JP6873456B2 - Cell transport system - Google Patents

Description

The present invention is a highly versatile system that can be arranged and used in a general microscope device, and is a work assuming one-cell analysis in which focus correction by autofocus control does not normally operate in a cell transport operation. The present invention relates to a cell transport system that enables efficient transport of target cells to a desired transport destination container in units of one cell even when used.

In recent years, the development of vibration-free pump technology that enables minute flow rate control has made it possible to realize suction and discharge technology with a very small amount of liquid that enables cell transfer operation in "one cell unit". The microfluidic volume manipulation technology is the basic technology of "1 cell analysis" that advances cancer research, stem cell research, iPS cell research, etc., and the improvement of operability and efficiency of the technology affects the progress of these researches. give.
In the current field of life science, there is a strong directivity of large-scale analysis technology such as high-throughput analysis, which correlates with the dramatic development of computer computing speed. Therefore, a microwell plate capable of mass processing of a sample is often used as a container for analysis, and it is expected that a microdevice container having microchip-shaped microholes and channels will be developed in the future.

When performing a cell transfer operation in "one cell unit" in such a microcontainer, in an actual work site, a dispensing operation from a dish to a microwell plate, a dispensing operation from a microwell plate to a multi-PCR tube, etc. There is a demand for cell transfer operation between containers having different shapes and thicknesses of the bottom surface of the container. In this operation, in each of the cell suction operation and the discharge operation into the microcapillary tube, microscopic observation through an objective lens arranged in the bottom surface direction or the top surface direction of the sample stage is performed to "to the target (bottom surface of the container)". It will be necessary to perform "focusing" and "confirmation of operation completion" each time. In this respect, the larger the sample analysis in which the dispensing operation is continuously performed, the more seriously the operation process affects the work efficiency.
Therefore, it is expected to develop a technique for efficiently executing a cell transport operation on a cell-by-cell basis, but its realization has been hindered by the following main problems.

1) Autofocus inhibition phenomenon As a technique for automating focusing in an optical system, a control means for correcting a focus shift by autofocus image processing is widely known, and the technique is also widely used in a general microscope system. This is an extremely useful technique.
As an autofocus control mechanism in a microscope, for example, it recognizes a deviation of the focal position from the observation target, quantitatively calculates a correction amount according to the deviation amount, and automatically corrects the focus according to the correction amount. A control mechanism to be executed can be mentioned (see, for example, Patent Document 1 and the like).

However, as a result of repeated research by the inventor of the present application on technological development related to cell transport, in the cell transport operation when performing "1 cell analysis", on the transport destination container side when "discharging" cells from the fine capillary tube, I faced a phenomenon in which the focus correction in autofocus control did not work properly. This autofocus inhibition phenomenon in the transport destination container occurs when the amount of the solution in the transport destination container is extremely small, such as several μL, and is caused by the fact that the trace liquid becomes a drop shape in the field of view due to surface tension. It is a phenomenon. In the drop-shaped liquid, a bright part and a dark part that become shadows are generated in the field of view, and extremely high contrast is generated, which is caused by the automatic recognition function of the focus correction amount not operating normally.
Such an autofocus inhibition phenomenon is likely to occur when the target cells are transported to a PCR tube or the like for analysis of genes or the like, and a reaction solution (expensive reagent) containing an enzyme for analysis or the like is made into a small scale. This is a phenomenon that occurs particularly frequently when it is performed. In this respect, it causes a serious work delay in a large-scale analysis that requires a large amount of reaction reagents containing expensive enzymes and the like.
In addition, in the analysis of genes, etc., it is required to transport the target cells to a microwell plate, PCR tube, etc., but since many reaction tubes are made of a material such as polypropylene, the transparency is lower than that of glass, etc. This point also acts in a direction that hinders the automatic recognition of autofocus.
Furthermore, when focusing is performed in a situation where the tip of the capillary tube is close to the bottom surface of the container, the contrast is further increased due to the influence of the reflection phenomenon of the tube tip that has entered the trace amount of liquid in the field of view. This makes autofocus control more difficult.

2) Confirmation of cell storage state in the fine capillary tube In the operation of transporting the target cells into the desired container in units of one cell, confirmation of cell "suction" in the transport source container and cell "discharge" in the transport destination container It is necessary to perform the operation surely while confirming. That is, in order to pinpoint the target cell into the desired container, the target cell is surely placed in the fine capillary tube as a requirement different from the problem of automating focusing mentioned in 1) above. An operation that reliably confirms that the cells have been aspirated and that the target cells have been reliably discharged from the fine capillary tube with a microscope through an objective lens arranged in the bottom surface direction or the top surface direction of the sample stage is performed. Desired.
However, in 1-cell analysis, it is required to perform analysis on a microscale, and in order to access the bottom surface of the container using a microcontainer such as a microplate, the microcapillary tube must be inserted almost vertically. Is required. In such a situation, the relative depth of focus is deep and the optical path is likely to be obstructed due to the arrangement position of the objective lens or the light source, and a phenomenon occurs in which it becomes difficult to confirm the state of the cells stored in the fine capillary tube. ..
This phenomenon tends to become apparent especially in the case of multi-sample processing using a minute microplate, which induces a problem of misidentifying the number of cells aspirated or ejected and causes a delay in work.

Japanese Patent No. 4815179

As shown above, in the one-cell transport technology, it is necessary to surely perform the cell aspiration operation and the cell ejection operation in the fine capillary tube while confirming with a microscope. I) Genes frequently used in one-cell analysis When performing a cell transfer operation into a trace solution for analysis such as, the normal operation of focus correction by autofocus control in the transfer destination container is hindered, so the focusing operation must be performed manually. is there. In addition, ii) when transporting cells using a microwell plate or the like, which is often used in 1-cell analysis, it is necessary to insert a fine capillary tube almost vertically to access the bottom surface of the container. Due to the arrangement position of the light source, it is difficult to confirm the state of the cells stored in the tip tube of the fine capillary tube.
These above-mentioned problems are recognized in the development of mass analysis technology for one cell, which is expected to develop dramatically, and are positioned as problems to be overcome in order to carry out "one-cell analysis" on a large scale. Be done.

The present invention has been made in view of the above-mentioned circumstances of the prior art, and a subject thereof is a work assuming a one-cell analysis in which focus correction by autofocus control does not normally operate in a cell transport operation (that is, cells). Technology that enables efficient cell transfer on a cell-by-cell basis even under conditions where the bottom structures of the transfer source container and the transfer destination container are different and the solution in the transfer destination container has high contrast). The purpose is to provide.

As a result of intensive research in the situation related to the above-mentioned prior art, the present inventor has made it possible to freely move in three-dimensional directions in the X-axis direction, the Y-axis direction, and the Z-axis direction on the microscopic sample stage surface in the cell transport system. The idea was to provide a driving means to be used as the above, and a control means capable of storing and automatically adjusting the three-dimensional coordinates of the microscope sample stage surface.
Here, when performing a cell transport operation on a trace scale required for single cell analysis or the like using a conventional general microscope system, the autofocus control does not function normally and the target cell transport source. The bottom shapes of the container and the destination container are different and manual focusing operation is indispensable. On the other hand, the microscope sample stage conceived by the present inventor copes with the difference between the bottom surfaces of the source container and the destination container. We have found that cell aspiration and cell ejection can be performed continuously and efficiently while automatically moving the focus position one after another.
Since the automatic movement and control can be performed only by automatically controlling the driving means of the microscope sample stage (1 article), it is a technique that can be realized by adding a simple control means.
Further, since the microscope sample stage conceived by the present inventor can be in an embodiment that can be arranged in a general commercially available microscope device, it can be used as a general-purpose member separate from the main body of the microscope device. Met. This is a mode that cannot be realized by a system that relies on the vertical movement of the objective lens, which is an article in which the focusing means is integrated with the microscope device, and can provide a great cost advantage in the introduction of the device by the consumer.

