effect to the condition setting unit 34. The condition setting unit 34 sets new acquisition conditions according to the culture vessel 50 used, as in the first embodiment.

First, the imaging control unit 33 images the culture vessel 50 based on the acquisition conditions set based on the input of the operator to acquire a phase difference image, and the display control unit 36 further generates a composite phase difference image. This retardation image is referred to as a first composite retardation image. On the other hand, the imaging control unit 33 images the culture vessel 50 based on the new setting conditions to acquire a new phase difference image, and the display control unit 36 further generates a new composite retardation image. This new composite retardation image is referred to as a second composite retardation image.

When the determination unit 35 determines that the culture vessel 50 corresponding to the set acquisition condition is not used, the determination unit 35 issues a warning as in the first embodiment. In this case, both the first composite retardation image and the second composite retardation image may be displayed on the warning screen.

Next, the process performed in the second embodiment will be described. FIG. 15 is a flowchart showing the processing performed in the second embodiment. First, the culture vessel 50 containing the cells to be observed is placed on the stage 51 (step ST21). Next, the condition setting unit 34 sets the acquisition conditions by receiving the input of the type of the culture container 50 to be used by the operator from the input unit 25 (step ST22). Then, the camera 90 images the culture container 50 and acquires the determination image 91 (step ST23).

Next, the determination unit 35 determines whether or not the culture vessel 50 corresponding to the set acquisition conditions is used based on the determination image 91 (step ST24). When step ST24 is affirmed, the imaging control unit 33 acquires a phase difference image based on the set acquisition conditions (step ST25), and the display control unit 36 generates a composite retardation image (step ST26). Then, the determination unit 35 saves the composite retardation image generated based on the set acquisition conditions in the storage 23 (step ST27), and ends the process.

When step ST24 is denied, the display control unit 36 generates a composite retardation image based on the set acquisition conditions as the first composite retardation image (step ST28). The process of step ST28 is the same as the process of steps ST25 to ST27. Further, the condition setting unit 34 sets a new acquisition condition corresponding to the culture vessel 50 used (step ST29), and the imaging control unit 33 acquires a new phase difference image based on the new acquisition condition. (Step ST30). Then, the display control unit 36 generates a second composite retardation image, which is a new composite retardation image (step ST31). Further, the determination unit 35 issues a warning (step ST32) and ends the process.

Note that some culture containers 50 have a fixed top and bottom. For example, in the 6-well well plate 52 shown in FIG. 16, the numbers 1 to 6 are engraved below the well W, and the 6 well Ws are unevenly arranged on the upper side of the well plate 52. When such a well plate 52 is placed on the stage 51 with the top and bottom turned upside down to generate a composite retardation image, as shown in FIG. 17, the composite retardation image 73 includes an image of originally necessary cells. It will not be possible. Therefore, in the first and second embodiments, it may be further determined whether or not the orientation of the culture vessel 50 is correct.

In the determination of whether or not the orientation of the culture vessel 50 is correct, first, as in the first and second embodiments, the culture vessel 50 corresponding to the set acquisition conditions is determined by using the template stored in the storage 23. Determine if is used. If the orientation of the culture vessel 50 is incorrect, it will not match any of the templates. Therefore, the determination unit 35 further reverses the top and bottom of the determination image to match with the template, and determines the culture container 50 used. It should be noted that the top and bottom of the template may be reversed to match with the composite phase difference image or the determination image. In this case, a warning may be given that the culture container 50 used is upside down.

When it is determined whether or not the orientation of the culture vessel 50 is correct as described above, it is possible to determine even if the culture vessel 50 corresponding to the set acquisition conditions and the culture vessel 50 used are different. .. When the culture container 50 corresponding to the acquisition conditions set in this way and the culture container 50 used are different, the same treatment as in the first and second embodiments may be performed.

Further, in the first embodiment, when the culture vessel 50 corresponding to the acquisition condition is not used, a new acquisition condition is set and a new synthetic retardation image is acquired, but the present invention is limited to this. It is not something that is done. It is possible to give only a warning without acquiring a new composite retardation image. In this case, the warning screen displays only the generated composite retardation image and the text for warning. It should be noted that the generated composite retardation image may not be displayed, and only the text for giving a warning may be displayed. In addition, the text for giving a warning and the text of the set acquisition condition may be displayed, and in addition to the text for giving a warning and the text of the set acquisition condition, the text of the new acquisition condition May be displayed. In this case, the operator can redo the imaging of the culture vessel 50 according to the warning. Therefore, loss of imaging opportunity can be prevented.

Further, in the second embodiment, when the culture vessel 50 corresponding to the acquisition condition is not used, the first synthetic retardation image corresponding to the acquisition condition and the second synthetic retardation image corresponding to the new acquisition condition are used. A composite retardation image is generated, but the present invention is not limited to this. Only one of the first composite retardation image and the second composite retardation image may be acquired. In this case, the warning screen displays only the first composite retardation image or the second composite retardation image and the text for warning.

Further, in each of the above embodiments, the well plate is used as the culture container 50, but the present disclosure can also be applied when a culture container 50 other than the well plate such as a flask and a petri dish is used. In this case, templates such as flasks and petri dishes that are expected to be used may be stored in the storage 23, and the determination unit 35 may perform the above determination by comparing the determination image with the template. ..

Further, in each of the above embodiments, the image sensor 16 is driven to acquire the phase difference image only at the position corresponding to the well W in the culture vessel 50 based on the acquisition conditions, but the present invention is limited to this. is not. A phase difference image may be acquired in the entire region of the culture vessel 50, and only the phase difference image at a position determined based on the acquisition conditions may be extracted to generate a composite phase difference image.

