JP2021157153A - Cell observation device and cell observation method - Google Patents

Abstract

To improve accuracy of a determination of a defective or non-defective cell, or a count of the number of cells.SOLUTION: A cell observation device 1 is provided with an objective lens 13 on a lower side of a stage 3, in which the objective lens 13 can ascend and descend by an elevation mechanism 14. As descending the objective lens 13 from a rise end to a descent end due to the elevation mechanism 14 by a given amount (a dimension), a cell C in a container 4 is photographed at each height position by imaging means 6. Then, control means 7 is configured to: obtain a length of an outer periphery of the cell C at each height position and a total sum value of the lengths thereof; and determine the height position where the total sum value is a minimum value as an in-focus position of the objective lens 13. Thereafter, the objective lens 13 is positioned at the height position obtained as the in-focus position by the elevation mechanism 14, and then, the cell C in the container 4 is photographed by the imaging means 6. As to the photographed cell C of the image, the control means 7 is configured to make a determination of a non-defective or defective cell, or count the number of cells C.SELECTED DRAWING: Figure 1

Description

The present invention relates to a cell observation device and a cell observation method, and more particularly to a cell observation device and a cell observation method capable of improving the quality determination of cells to be observed and the accuracy of cell counting.

Conventionally, when observing cells with an image observation device such as a microscope, the cells are imaged by an imaging means such as a digital camera, and the image of the captured cells is image-processed to determine the contrast, thereby autofocusing. A device that operates is known (for example, Patent Document 1).

Japanese Unexamined Patent Publication No. 2008-20298

However, when observing cells or counting cells using a device that performs an autofocus operation for determining contrast as described above, the focus position of the imaging means is the position that the operator visually focuses on. In some cases, the cells were misaligned, resulting in errors in cell quality judgment and cell counting. Therefore, the automation of cell culture has not progressed, which has hindered the spread of regenerative medicine as a factor that causes the cost of regenerative medicine products to increase, and there has been a demand for a cell observation device and a cell observation method that can set an appropriate focus position. It is a thing.

In view of the above circumstances, the present invention according to claim 1 comprises a stage on which a container containing cells is placed, an imaging means for imaging cells in the container, a height position of the stage, or the imaging. It is provided with an elevating means for changing the height position of the focal point of the means and a control means for detecting cells in the image captured by the imaging means and obtaining a total value of calculated values for the detected cells.
While the height position of the stage or the height position of the focal point of the imaging means is changed by the elevating means, the cells in the container are imaged by the imaging means at each predetermined height position.
The control means calculates the total value of the calculated values for the cells in the image of each height position imaged by the image pickup means, and determines the position where the total value becomes the minimum value as the in-focus position of the image pickup means. ,
It is characterized in that the container placed on the stage is positioned at the in-focus position by the elevating means.
Further, according to the third aspect of the present invention, there is a stage in which a container containing cells is placed, an imaging means for imaging cells in the container, and a height position of the stage or a focus of the imaging means. The elevating means for changing the height position and the cell in the image captured by the imaging means are detected, and the length of the outer circumference of the detected cell, the diameter of the approximate circle of the outer circumference of the cell, the diameter of the circumscribed circle of the cell, Alternatively, it is equipped with a control means for calculating any of the diameters of the inscribed circles of the cell.
The focus of the imaging means is the container based on any of the length of the outer circumference of the cell, the diameter of the approximate circle of the outer circumference of the cell, the diameter of the circumscribed circle of the cell, or the diameter of the inscribed circle of the cell calculated by the control means. The focus position is determined according to the height position of the cells inside.
The elevating means adjusts the container placed on the stage to a determined focusing position.
Further, in the present invention according to claim 4, the height position of the container in which the cells are housed or the height position of the focal point of the imaging means for imaging the container is changed, and the imaging means is used for each predetermined height position. Get an image of the cells in the container,
A cell is detected in the acquired image of each cell, and the outer circumference length of the detected cell, the diameter of the approximate circle of the outer circumference of the cell, the diameter of the circumscribed circle of the cell, the diameter of the inscribed circle of the cell, or the diameter of the cell Calculate the total value of any of the areas and
It is characterized in that the height position where the total value becomes the minimum value is determined as the in-focus position where the focus of the imaging means matches the position of the cells in the container.

According to such a configuration, it is possible to provide a cell observation device and a cell observation method having an appropriate autofocus function, and it is possible to improve the accuracy of cell quality determination and cell counting.