On the other hand, when cells are transported using a conventional general microscope system, the microscope sample stage is usually capable of moving only in two-dimensional directions in the X-axis direction and the Y-axis direction. Focusing is required each time according to the difference in the shape of the bottom surface of each container. In such a general microscope system, it is necessary to perform focusing by moving the objective lens up and down, but when discharging cells in a microfluidic system, the focusing operation is manually performed due to the autofocus inhibition phenomenon. I have to do it every time.
Further, when carrying out cells with a general microscope system, it is necessary to move the capillary tube up and down to avoid contact with the container wall surface every time the microscope sample stage is moved. In this regard, if a general microscope system is constructed with control means capable of storing and automatically adjusting three-dimensional coordinates, the microscope stage, the objective lens, and the driving means of the capillary tube (three articles) are used. It is necessary to automatically control after interlocking three-dimensionally. This is a complicated device configuration as compared with the system according to the present invention, and is a complicated device system that imposes on the user the adjustment confirmation of the precise interlocking operation between the separated articles at the time of use. Further, since it is necessary to have an interlocking control system including an objective lens, the product form must be integrated with the main body of the microscope device, which imposes a large cost burden on the introduction of the device by the consumer.

Further, as a result of intensive research, the present inventor has found a fine capillary tube shape including a bent shape in the range of 90 to 150 ° as a fine structure near the tip of the fine capillary tube for sucking and discharging cells. I was inspired.
The present inventor uses a fine capillary tube having the tip microstructure so that even when the microwells of the microwell plate are accessed vertically to the fine capillary tube, the tip of the microstructure can be formed. It has been found that the bottom surface of the container can be accessed horizontally or at an appropriate inclination angle. As a result, the present inventor has found that the optical path is not blocked even when the objective lens or the light source is arranged in the vertical direction, and the inside of the tip tube of the fine capillary tube can be easily visually recognized.

Here, Patent Document 1 is a document that discloses a microscope focus correction technique using autofocus control as described above. In paragraph 0037 and the like of the document, there is another aspect of the automatic focus position correction means by autofocus control. As an example, it is described that focus correction by autofocus control is possible by moving the microscope table up and down instead of the objective lens.
However, since Patent Document 1 is merely a document that discloses a focus correction technique based on autofocus control, a "fine liquid amount" solution found in a cell transport technique premised on one-cell analysis is used. There is no description that discloses or suggests the problem of focusing. In this regard, as a solution for overcoming the problem, a driving means that enables free movement in the three-dimensional direction on the microscope sample stage surface "without using autofocus control" and a control means for the microscope sample stage surface are provided. The idea to be provided cannot be conceived from the description of Patent Document 1.
Further, Cited Document 1 does not disclose the fine capillary tube relating to cell transport, nor does it disclose the technical features relating to the tube tip microstructure of the microcapillary tube relating to the improvement of visibility of stored cells in the capillary tube. ..

Further, in the "problem to be solved by the invention" (paragraph 0009) according to Patent Document 1, "providing a drive system in the Z-axis direction for automatically correcting the focal position is practical due to problems such as cost. It is stated that "it is not", and this point is consistent with the fact that the focus of the products in the market of conventional general microscope systems is usually performed by moving the "objective lens" side up and down. Are listed. Regarding these points, in the technical field related to the present invention, there is an obstructive technical common sense in the idea of providing a driving means and a control means for moving the "microscope sample stage" side up and down as focusing means for the microscope product. It is recognized that it indicates that.

The present inventor has completed the present invention based on the above findings. The present invention specifically relates to the invention described below.
[Item 1]
It is a cell transfer system that enables transfer of target cells to a desired destination container in units of one cell;
(A) A microscope sample stage provided with a sample container holding means on the surface of the sample stage and a driving means capable of automatically moving in the X-axis, Y-axis, and Z-axis directions.
(B) A control means that can store the three-dimensional coordinates of the sample stage surface and automatically moves the sample stage surface to an arbitrary set three-dimensional coordinate based on the stored relative position information, and
(C) A fine capillary tube disposed above the sample stage surface, the tip of which microstructures for cell suction and cell ejection include a bent or curved shape in the range of 90 to 150 °. It is equipped with a capillary tube,;
A cell transport system that is arranged and used in a microscope device.
[Item 2]
Item 1 allows automatic focusing operation to be performed even in situations where focus correction by autofocus control does not work properly for a solution whose microscopic image has a high contrast with a very small amount in the transport destination container. The cell transport system described.
[Item 3]
Item 1 or 2 that the tip microstructure of the fine capillary tube is a tube structure having an inner diameter of 5 to 500 μm and includes a bent or curved shape portion within a range of 5 mm or less from the tube tip. The cell transport system according to.
[Item 4]
The driving means provided in the microscope sample stage has a driving unit capable of moving in units of 0.1 to 100 μm, and the first to third actuators have different operating axes in three-dimensional directions. Including a structure, which is arranged such that;
The support portion of the first actuator is fixedly held by the microscope sample stage support means, the support portion of the second actuator is fixedly held by the drive portion of the first actuator, and the drive portion of the second actuator is fixedly held. The support portion of the third actuator is fixedly held, and the stage surface of the microscope sample stage is fixed to the driving portion of the third actuator;
Item 6. The cell transport system according to any one of Items 1 to 3.
[Item 5]
Item 4. The cell transport system according to any one of Items 1 to 4, further comprising a microscope device.
[Item 6]
Item 5. A method for transporting target cells to a transport destination container in units of one cell, which comprises using the cell transport system according to Item 5.

By using the cell transfer system according to the present invention, it is possible to simultaneously execute the moving operation and the focusing operation of the microscope sample stage surface, and the bottom surfaces of the transfer source container and the transfer destination container are accessed. It is possible to easily confirm the cell storage state in the tip tube of the fine capillary tube.
As a result, in the present invention, the work assuming one-cell analysis in which the focus correction by the autofocus control does not normally operate in the cell transport operation (that is, the bottom structures of the cell transport source container and the transport destination container are different, and the transport destination container is different. It is possible to provide a technique that enables efficient cell transport on a cell-by-cell basis even under the condition that the contrast of the solution is high).

As described above, in the present invention, for example, it is possible to efficiently perform a continuous cell distribution operation of a large number of samples on a microwell plate or the like in one cell analysis or the like.
Further, in the present invention, for example, it is possible to provide a highly versatile cell transfer system that can be arranged and used in a conventional general microscope device.

It is a structural drawing which showed the structure about the microscope sample stage which enables three-dimensional space movement about the cell transport system manufactured in Example 1 from the front view direction. The broken line in the figure shows the structure of the main body of the microscope device.

It is a structural drawing which showed the appearance of each actuator about the microscope sample stage manufactured in Example 1. FIG. FIG. 2A: Top view of the actuator for moving the X-axis. FIG. 2B: Top view of the Y-axis moving actuator. FIG. 2C: Front view of the Z-axis moving actuator.

FIG. 5 is a structural diagram showing the structure of the sample stage surface near the sample container holding means on the surface of the microscope sample stage manufactured in Example 1 from the top view direction. The broken line in the figure shows the structure of the main body of the microscope device.