Further, in each of the above embodiments, the observation position in the culture vessel 50 is scanned by moving the stage 51, but the present invention is not limited to this. The image pickup system including the image pickup optical system 14 and the image pickup element 16 may be moved. Further, both the stage 51 and the imaging system may be moved.

Further, although each of the above embodiments applies the present disclosure to a phase-contrast microscope, the present disclosure is not limited to a phase-contrast microscope, but is applied to other microscopes such as a differential interference microscope and a bright-field microscope. You may.

Further, in the second embodiment, the phase difference image formed by the imaging optical system 14 is imaged by the image pickup device 16, but the imaging optical system 14 does not provide the image pickup element 16. An observation optical system or the like may be provided so that the operator can directly observe the imaged phase difference image of the observation target.

Further, in each of the above embodiments, for example, a processing unit (Processing) that executes various processes such as an autofocus control unit 31, a scanning control unit 32, an imaging control unit 33, a condition setting unit 34, a determination unit 35, and a display control unit 36. As the hardware structure of the Unit), various processors (Processors) shown below can be used. As described above, the various processors include a CPU, which is a general-purpose processor that executes software (program) and functions as various processing units, and a circuit after manufacturing an FPGA (Field Programmable Gate Array) or the like. Dedicated electricity, which is a processor with a circuit configuration specially designed to execute specific processing such as programmable logic device (PLD), ASIC (Application Specific Integrated Circuit), which is a processor whose configuration can be changed. Circuits and the like are included.

One processing unit may be composed of one of these various processors, or a combination of two or more processors of the same type or different types (for example, a combination of a plurality of FPGAs or a combination of a CPU and an FPGA). ) May be configured. Further, a plurality of processing units may be configured by one processor.

As an example of configuring a plurality of processing units with one processor, first, as represented by a computer such as a client and a server, one processor is configured by combining one or more CPUs and software. There is a form in which this processor functions as a plurality of processing units. Second, as typified by System On Chip (SoC), there is a form that uses a processor that realizes the functions of the entire system including multiple processing units with a single IC (Integrated Circuit) chip. is there. As described above, the various processing units are configured by using one or more of the various processors as a hardware structure.

Further, as the hardware structure of these various processors, more specifically, an electric circuit (Circuitry) in which circuit elements such as semiconductor elements are combined can be used.

10 Microscope device 11 White light source 12 Condenser lens 13 Slit plate 14 Imaging optical system 15 Imaging optical system drive unit 16 Imaging element 17 Horizontal drive unit 18 Detection unit 20 Observation control device 21 CPU
22 Memory 23 Storage 24 Display 25 Input 31 Autofocus control 32 Scanning control 33 Imaging control 34 Condition setting 35 Judgment 36 Display control 50 Culture container 51 Stage 51a Aperture 60 Condition setting screen 61A to 61C, 62A ~ 62C, 63A ~ 63D Command 70-73, 82-83 Composite phase difference image 80 Warning screen 81 Text 90 Camera 91 Judgment image E Scanning end point L Illumination light M Solid line S Scanning start point T0 Template W

Claims

A condition setting unit that sets acquisition conditions for acquiring an observation image by imaging the observation target housed in the container with the imaging unit, and
An observation control device including a determination unit that determines whether or not the container corresponding to the set acquisition condition is used based on the determination image including the observation target.
The observation control device according to claim 1, wherein the determination unit gives a warning when the determination is denied.
An imaging control unit that acquires the observation image by imaging the observation target with the imaging unit based on the set acquisition conditions is further provided.
If the determination is denied, the condition setting unit sets a new acquisition condition corresponding to the container used.
The observation control device according to claim 1 or 2, wherein the imaging control unit acquires a new observation image based on the new acquisition condition.
An imaging control unit that acquires the observation image by imaging the observation target with the imaging unit based on the set acquisition conditions is further provided.
If the determination is denied, the condition setting unit sets a new acquisition condition corresponding to the container used.
The observation control device according to claim 1 or 2, wherein the imaging control unit acquires both the observation image based on the acquisition conditions and the new observation image based on the new acquisition conditions.
An imaging control unit that acquires the observation image by imaging the observation target with the imaging unit based on the set acquisition conditions is further provided.
The observation control device according to any one of claims 1 to 4, wherein the determination image is the observation image acquired based on the set acquisition conditions.
Based on the acquisition conditions, the imaging control unit acquires the observation image including only the region in which the observation target exists in the container.
By determining whether or not the observation target is included in the region where the observation target should exist in the observation image, the determination unit determines whether the container corresponding to the set acquisition condition is used. The observation control device according to claim 5, wherein it determines whether or not.
The observation control device according to any one of claims 1 to 4, further comprising a determination image acquisition unit that captures the observation target and acquires the determination image.
The observation control device according to any one of claims 1 to 7, wherein the determination unit further determines whether or not the orientation of the container included in the image to be observed is correct.
The observation control device according to any one of claims 1 to 8, wherein the container is a well plate.
A process of setting acquisition conditions for acquiring an observation image by imaging an observation target housed in a container with an imaging unit, and
An observation control method including a step of determining whether or not the container corresponding to the set acquisition condition is used based on the determination image including the observation target.
The procedure for setting the acquisition conditions for acquiring the observation image by imaging the observation target contained in the container with the imaging unit, and
An observation control program that causes a computer to execute a procedure for determining whether or not the container corresponding to the set acquisition condition is used based on the determination image including the observation target.