The vertical sectional view which shows one Example of this invention. The graph which shows the total value of the calculated value about a cell in the image of the cell imaged by the imaging means at the different height positions of the Z axis (vertical direction) of FIG. It is a figure which shows the relationship between the image of a cell photographed by the imaging means of FIG. 1 and the calculated value about a cell, and FIG. When the diameter of the approximate circle is the calculated value, FIG. 3 (C) is the calculated value of the diameter of the circumscribed circle, and FIG. 3 (D) is the calculated value of the diameter of the inscribed circle. ) Indicates the case where the area is used as the calculated value.

Hereinafter, the present invention will be described with reference to the illustrated examples. In FIG. 1, reference numeral 1 denotes a cell observation device, which has substantially the same configuration as a microscope, and is adjacent to an isolator 2 as a sterile cell culture system. Is located in.
An observation unit 2A communicating with the inside of the isolator 2 is projected outward from a part of the wall surface of the isolator 2 facing the cell observation device 1. The stage 3 is horizontally arranged on the bottom of the observation unit 2A, and the container 4 to be observed is placed on the stage 3. As the container 4, a dish-shaped hemocytometer having an open upper surface is used.
A robot hand or an operator (not shown) arranged in the isolator 2 uses a pipette to supply the cell suspension from the culture vessel into the container 4 in the isolator 2 and accommodates the cell suspension in the container 4. The cell C is observed by the cell observation device 1. The observation unit 2A, the stage 3 and the container 4 are made of a transparent material such as glass, and the cells C in the container 4 placed on the stage 3 can be observed by the cell observation device 1.
Before supplying the cell suspension to the container 4, a test solution such as trypsin may be added to the culture container in the isolator 2, and the cell suspension is placed in the container to facilitate counting of cells C. A test solution such as a diluted trypan blue solution may be added to the cell suspension before supplying to 4.

The cell observation device 1 irradiates the white light L1 from the white light source 5 toward the container 4 on the stage 3, and in a state where the white light L1 is irradiated, the cells C in the container 4 are imaged from below by the imaging means 6. Based on the image taken by the imaging means 6, the control means 7 determines the quality of the cell C and counts the number (number) of the cells C.
Here, as the cells C to be observed by the cell observation device 1, stem cells such as iPS cells and ES cells, cells differentiated from the stem cells, cells collected by biopsy and the like, and collected cells were cultured. It is supposed to be a cell.
The cell observation device 1 is arranged above the stage 3, the frame 8, and the frame 8 on which the container 4 containing the cells C is placed, and the white light L1 is directed toward the inside of the container 4 on the stage 3. The white light source 5 that irradiates the white light source 5, the first optical system 11 that guides the white light L1 emitted from the white light source 5 to the container 4 on the stage 3, and the cells C in the container 4 that are arranged at the lower part of the frame 8. The image pickup means 6 that images the image from the lower side, the second optical system 12 arranged between the image pickup means 6 from the position on the lower side of the stage 3, and the elevating mechanism 14 that raises and lowers the objective lens 13 of the second optical system 12. The control means 7 is provided for controlling the operation of the white light source 5, the elevating mechanism 14, and the like, as well as determining the quality of the cell C and counting the number (number) of particles of the cell C.
The frame 8 in which each of the above components is arranged is supported by a horizontal moving mechanism 15 (not shown), and the horizontal moving mechanism 15 enables the entire frame 8 to move in the X and Y directions orthogonal to each other in the horizontal plane. ing. As a result, the entire cell observation device 1 excluding the stage 3 can be moved in the XY direction on the horizontal plane, and the imaging means 6 can adjust the horizontal position and the observation area when the container 4 on the stage 3 is imaged. Can be changed.