It is a structural drawing which showed the appearance of the fine capillary tube manufactured in Example 1. FIG.

It is a schematic diagram which showed the appearance structure of the microscope sample stage manufactured in Example 1 from the perspective direction.

It is a schematic diagram which arranged the cell transport system manufactured in Example 1 in a microscope apparatus, and showed the appearance structure as a whole from the perspective direction.

It is a schematic diagram which showed the appearance structure of the constituent article attached to the cell transport system manufactured in Example 1 from the perspective direction.

FIG. 5 is a photographic image showing a state in which the fine capillary tube manufactured in Example 1 is arranged above the sample stage surface.

It is a schematic diagram which showed the moving state of the microscope sample stage surface with time in the cell transport operation using the system which concerns on this invention. The thick black arrow in the figure indicates the moving direction of the sample stage surface. FIG. 9A: Cell aspiration operation in the transport source container. FIG. 9B: Downward movement of the sample stage surface. FIG. 9C: Movement of the sample stage surface in the XY direction. FIG. 9D: Upward movement of the sample stage surface and cell ejection operation in the destination container.

Hereinafter, embodiments of the present invention will be described in detail. In addition, the code number in the following description means the code number used in the drawing.

[Explanation of terms]
In the present specification, the terms X-axis, Y-axis, and Z-axis are used as terms for the axial directions of the three-dimensional space. Here, "X-axis" is used as a term indicating a horizontal axis in the left-right direction in the front view direction of the cell transport system according to the present invention. The term "Y-axis" is used to refer to the horizontal axis in the front-back direction in the same front-view direction. Further, "Z axis" is used as a term indicating a vertical axis in the vertical direction in the same front view direction.
In the present specification, the "front view" of the cell transport system refers to a viewpoint from the direction in which the eyepiece of the microscope is provided in a state of being arranged in a general microscope device.

1. 1. Configuration of Cell Transport System The present invention relates to a cell transport system that enables transport of target cells to a desired transport destination container in units of one cell.
Specifically, the present invention is a cell transport system that enables transport of target cells to a desired transport destination container in units of one cell;
(A) A microscope sample stage having a sample container holding means on the surface of the sample stage and a driving means capable of automatically moving in the X-axis, Y-axis, and Z-axis directions.
(B) A control means that can store the three-dimensional coordinates of the sample stage surface and automatically moves the sample stage surface to an arbitrary set three-dimensional coordinate based on the stored relative position information, and
(C) A fine capillary tube disposed above the sample stage surface, the tip of which microstructures for cell suction and cell ejection include a bent or curved shape in the range of 90 to 150 °. It is equipped with a capillary tube,;
The present invention relates to a cell transport system that is arranged and used in a microscope device.
The technical scope of the cell transport system according to the present invention is not limited to the mode including all of the features described below with respect to the features other than the essential features.

[Microscope sample stage]
The cell transport system (1) according to the present invention includes a microscope sample stage (2) as a constituent article thereof. The microscope sample stage (2) according to the present invention is characterized by including a driving means (21) capable of automatically moving in the X-axis, Y-axis, and Z-axis directions. That is, in the microscope sample stage (2) according to the present invention, the automatic free movement of the sample stage surface (20) to the arbitrarily set coordinates in the three-dimensional coordinate space is realized by providing the driving means.

Basic structure of sample stage surface As the microscope sample stage (2) according to the present invention, a sample stage surface for a microscope provided with a sample container holding means (201) on the stage surface (20) on which the sample container is arranged is adopted. be able to. Here, as the sample container holding means (201), any structure that can be adopted for the sample stage surface for an optical microscope is used as long as it is a means capable of arranging a container or the like for storing or placing the target cells. However, it is desirable that the structure is capable of holding two or more sample containers corresponding to the cell transport source container and the cell transport destination container.
As a specific structure for realizing the means, for example, by pushing force of a semi-frame type structure having a holding means by two or more members arranged on the upper surface side of the sample stage surface, an elastic body, or the like. Examples include, but are not limited to, a structure to which a container or the like can be fixed, a structure having a frame that can be fitted with a sample container, and the like.

The microscope sample stage (2) according to the present invention is a sample container holding capable of holding two or more, preferably two sample containers, depending on the structure of one, in order to hold the cell transport source container and the cell transport destination container. It is possible to adopt an embodiment provided with the means (201). Further, it is also possible to adopt an embodiment in which two or more, preferably two sample container holding means, which enable holding of one sample container, are provided.

As the structure constituting the stage surface (20) in the microscope sample stage according to the present invention, it is possible to adopt the structure of a sample stage for a normal optical microscope except that the sample container holding means is provided. Is. As one aspect of the microscope sample stage according to the present invention, it is desirable that the portion of the structure on the upper surface side where the sample container is arranged has a flat shape or a substantially flat shape.
Here, as an aspect of the sample stage suitable for a normal upright microscope or an inverted microscope, the sample stage surface (20) is arranged at a position sandwiched between the objective lens and the light source when transmitted illumination is used. Therefore, it is preferable that the stage portion corresponding to the portion of the sample container in which the sample is stored or placed is provided with an optical path transmission structure for securing an optical path between the objective lens side and the light source side. As a structure for ensuring such an optical path, for example, an opening structure, a window structure, a structure formed of a translucent member, or the like can be adopted. When a translucent member is used as the member for molding the optical path securing portion, glass, a transparent resin, or the like having excellent light transmittance and strength to withstand the installation of a container can be mentioned.
Further, in one aspect of the microscope sample stage according to the present invention, in the case of a microscope device that uses epi-illumination in an upright microscope, it is essential that the sample stage surface (20) has the above-mentioned opening structure. Absent. In this embodiment, it is possible to adopt a flat sample stage in which the portion where the sample container is arranged is suitable for installing the bottom surface of the container.

Further, as one aspect of the microscope sample stage according to the present invention, it is also possible to incorporate or incorporate the lighting means into the stage surface (20) itself of the sample stage. In this aspect, it is preferable to adopt a structure formed of a translucent member or the like for the stage portion in contact with the portion of the sample container in which the sample is stored or placed. The light from the lighting means mounted inside can irradiate the upper surface of the sample container via the translucent member. As the translucent member, the same material as that described in the above paragraph can be adopted. As the lighting means, it is possible to adopt existing lighting for a microscope such as an LED. It is also possible to use a lighting means equipped with a fluorescent filter or the like.

Driving means in the X-axis, Y-axis, and Z-axis directions As the microscope sample stage (2) according to the present invention, a driving means (21) capable of automatically moving in the X-axis, Y-axis, and Z-axis directions is used. Be prepared.
As the driving means (21) provided in the microscope sample stage according to the present invention, the first to third actuators having a driving unit capable of moving in minute units have their respective operating axes in a three-dimensional direction. It includes structures that are arranged at different angles. By providing the driving means, the automatic movement of the sample stage surface (20) to the arbitrarily set coordinates in the three-dimensional coordinate space is realized.

In the present invention, as the driving means (21) that enables automatic movement in the X-axis, Y-axis, and Z-axis directions, interlocking control that enables coarse movement and fine movement in each of the three-dimensional directions is used. It is possible to adopt a structure including three or more actuators, but in a preferred embodiment, the structure includes three actuators, a first actuator, a second actuator, and a third actuator. The structure can be adopted.
One aspect of the microscope sample stage (2) according to the present invention is: an actuator capable of freely moving in the X-axis direction, an actuator capable of freely moving in the Y-axis direction, and an actuator capable of freely moving in the Z-axis direction. It can be provided with an actuator, which can be controlled in conjunction with the actuator, and can be used as a driving means for enabling free automatic movement in a three-dimensional direction.
Further, as another aspect,; an actuator capable of freely moving in one direction on the XY plane, an actuator capable of freely moving in an angle direction different from the one direction on the XY plane, and a ZX plane or a ZZ plane. An actuator that enables free movement in one direction is provided; and these can be controlled in conjunction with each other to serve as a driving means that enables free movement in three-dimensional directions.

Actuators (22, 23, 24) that can be used as the driving means according to the present invention include a support portion (221, 231 and 241), a power unit held by the support portion (222, 232, 242), and the like. Examples of the structure include a drive unit (223, 233, 243) that moves along the axis by a force from the power unit, and the like as a main member. In the structure, the drive unit can be freely moved in the axial direction by the force from the power unit, and the moving body fixed to the drive unit can be freely moved in the axial direction.
Here, the power unit and the drive unit can be used without particular limitation as long as they have specifications that enable the realization of the rough movement and the fine movement described below.
As the power unit (222, 232, 242) of the actuator according to the present invention, one using an electrically driven motor is preferable in order to realize automatic operation by the control means. As the power unit and drive unit of the actuator according to the present invention, it is preferable to use a servo motor or a stepping motor using AC (alternating current power supply) or DC (direct current power supply).

It is preferable that the first to third actuators (22, 23, 24) according to the present invention have different specifications depending on each axial direction. For example, as an actuator that realizes movement in the X-axis direction and the Y-axis direction, it is preferable that the actuator has a large movement width during rough movement. Further, as the actuator that realizes the movement in the Z-axis direction, it is preferable that the actuator has a small movement unit at the time of fine movement in order to execute the focusing operation more precisely.

As the driving means (21) of the microscope sample stage according to the present invention, in order to enable free automatic movement in the space of the microscope field of view and its surroundings, the first to third actuators (22, 23, It is necessary to construct a composite structure in which each actuator is three-dimensionally combined so that the structure is such that 24) operates in conjunction with each other.
Here, as the operation axes of the first to third actuators, the three-dimensional space movement of the microscope sample stage surface (20) can be realized by combining the operation axes so that the respective operation axes have different angles in the three-dimensional direction. It becomes. For example, the movement can be realized by arranging and combining the operating axes of each other so as to have different angles of about 30 ° to 150 °, preferably about 45 ° to 135 °.
As a preferred embodiment of the system according to the present invention, from the viewpoint of device design and movement efficiency, the three operation axes are orthogonal to each other and combined as an X-axis movement actuator, a Y-axis movement actuator, and a Z-axis movement actuator. It is preferable to use the aspect.

As one aspect of the structure for realizing the above operation, the support portion of the first actuator is fixedly held by the microscope sample stage support means (25), and the drive portion of the first actuator holds the support portion of the second actuator. The support portion is fixedly held, the support portion of the third actuator is fixedly held by the drive portion of the second actuator, and the stage surface (20) of the microscope sample stage is fixed to the drive portion of the third actuator. Can be mentioned as an actuator composite structure.
Here, the order of combining the first to third actuators is the sample stage supporting means, the X-axis moving actuator, the Y-axis moving actuator, and the Z, assuming application to a normal microscope system. The combination of the shaft moving actuator and the sample stage surface can be adopted in this order (see FIGS. 1 and 5), but it is also possible to change the order of the actuators and adopt the combination mode. For example, it is also possible to construct an actuator composite structure in which the sample stage supporting means, the Y-axis moving actuator, the X-axis moving actuator, the Z-axis moving actuator, and the sample stage surface are combined in this order. Further, depending on the device mode, a sample stage supporting means, a Z-axis moving actuator, an X-axis moving actuator (or a Y-axis moving actuator), a Y-axis moving actuator (or an X-axis moving actuator), and a sample stage surface. It is also possible to construct an actuator composite structure in which the order of, is combined.
Further, as a fixing mode between the microscope sample stage supporting means (25), the first to third actuators (22, 23, 24), and the stage surface (20) of the sample stage, each member is directly fixed. Indirect fixing mode via a support member, a holding member, or the like is also included.

The microscope sample stage support means (25) in the present invention is a member capable of fixing and holding one support portion (221, 231 and 241) of the actuators, and is the first to third third. Any structure is not particularly limited as long as it supports the weights of the actuators (22, 23, 24) and the sample stage surface (20) and the drive device has a structure and strength capable of operating normally as described above. It is possible to adopt the one.
One aspect of the microscope sample stage supporting means (25) is, for example, a self-standing structure provided with two or more pedestals (251), and a structure in which the pedestals are bridged by a supporting member (252). Can be mentioned. In this aspect, when the microscope device (6) is used, it is installed at a position where the objective lens portion (62) is sandwiched from both the top view and the front view of the microscope device. It is preferable to use a structure in which the gantry is arranged as described above. As the gantry, it is also possible to provide a height adjusting means on the support column of the gantry so that the height of the support member bridging between the gantry can be adjusted.

As the support member (252) for bridging between the gantry, one designed to secure a space capable of ensuring the movement of the microscope sample stage surface (20) in the three-dimensional direction is preferable. In particular, those designed so as to secure a working space that does not come into contact with the objective lens or the lighting means are preferable so that the movement in the Z-axis direction is guaranteed. Here, as the support member (252) for cross-linking between the pedestals, a plate-shaped or rod-shaped member can be adopted, but the cross-linked state between the pedestals is horizontally bridged. Suitable. Further, as the support member (252) for bridging between the pedestals, it is also possible to provide a length adjusting means.

In the present invention, each component such as the stage surface (20) and the actuators (22, 23, 24) constituting the microscope sample stage, and the microscope sample stage support means (25) is separated from each other. It is possible to have a possible mode, but it is also possible to have a configuration mode integrated as a product.

Each actuator (22, 23, 24) according to the present invention is required to be a driving means capable of executing both "coarse movement" and "fine movement".

Here, as the "coarse movement" that can be performed by each actuator (22, 23, 24) according to the present invention, when a "reference point" having three-dimensional coordinates is placed, a few cm to a number from the reference point. It refers to an operation that realizes movement in a wide range of 10 cm unit while maintaining the state of the sample and moving at an appropriate speed.
The rough movement according to the present invention can be determined by the design of the device mode, and one aspect may include the following movement width. For example, as the actuator (22) that drives in the X-axis direction, the movement width in the range of 100 cm or less with respect to the reference point, preferably the movement width in the range of 50 cm or less with respect to the reference point, more preferably 30 cm. The movement width in the following range can be mentioned.
Further, as the actuator (23) for driving in the Y-axis direction, a movement width within a range of 100 cm or less with respect to the reference point, preferably a movement width within a range of 50 cm or less with respect to the reference point, more preferably. The movement width in the range of 30 cm or less can be mentioned.
Further, as the actuator (24) for driving in the Z-axis direction, for example, a movement width within a range of 30 cm or less with respect to the reference point, preferably a movement width within a range of 15 cm or less with respect to the reference point can be mentioned. Can be done.
The moving speed in the rough movement is not particularly limited as long as it is within a speed range in which the liquid of the sample does not scatter and does not cause a work delay. It is preferable that the moving speed is about 20 cm / sec. Further, when moving in the Z-axis direction, a moving speed of about 0.01 to 5 cm / sec is preferable.