A storage recess 8A is formed in the central portion of the front surface of the frame 8 in accordance with the position of the observation portion 2A in the isolator 2, and the cells in the container 4 are stored in the storage recess 8A. Is supposed to be observed.
The white light source 5 and the first optical system 11 are arranged on the upper side of the frame 8, and the imaging means 6 and the second optical system 12 are arranged on the lower side of the frame 8.
The first optical system 11 is arranged on the upper side of the accommodating recess 8A of the frame 8, and is composed of the first reflection mirror 17, the retardation ring 18, and the condenser lens 19 in order from the upper side. The constituent members of the first optical system 11 are fixedly arranged at a predetermined height position of the frame 8.
The operation of the white light source 5 is controlled by the control means 7, and when the control means 7 operates the white light source 5, the white light L1 emitted from the white light source 5 is lowered by the first reflection mirror 17. After being reflected toward, the container 4 on the stage 3 is irradiated via the retardation ring 18 and the condenser lens 19.
On the other hand, the second optical system 12 is arranged on the lower side of the accommodating recess 8A in the frame 8, the objective lens 13 horizontally arranged at a position directly below the accommodating recess 8A, and the second reflection arranged below the objective lens 13. It includes a mirror 21, a retardation plate 22 and a condenser lens 23 which are sequentially arranged between the second reflection mirror 21 and the image pickup means 6.
The second reflection mirror 21, the retardation plate 22, and the condenser lens 23 are fixedly arranged at predetermined height positions, but the objective lens 13 is supported by the elevating mechanism 14 and is supported in the Z direction (vertical direction). It can be raised and lowered.
The operation of the elevating mechanism 14 is controlled by the control means 7, and the control means 7 moves the objective lens 13 up and down within a movable vertical range by the elevating mechanism 14, so that the imaging means 6 moves the container. The focal position of the objective lens 13 can be adjusted when the cell C in 4 is imaged.
In this embodiment, the white light L1 is emitted from the white light source 5, the container 4 on the stage 3 is irradiated with the white light L1 via the first optical system 11, and the white light L1 is irradiated in that way. The cells C in the container 4 in the state are imaged by the imaging means 6 from the lower side via the second optical system 12.
A digital camera is used as the imaging means 6, and the image of the cells C in the container 4 captured by the imaging means 6 is transmitted to the control means 7. Then, the control means 7 counts the quality of the cells and the number of cells based on the image of the cells C in the container 4 imaged by the imaging means 6.
As will be described in detail later, in this embodiment, when the imaging means 6 images the cells C in the container 4, the focus of the objective lens 13 can be autofocused to the in-focus position that matches the position of the cells C in the container 4. The device and method are proposed, and once the in-focus position is obtained, the container 4 is imaged again by the imaging means 6 at that position, and the quality of the cell C is determined based on the captured image. It has become.

Hereinafter, a specific autofocus method and determination processing by the cell observation device 1 will be described.
First, a robot hand (or an operator) adds a test solution such as trypsin into a culture vessel (dish, plate, flask, etc.) (not shown) in which cells are cultured in the isolator 2, if necessary.
Next, in the isolator 2, a robot hand (or an operator) takes out the cell suspension from the culture vessel using a pipette, and then supplies the cell suspension into the vessel 4 on the stage 3. If necessary, a test solution such as a diluted trypan blue solution is added to the cell suspension taken out from the culture vessel in the isolator 2 to count the number of cells C by the cell observation device 1. It will be easier to do.
After that, in the cell observation device 1, the height region where the in-focus position of the objective lens 13 when the imaging means 6 images the container 4 is first obtained by a rough search, and then the above-mentioned height region is obtained by a detailed search. The focus position in is calculated.
More specifically, the coarse search is performed as follows. That is, first, in a state where the white light L1 is irradiated from the white light source 5 toward the container 4, the height position of the ascending end to the height position of the descending end (Z) within the range in which the objective lens 13 can be moved up and down by the elevating mechanism 14. While lowering by a predetermined amount (for example, by 30 μm) from 0 to X) on the axis, the cell C in the container 4 is imaged by the imaging means 6 at each height of lowering by the predetermined amount, and the height position to be imaged is set. Get multiple different images. Instead of lowering the container 4 intermittently in this way, the container 4 may be imaged by the imaging means 6 each time the container 4 is lowered by a predetermined amount while being continuously lowered.
Since the image of the cells C in the container 4 captured by the imaging means 6 is transmitted to the control means 7, the control means 7 takes each image in the image at each height position imaged by the imaging means 6 by a predetermined amount. After calculating the outer peripheral lengths of all the cells C or cell regions in the image as calculated values, the total value of the outer peripheral lengths as those calculated values is further calculated. In this example, the length of the outer circumference of the cell C obtained for each image is used as a calculated value (see FIGS. 2 and 3 (A)).
For ease of understanding, only one cell C is shown in FIG. 3 (A), but in reality, a plurality of cells C or cell regions are detected, and all of these cells are detected. After calculating the length of the outer circumference with respect to C or the cell region, the total value is calculated.
As the calculated value, instead of using the length of the outer circumference of the cell C or the cell region, the diameter of the approximate circle of the outer circumference of each cell C or the cell region in each image is obtained as the calculated value, and the diameter of each cell is calculated. The total value of the diameters of the approximate circles on the outer circumference may be calculated (see FIG. 3B). Also, instead of the diameter of the approximate circle on the outer circumference of the cell C, the diameter of the circumscribed circle of the cell C (FIG. 3 (C)) or the diameter of the inscribed circle of the cell C (FIG. 3 (D)), or the cell C. (Fig. 3 (E)) may be obtained as a calculated value, and the total value for them may be calculated.
In this way, when the captured image of the cell C at each height is acquired by the imaging means 6 when the objective lens 13 is lowered by a predetermined amount from the ascending end to the descending end, the control means 7 calculates the calculated image. A rough height region A1 at which the total value of the values is the minimum value A4 (the value that is the valley between the peaks A2 and A3) is obtained (see FIG. 2).
The "minimum value A4" here means the local minimum value in FIG. 2, and is different from the simple minimum value. That is, paying attention to the fluctuation of the total value of the outer circumference of the cell C as the calculated value, the valley portion A4 of the peak A2 and the peak A3 (the minimum value at which the decreasing tendency is switched to the increasing tendency) is referred to as the "minimum value A4". The control means 7 recognizes the above.
In this way, in this embodiment, the control means 7 obtains the height region A1 including the minimum value A4 corresponding to the in-focus position AX by first performing a rough search.