The actuators (22, 23, 24) according to the present invention are required to be capable of realizing fine movement to the extent that focusing operation can be ensured in addition to the above-mentioned wide-range and smooth rough movement function. Be done.
Here, the "fine movement" refers to a movement of a minute distance in units of μm, and specifically, a movement of a minute distance in units of 0.1 to 100 μm can be realized. The movement operation of the minute distance is realized by the first to third actuators having a drive unit that enables the movement operation in minute units.
It is preferable that the movement is a fine distance of 50 μm or less, more preferably 20 μm or less, and further preferably 10 μm or less. The lower limit of the value is not particularly limited as long as the technical and cost limitations can be overcome, but for example, it is preferably 0.2 μm or more, preferably 0.5 μm or more, and more preferably 1 μm or more. is there.

Since the actuators (22, 23, 24) according to the present invention can perform fine movement with the above range as the minimum unit, a predetermined position of the cell transport source and / or cell transport destination container (in a specific embodiment, the bottom surface of the container). The upper surface) or a point slightly above this can be moved to coincide with the focal position (54) of the objective lens.
In the present invention, the smaller the unit of the fine movement, the more a high-end device embodiment capable of dealing with fine wells, microchips, and the like is realized. Further, in the present invention, even if the unit of fine movement is relatively large, it is possible to adopt an apparatus mode with inexpensive specifications, and it is possible to adopt an embodiment according to the product application.

As a mode of moving the microscopic sample stage surface (20) according to the present invention in the three-dimensional direction, only the position of the sample stage surface is freely moved in the three-dimensional direction while maintaining the posture of the sample stage surface in the same direction. It becomes possible.
As the sample stage (2) according to the present invention, it is preferable to design the microscope sample stage surface (20) horizontally so that the direction on which the sample container is arranged faces the upward vertical direction. Depending on the situation, it is also possible to adopt an embodiment in which the sample stage surface forms a surface having an angle with respect to the Z-axis direction. Further, it is also possible to provide a means for adjusting the angle of the sample stage surface (20).

In the cell transport system (1) according to the present invention, it is necessary to realize precise automatic focusing operation by the driving means (21) of the sample stage, so that each actuator drive control described above is based on three-dimensional coordinate information. It is precisely controlled and executed by electric drive.

[Control means]
The cell transport system (1) according to the present invention can store three-dimensional coordinates with respect to the microscope sample stage surface (20), and to arbitrarily set three-dimensional coordinates based on the stored relative position information. It is provided with a control means for automatically moving the microscope sample stage surface (20).

The control means (3) of the microscope sample stage surface (20) according to the present invention includes a means for storing position information of three-dimensional coordinates, and is a three-dimensional space that can be moved by the driving means (21) of the microscope sample stage. Within, the position information of the three-dimensional coordinates can be stored. Here, the coordinates that can be stored in the three-coordinate space are the coordinates in the three-dimensional space defined in the driving range of the microscope sample stage.

As the coordinate point that is the movement source, it is possible to store the initially set coordinate point in advance and use this as the origin coordinate. Further, as the origin coordinates, it is possible to newly store and use the coordinate points set arbitrarily. Further, the position coordinates of the current sample stage surface (20) can be used as the origin. These coordinates can be used as "reference points" to calculate the positional relationship with the relative coordinates.

As the coordinate point to be moved, the position coordinates can be arbitrarily set and stored as long as it is in a three-dimensional space that can be moved by the driving means (21) of the microscope sample stage. As the position coordinates of the moving destination to be stored, it is possible to directly input the coordinate values as numerical values representing the relative positional relationship from the reference point (origin or the like) and store them. It is also possible to input and store the moving distance corresponding to the difference from the reference point (origin or the like).
As a mode for storing the position coordinates in the control means (3) according to the present invention, it is possible to perform the mode for directly storing the predetermined coordinates. It is also possible to adopt an embodiment in which the microscope sample stage surface (20) is moved to an arbitrary point within the three-dimensional coordinates, the coordinates of the point are displayed, input and stored, and / or automatically stored.
Here, as one aspect of the operation of moving the microscope sample stage surface (20) to an arbitrary position, a manual operation mode is provided in the following control means, and the operation is finely performed at an arbitrary position by electric drive by button operation or the like. It can be adjusted and moved. It is also possible to adopt a mode in which a position adjusting means by driving using a separate power unit (for example, a human-powered rotational movement of a knob or a handle) is provided. As such an auxiliary power unit, operating members such as knobs and handles linked with the actuator drive unit (223, 233, 243) are arranged in the lateral direction or downward of the microscope sample stage (2). However, it is possible to adopt a device mode that enables the manual rotation operation or the like via the operating member to be converted into a moving motion along an axis in the driving means.

Here, as the points shown as the relative coordinates from the reference point (origin, etc.), for example, one point on the bottom surface when the sample container is installed and the position where the objective lens is in focus are specified, and the coordinates are set. It is possible to set. It should be noted that the position coordinates can be set without particular limitation as long as the position coordinates can express the relative positional relationship.

As the control means according to the present invention, any semiconductor circuit, device, device, medium, or the like capable of ordinary data storage as a storage means of position information can be used, and is not particularly limited. In the control means according to the present invention, it is possible to store a large amount of position information in the storage means.

In the control means (3) according to the present invention, the operation of the drive means is controlled based on the storage information of the three-dimensional coordinates stored in the storage means, and the precise automatic movement operation of the microscope sample stage surface (20) is executed. Will be done.
As an operation related to the automatic movement, as one aspect, the control means (3) calculates the relative movement amount from the reference point and / or the current position, and constitutes the driving means (21) of the microscope stage. By accurately controlling the drive amount of each actuator corresponding to the axial direction, the movement of the microscope sample stage surface (20) to a predetermined position is achieved.
In the control means according to the present invention, a cell transport system for sequentially and automatically moving the microscope sample stage surface (20) to an arbitrary position coordinate based on the stored position memory information is realized.

In the control of each actuator in the present invention, the sample stage surface (2) is driven so as to achieve three-dimensional automatic movement. In the configuration of the system according to the present invention, the fine capillary tube (4) is the sample stage surface. It is a usual mode that it is arranged above (2). Further, in the usual specification aspect of the cell transport system according to the present invention, the position where the focus focus from the objective lens exists (54) and the tip position (42) of the fine capillary tube are fixed, and the sample stage surface is fixed. The movement operation (2) is usually executed (see FIG. 9).
Therefore, as a mode for moving cells from the cell transport source container (51) to the transport destination container (53) using the system according to the present invention, the sample stage is moved to a position where there is no contact between the fine capillary tube and the container wall. After performing the downward movement in the Z-axis direction, it is preferable to perform the movement control in the XY plane direction, and then perform the operation control so as to have a trajectory for performing the upward movement in the Z-axis direction (see FIG. 9).
Since the control and drive operations in the cell transport system (1) according to the present invention are a series of operations that take into consideration the positional relationship with the fine capillary tube and enable focusing, it is necessary to control other members and the like. This is an operation that can be performed by independent control of the microscope sample stage alone. Therefore, it is not necessary to control the objective lens or the like and to perform interlocking operations such as fine movement, and it is possible to construct a cell transport system by configuring the control means and the drive means related to the sample stage.

The control means (3) in the cell transport system according to the present invention can be arranged by incorporating a semiconductor circuit, a chip, or the like in the support of each actuator. It is also possible to provide a housing separate from each actuator and arrange the control means inside the housing. It is also possible to use an electronic computing device such as a PC incorporating application software or the like that functions as a control means as a control means.

As one aspect of the cell transport system according to the present invention, it is preferable to provide interface means (31, 32) as an input means for the above control. For example, as one aspect, it is preferable to employ input means such as key operation on the device, keyboard operation by PC operation, touch panel operation, and the like. Further, it is preferable that the mode is provided with a display means including a monitor, a display, and the like. When such an interface mode is adopted, it is possible to easily execute input settings such as arbitrary three-dimensional coordinates and / or movement width. Further, in the mode in which the manual operation mode is provided, the interface means can be used as an operation unit.

[Fine capillary tube]
The cell transport system (1) according to the present invention includes a fine capillary tube (4) arranged above the microscope sample stage surface (20).
Here, the system according to the present invention is not limited to a system in which fine capillary tubes are "arranged" upward in aspects such as product sales, and is used in combination with a microscope sample stage (2). It includes a product form such as a packed state in which the fine capillary tube (4) to be formed is provided as a constituent article.
Further, even if the microcapillary tube (4) is packaged separately from the microscope sample stage (2) or shipped separately, the cell transport system (1) according to the present invention is substantially configured. When it is recognized as a product form inseparable from the article, it is included in the scope of the cell transport system according to the present invention.

Tube tip microstructure As the microcapillary tube (4) according to the present invention, a structure having a tip microstructure (41) suitable for cell suction and cell ejection in units of cells is adopted as a structure constituting the vicinity of the tube tip. Will be done. Here, as the tip microstructure (41) of the tip of the fine capillary tube, a bent or curved shape (43) in which the tip microstructure that performs cell suction and cell ejection is within a certain angle range (in the present specification, It is preferable to have a microstructure including the first bent or curved shape). Here, the bent or curved shape (43) in the tip microstructure is specifically accompanied by a sharp angle change, an angle change due to a gentle curve, an angle change due to continuous repetition of a shallow angle change, and the like. The shape can be mentioned.

In the fine capillary tube according to the present invention, the constant angle of the bent or curved shape (43) of the tip microstructure may specifically include a bent or curved shape in the range of 90 to 150 °. As a preferred embodiment, the angle range is preferably 95 to 140 °, more preferably 100 to 120 °. Here, these angles are expressed as the angles of the intersections in the direction in which the pipe tip is bent when the intersection of the straight pipe axis on the start point side and the pipe length axis on the end point side of the bent or curved shape is set. It is possible to do.
Here, if the angle of the bent shape (43) included in the tip microstructure (41) is too obtuse, it becomes difficult to capture the cellular state in the tube in the field of view, while if it is too acute. Since the suction and discharge operation of cells is likely to be hindered, it is preferable to appropriately set the angle so as to be within the above range.

In the present invention, by adopting the above structure as the pipe tip (42) of the fine capillary pipe, the fine structure is obtained even when the fine wells and the like are accessed vertically or substantially vertically to the fine capillary pipe. It is possible to access the bottom surface of the sample container (5) so that the tube tip portion (42) is horizontal or has an appropriate inclination angle. As a result, in the system according to the present invention, even when the objective lens or the light source is arranged in the vertical direction, the optical path is not blocked by the tube base side of the fine capillary tube, so that the cells in the tip tube of the fine capillary tube are not blocked. It becomes possible to visually recognize the state of. That is, it is possible to visually confirm the state of cell suction into the tube at the tube tip (42) and cell discharge from the tube.

The structure on the tip side of the bent or curved shape (43) in the fine capillary tube according to the present invention is the first bent or curved structure as long as there is no significant influence on the visibility of the contents, operability, and the like. Separately, it is also possible to adopt a structure having a gentle curve or the like. However, considering the ease of visually confirming the state of cell suction and discharge in the tube tip tube, it is preferable that the tip of the tube tip (42) has a straight tube shape or a substantially straight tube shape. More preferably, the tip of the pipe tip has a straight pipe shape.

As the tip microstructure (41) of the fine capillary tube according to the present invention, the bent or curved shape (43) is formed within a certain range from the tip of the tube in consideration of application to a microplate or the like having fine wells. It is preferable that the product is a product. Specifically, the range of the position where the bent or curved shape is formed is within 5 mm, preferably 4 mm or less, more preferably 4 mm or less from the tip of the pipe tip (42) in consideration of use for a fine well such as a microplate. It is preferably 3 mm or less.

The tip microstructure (41) of the microcapillary tube according to the present invention preferably has a shape and size suitable for realizing cell suction and ejection in cell units. Specifically, as the tube shape constituting the tip microstructure (41), it is preferable that the inner diameter of the cross section is 500 μm or less, preferably 200 μm or less, more preferably 100 μm or less, and further preferably 80 μm or less. .. The lower limit of the inner diameter of the tube tip can be set according to the intended use of the target cell, and examples thereof include 5 μm or more, preferably 10 μm or more, and more preferably 15 μm or more.
The pipe wall thickness of the pipe tip is not particularly limited as long as it can secure the strength of the pipe tip, but may be in the range of 1 to 100 μm, preferably 3 to 50 μm.
Further, the tube shape constituting the tip microstructure (41) is preferably a circular tube or a substantially circular tube having a circular or substantially circular cross section.

As the material constituting the fine capillary pipe (4) according to the present invention, at least the portion constituting the tip microstructure (41) is preferably at least from the pipe tip so that the inside of the pipe can be visually recognized. It is preferable that the portion having a pipe length of 10 mm or less, more preferably at least the portion having a pipe length of 20 mm or less from the pipe tip, is formed of a material having light transmission and sufficient strength. Specifically, it is preferable that these portions are molded of a transparent glass material or a transparent resin. As the material, it is preferable to use a transparent glass material in consideration of strength, durability, cost and the like.

Structures other than the pipe tip The structure (44) on the tube tip side of the fine capillary pipe according to the present invention is different from the above-mentioned tip microstructure (41) and is suitable for maintaining the strength of the entire pipe to some extent. It is possible to adopt the one having the pipe diameter of. As one aspect of the fine capillary tube according to the present invention, for example, the outer diameter of the tube (44) on the side of the tube (44) from the bent or curved shape is 0.5 mm or more, preferably 0.6 mm or more, more preferably 0. Those having a diameter of 7 mm or more can be adopted. The upper limit is preferably 3 mm or less, preferably 2 mm or less, and more preferably 1.5 mm or less in consideration of the size suitable for a small amount of operation and the correspondence to the fine wells of the sample container. ..
As the capillary pipe structure (44) on the Sugamoto side, a structure having a gentle curve or the like can be adopted as long as the contents visibility and operability are not significantly affected, but fine wells and the like can be adopted. Considering the access to the bottom surface, the shape is preferably a straight tubular shape or a substantially straight tubular shape within a range of at least 10 mm, preferably at least 15 mm or less on the side of the tube from the first bent or curved shape. Is preferable. More preferably, the portion has a straight pipe shape.
The material constituting the capillary tube (44) on the side of the tube tip microstructure is not particularly limited as long as it can function as a capillary tube, such as glass, resin, or metal. It is preferably made of a glass material or a transparent resin having light transmittance. As the material, it is preferable to use a transparent glass material in consideration of strength, durability, cost and the like.

The fine capillary pipe (4) according to the present invention is preferably a member in which the fine structure at the tip of the pipe and the pipe structure on the base side of the pipe are integrally formed in consideration of strength, cost and the like. Preferably, it is integrally molded with a transparent glass material.