A detailed search is performed after the above-mentioned rough search. That is, after that, with respect to the height region A1 including the minimum value A4, the height of the rising end in the height region A1 is set for each predetermined amount (for example, 6 μm) which is smaller than the case of the above-mentioned rough search. The objective lens 13 is lowered by the elevating mechanism 14 to the height of the descending end, and the image of the cells C in the container 4 is imaged by the imaging means 6 each time.
Then, based on the image of the cells C in the container 4 at each height position imaged by the imaging means 6 in the height region A1, the control means 7 has each height as in the case of the above-mentioned rough search. After obtaining the length of the outer circumference of the cell C in the image of the position as a calculated value, the total value thereof is calculated.
Again, as calculated values, instead of the length of the outer circumference of the cell C, as shown in FIGS. 3 (B) to 3 (E), the diameter of the approximate circle of the outer circumference of the cell C and the circumscribed circle of the cell C. The diameter of the cell, the diameter of the inscribed circle of the cell C, or the area of the cell may be determined, and they may be detected and used.
After that, the control means 7 focuses the objective lens 13 when the imaging means 6 photographs the container 4 at a height position where the total value of the calculated values becomes the minimum value A4 (minimum value) in the height region A1. It is determined as the in-focus position AX that matches.
In this embodiment, as described above, the control means 7 lowers the objective lens 13 from the ascending end to the descending end by a predetermined amount via the elevating mechanism 14, and the cell C in the container 4 is brought down by the imaging means 6 accordingly. The images of the above are sequentially imaged by the imaging means 6, first, the height region A1 having the minimum value A4 is obtained in the rough search, and then the position of the minimum value A4, that is, the focus of the objective lens 13 is the container 4 by the detailed search. The in-focus position AX that matches the height of the cell C inside is obtained.
Then, after that, the objective lens 13 is moved by the elevating mechanism 14 to the height position of the minimum value A4 detected as the in-focus position AX, the objective lens 13 is stopped at that height position, and then imaging is performed again. The cell C in the container 4 is imaged by the means 6. As a result, the cells C in the container 4 can be imaged by the imaging means 6 in a state where the cells C in the container 4 are accurately focused.
After that, the control means 7 determines the quality of the cells and counts the cell particles based on the image of the cells C in the container 4 taken by the imaging means 6 at the in-focus position AX. There is.

As described above, according to the present embodiment, the operator visually raises and lowers the objective lens 13 to obtain the focus position of the imaging means 6 that matches the cells in the container 4 with the same accuracy as when searching for the focus position. The in-focus position AX can be detected by autofocus. Therefore, it is possible to provide the cell observation device 1 having an optimum autofocus function for imaging the cells C in the container 4 with the imaging means 6. In addition, it is possible to provide a cell observation device 1 capable of improving the accuracy of determining the quality of cells C and counting the number of cells.
Further, the cell observation device 1 of the present embodiment can be mounted on the cell automatic culture device, which can greatly contribute to the spread of regenerative medicine.