Further, in the Sugamoto side structure (44), in order to avoid contact with a member on the microscope side in the space above the microscope stage surface (20), a second bent or curved shape is provided separately from the above microstructure. The structure (45) provided with the above can also be adopted. The second bent or curved shape (45) is preferably formed at a position at least 10 mm or more, preferably at least 15 mm or more away from the first bent or curved shape (43) on the side of the tube. In the fine capillary tube having such a second bent or curved shape (45), the upper space of the sample stage surface (20) is well secured, and the upper part of the microscope device (6) in the working space is improved. This is a preferred embodiment in terms of avoiding contact with the structure.

In one aspect of the use of the cell transport system (1) according to the present invention, the microcapillary tube (4) according to the present invention has a microscopic drive and control of cell suction and protrusion on the Sugamoto side (44). It can be used by connecting to a solution supply pipe (47) or the like connected to a liquid volume pump (46). Here, as the minute liquid amount pump (46), the pump vibration is remarkably reduced, and a pump capable of sucking and discharging a solution having a minute amount of 1 μL or less, preferably 0.1 pL to 0.1 μL. Can be adopted. As such a minute liquid amount pump, for example, a pump using the principle of an electroosmosis flow pump, an oil-filled vibration-free pump, a piezo-driven pump, an instantaneous high temperature cooling pump in a capillary, or the like can be used. However, a technique using another principle can be used as long as it can exhibit the function of controlling the liquid amount.

In the usage mode of the cell transport system according to the present invention, the microcapillary tube (4) according to the present invention can be used by being directly or indirectly fixed or retained by the microcapillary tube supporting means (48). Fixing to the fine capillary pipe supporting means (48) includes a mode in which the pipe base portion of the fine capillary pipe, the solution supply pipe connected to the pipe base portion, the minute liquid amount pump main body, and the like are fixed or retained and held. However, the specific embodiment is not particularly limited.
As the structure of the fine capillary pipe supporting means (48), any structure can be adopted without particular limitation as long as it is a support member capable of fixing the position of the pipe tip of the fine capillary pipe. For example, as the support means, it is possible to adopt a self-supporting support member having its own pedestal. Further, as the support member, a member capable of fixing the position of the fine capillary tube by connecting to another device member or the like (for example, the main body of the microscope device, the microscope sample stage support means, etc.) may be adopted. It is possible.

The fine capillary tube supporting means (48) according to the present invention may be provided with a driving means capable of moving the fine capillary tube (4) in the Z-axis direction. Further, as the fine capillary pipe supporting means according to the present invention, it is also possible to use an operating means (49) such as Joyce Tech to enable the position adjusting operation of the pipe tip.

[Other components]
The cell transport system (1) according to the present invention excludes a system including constituent articles other than the microscope sample stage (2), control means (3), fine capillary tube (4), etc. described above. It's not something. For example, a system aspect including a sample container, each support means, a minute amount pump, an input means, a display means, and the like is also included in the system according to the present invention. Further, the cell transport system according to the present invention also includes a device that has already been arranged in the main body (6) of the microscope device and is in a usable state.

2. Functions exhibited by the cell transport system In the cell transport system (1) according to the present invention, the above paragraph 1. By disposing and using the system constituent member described in the above in the microscope device (6), it becomes possible to use it as a high-performance cell transport system that can be used for cell transport in units of cells.

[Automatic focusing operation and visibility]
In the cell transfer system (1) according to the present invention, by the action of the configuration described in the above paragraph, the focusing operation on the bottom surface of the sample container (5) required in the one cell transfer technique can be performed on the microscope sample stage surface (20). ) (1 article) can be realized by the automatic movement operation "only". As a result, in the system according to the present invention, it is possible to automatically perform the focusing operation even for a high-contrast solution in which the focus correction by the autofocus control does not operate normally, and the transfer source container and the transfer destination container can be executed. It is possible to continuously and efficiently perform cell aspiration and cell ejection while moving back and forth between the focus positions corresponding to the differences in the bottom surfaces of the above.
Here, the autofocus inhibition phenomenon caused by the high contrast in the field of view of the microscope is a phenomenon that frequently occurs during transportation to a very small amount of solution filled in the destination container, such as one-cell analysis involving a large amount of gene analysis. is there. Therefore, the cell transport system (1) according to the present invention, which does not require focusing depending on autofocus control, can be a technique for providing efficient workability in the said application.

The cell transfer system (1) according to the present invention is a cell transfer system having a high function that enables transfer of target cells in a transfer source container to a desired transfer destination container in units of one cell. The cell transport system (1) according to the present invention has a function of enabling suction and discharge of target cells in units of one cell using a fine capillary tube, but depending on setting conditions and / or device configuration. It is also possible to perform a suction or discharge operation of two or more cells. Similarly, the aspect of the cell transport system (1) according to the present invention does not exclude a system that enables a suction or discharge operation of a cell mass, a tissue piece, or the like.

In the cell transport system (1) according to the present invention, the transport source container (51) in which the target cells are stored or arranged before the operation, the transport destination container (53) which is the transfer destination container, and the like are stored in the sample container holding means. It is installed and used in (201). When installing these sample containers in the sample container holding means, it is also possible to use an adapter member or the like.

Here, as the “transport source container” (51), a sample container having an open top surface in which the target cells can be stored in the container or placed on the container can be mentioned. In the normal use mode of the system (1) according to the present invention, cells are stored or arranged in the container (51) together with a solution such as a medium. The shape of the container is not particularly limited, but it is possible to use a container in which cells are individually dispensed, a container containing a culture solution of the cultured cells of the dispensing source, or the like. Specific embodiments of the container include containers such as multi-well plates, culture dishes, PCR tubes, medium reservoirs, slide glasses, microchips, and the like. In addition, in this specification, a slide glass and a microchip can also be used as those contained in a sample container.

Further, the “transportation destination container” (53) refers to a sample container to which the target cells move. As the container, the same container as that mentioned in the transport source container can be used, but a fine container into which the analytical reagent and the culture medium have been dispensed can be used. Further, when performing a cell transport operation in 1-cell analysis or the like, a microwell plate having 48 to 384 holes provided with fine fine wells, a PCR tube plate, a PCR cap, and a microdevice provided with microholes. , Hydrophilization spots on slide glass-like plates, etc. can be mentioned.
In view of the technical features of the system according to the present invention, it does not mean that the usable destination container (53) is limited to these. In the system (1) according to the present invention, in addition to being able to use a sample container having a large diameter, it is efficient with cell ejection into fine wells and the like, which was difficult to apply by conventional microscopy techniques and the like. Cell transport is possible.

In the cell transport system (1) according to the present invention, it is possible to confirm the cell storage state in the tip (42) tube of the fine capillary tube that has accessed the bottom surfaces of the transport source container, the transport destination container, and the like.
Specifically, in the cell transport system (1) according to the present invention, when the microwells and the like are accessed vertically or substantially vertically to the fine capillary tube due to the unique shape of the tip microstructure (41) of the fine capillary tube. Even if there is, it is possible to access the bottom surface of the sample container (5) so that the tube tip portion having a fine structure is horizontal or has an appropriate inclination angle. As a result, in the system (1) according to the present invention, even when the objective lens or the light source is arranged in the vertical direction, the optical path is not blocked by the tube base side (44) of the fine capillary tube, so that the fine capillary is formed. Tip of tube (42) It becomes possible to visually recognize the state of cells in the tube. That is, it is possible to visually confirm the state of cell suction into the tube and cell discharge from the tube in the tube tip microstructure.
Here, the fact that the tip (42) of the fine capillary tube "accesses" the bottom surface of the sample container (5) means that not only the state of contact with the upper surface of the bottom surface of the container but also the cells whose tip is on the bottom surface can be sucked. The state of close proximity is also included. Expressed as a specific numerical value of the distance, for example, a state of being close to each other within a range of 2 mm or less, preferably 1 mm or less, and more preferably 0.5 mm or less is included.