In this example, as the cells C to be observed, stem cells such as iPS cells and ES cells, cells differentiated from stem cells, cells collected by biopsy, and cells in which the collected cells are cultured are assumed. However, it is not particularly limited to these.
In addition, depending on the type of cell and the course of culture (early, middle, late), the target (the length of the outer circumference of the cell, which is the basis of the calculated values shown in FIGS. 3 (A) to 3 (E), The diameter of the approximate circle on the outer circumference of the cell, the diameter of the circumscribing circle of the cell, the diameter of the inscribed circle of the cell, the area of the cell) may be appropriately used.
Further, in the above-described embodiment, the objective lens 13 is lowered by a predetermined amount (dimensions) from the ascending end to the descending end via the elevating mechanism 14 in the rough search and the detailed search. The calculated value may be obtained by raising the lens 13 from the descending end to the ascending end by a predetermined amount (dimension). Further, it does not necessarily have to be lowered from the rising end to the falling end, or raised from the falling end to the rising end, and may be lowered or raised within a predetermined range.
Further, instead of raising and lowering the objective lens 13 by the raising and lowering mechanism 14, a configuration in which the stage 3 itself is raised and lowered may be adopted, and further, a configuration in which the imaging means 6 and the second optical system 12 are integrally raised and lowered is adopted. You may.
Further, in the observation unit 2A, instead of the stage 3, the robot hand may hold the container 4 and move the container 4 horizontally or vertically.

1 ‥ Cell observation device 3 ‥ Stage 4 ‥ Container 6 ‥ Imaging means 7 ‥ Control means 13 ‥ Objective lens 14 ‥ Elevating mechanism AX ‥ Focusing position A4 ‥ Minimum value C ‥ Cell

Claims (4)

A stage on which a container containing cells is placed, an imaging means for imaging cells in the container, an elevating means for changing the height position of the stage or the focal height position of the imaging means, and the imaging A control means for detecting cells in an image captured by the means and obtaining a total value of calculated values for the detected cells is provided.
While the height position of the stage or the height position of the focal point of the imaging means is changed by the elevating means, the cells in the container are imaged by the imaging means at each predetermined height position.
The control means calculates the total value of the calculated values for the cells in the image of each height position imaged by the image pickup means, and determines the position where the total value becomes the minimum value as the in-focus position of the image pickup means. ,
A cell observation device characterized in that a container placed on the stage is positioned at a focal point position by the elevating means. The calculated value for the cell is characterized by being any one of the length of the outer circumference of the cell, the diameter of the approximate circle of the outer circumference of the cell, the diameter of the circumscribed circle of the cell, the diameter of the inscribed circle of the cell, or the area of the cell. The cell observation device according to claim 1. A stage on which a container containing cells is placed, an imaging means for imaging cells in the container, an elevating means for changing the height position of the stage or the focal height position of the imaging means, and the imaging A cell is detected in an image imaged by the means, and either the length of the outer circumference of the detected cell, the diameter of the approximate circle of the outer circumference of the cell, the diameter of the circumscribed circle of the cell, or the diameter of the inner circle of the cell is determined. Equipped with a control means to calculate
The focus of the imaging means is the container based on any of the length of the outer circumference of the cell, the diameter of the approximate circle of the outer circumference of the cell, the diameter of the circumscribed circle of the cell, or the diameter of the inscribed circle of the cell calculated by the control means. The focus position is determined according to the height position of the cells inside.
A cell observation device, characterized in that the container placed on the stage is adjusted to a determined in-focus position by the elevating means. The height position of the container containing the cells or the height position of the focal point of the imaging means for imaging the container is changed, and an image of the cells in the container is acquired by the imaging means at each predetermined height position.
A cell is detected in the acquired image of each cell, and the outer circumference length of the detected cell, the diameter of the approximate circle of the outer circumference of the cell, the diameter of the circumscribed circle of the cell, the diameter of the inscribed circle of the cell, or the diameter of the cell Calculate the total value of any of the areas and
A cell observation method, characterized in that a height position at which the total value becomes a minimum value is determined as a focused position where the focus of the imaging means matches the position of cells in the container.

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