In the usage mode of the system (1) according to the present invention, the microcapillary tube (4) is arranged above the microscope sample stage surface (20), and the microscope sample stage surface (20) is moved three-dimensionally. By executing this, the access operation of the sample container (5) is executed by moving the tube tip (42) from below.
Here, in the access operation according to the present invention, the position (54) where the focus focus from the objective lens (62) exists and the position of the tube tip (42) of the fine capillary tube are fixed, and the microscope sample. It can be executed by the automatic movement operation "only" of the stage surface (20). In this operation, the pipe tip (42) of the fine capillary tube is left in a fixed state, and the access operation of the pipe tip (42) from the upper surface opening of the sample container (5) toward the bottom surface is executed.
In the access operation according to the present invention, it is also possible to perform an operation of finely adjusting the position of the tip (42) of the fine capillary tube in order to perform cell suction or discharge after the movement operation of the sample stage surface (20). However, it is not essential to actively move the pipe tip side as a basic operation.

In the specification aspect of the system (1) according to the present invention, the posture of the fine capillary tube (4) in the access operation is such that the tube tip (42) is horizontally or appropriately tilted with respect to the bottom surface of the sample container (5). It is preferable that the components are arranged so as to be. Specifically, the inclination of the pipe tip with respect to the transport source container and the bottom surface of the transport destination container is within 45 °, preferably within 30 °, more preferably within 20 °, and further preferably within 10 ° with respect to the horizontal plane. Is preferable.
Here, in the access operation in the present invention, when a microplate or the like provided with a fine well is used, the structure portion on the side of the tube is perpendicular to (or large) with respect to the container wall surface with respect to the fine structure of the fine capillary tube. It is necessary to perform the access operation within 20 °, preferably within 10 °). However, due to the feature of the tip microstructure (41) of the fine capillary pipe according to the present invention, the pipe tip can be set horizontally or at an appropriate inclination angle. It is possible to access the bottom surface of the container in this way.

[Use for placement in microscope equipment]
In the cell transport system (1) according to the present invention, the automatic access operation to the tip (42) of the fine capillary tube having the focusing operation can be executed by the self-sustaining control of only the microscope sample stage surface (20). Become. Therefore, the cell transport system (1) according to the present invention has a device configuration integrated with the microscope without controlling or interlocking operations of other members such as the fine capillary tube (4) and the objective lens (62). It is possible to construct a cell transport system that does not require this.

The cell transport system (1) according to the present invention includes a system component composed of a component article arranged in a normal microscope device (6) and capable of use. Therefore, the above paragraph 1. By arranging the system component described in the above in the conventional general microscope device (6), it becomes possible to use it as a high-performance cell transfer system that can be used in the one-cell transfer technique.

The cell transport system (1) according to the present invention is described in paragraph 1. By designing the arrangement of the microscope sample stage surface (20) and the sample stage support means (25) in such a manner that the positional relationship with the objective lens (62) is appropriate, as described in the above, a general microscope apparatus. Can be used for placement in. That is, the cell transport system according to the present invention can be used as a general-purpose member for the existing microscope device (6).
As an embodiment of the arrangement of the cell transport system (1) according to the present invention on the microscope device (6), one aspect is a sample stage in which the microscope device main body originally includes the microscope sample stage (2) according to the present invention. An embodiment in which the sample stage (61) is arranged above the sample stage (61) or in place of the sample stage (61) can be mentioned.
Further, as an arrangement mode of the fine capillary tube supporting means (48) according to the present invention, it is fixed to the main body of the microscope device (6) or to an immovable portion of a member constituting the microscope sample stage (2) according to the present embodiment. Further, an embodiment in which the fine capillary tube (4) is arranged above the sample stage surface (20) and used can be mentioned.

Here, as the microscope device (6) capable of disposing and using the cell transport system according to the present invention, it can be applied to a normal optical microscope device. As the microscope device (6) that can be used in this arrangement, it can be applied to any of an upright microscope, an inverted microscope, and the like. It can also be placed and used in a stereomicroscope. It can also be used in fluorescence microscopy equipment.

As the cell transport system according to the present invention, the cell transport system (1) according to the present invention also has a system embodiment already arranged in the main body of the microscope device (6) described above and is in a usable state. include. That is, in the present invention, the above paragraph 1. In addition to the cell transport system including the constituent articles described in the above, the cell transport system (1) according to the present invention also includes a cell transport system including a microscope device. In addition, the aspect of the cell transport system (1) according to the present invention also includes an aspect in which the product is integrated with the main body of the microscope device from the beginning.

[Comparison of operability]
The cell transport system (1) according to the present invention is a work assuming one-cell analysis as in Example 3 (that is, the bottom structures of the cell transport source container and the transport destination container are different, and the solution of the transport destination container is different. When used in a situation where the contrast is high), the operation steps required from cell aspiration to cell ejection can be performed only by the following four operation steps.
That is, i) automatic movement and automatic focusing operation of the microscope sample stage surface (downward movement in the Z-axis direction, movement in the XY direction, upward movement in the Z-axis direction), ii) cell suction operation in the transport source container. , Iii) Automatic movement and automatic focusing operation of the microscope sample stage surface (downward movement in the Z-axis direction, movement in the XY direction, upward movement in the Z-axis direction), iv) Cell discharge operation in the destination container, It is possible to carry out cell transport with only simple operations by only the "4" operation steps of (see FIG. 9).
In this embodiment, it is assumed that the bottom surface shape is different between the transport source container and the transport destination container, but since the focusing operation is performed at the same time when the microscope stage surface is moved, the container bottom is moved to the pipe tip. The operation and the focusing operation can be achieved by "only one operation". Further, as the solution in the transport destination container, it is assumed that the autofocus control does not function due to high contrast, but in the system according to the present invention, the automatic focusing operation is executed by a principle completely different from the autofocus control. It will be possible.

On the other hand, the operation steps required when the same operation content is executed by using the XY plane moving microscope stage, which is a conventional technique, are as follows. That is, i) automatic movement of the microscope sample stage surface (movement in the XY direction), ii) downward movement operation of the fine capillary tube, iii) manual focusing, iv) cell suction operation in the transport source container, v) fine Upward movement operation of capillary tube, vi) Automatic movement of microscope sample stage surface (movement in XY direction), vii) Downward movement operation of fine capillary tube, viii) Manual focusing, ix) Cell discharge in destination container Operation, "9" operation steps are required.
Here, in the comparison mode, a "manual" focusing operation is required to compensate for the inability of autofocus. Further, in the comparative aspect, in order to avoid contact between the fine capillary tube and the container wall, an operation of moving the fine capillary tube upward and downward is always required.

The difference in the required number of steps from the conventional cell transfer device provided with the XY plane moving microscope stage is that the more the cell transfer operation needs to be repeated, the more remarkable the difference in work efficiency becomes. That is, it is recognized that the cell transport system (1) according to the present invention is a system having an advantage in work efficiency as the analysis is performed on multiple samples or on a large scale.

3. 3. Cell Transport Method The present invention includes an invention relating to a method for transporting a target cell, which is characterized by using the cell transport system (1), to a desired transport destination container in units of one cell. As the steps constituting the method, the operation procedure and the like described in the above paragraph are referred to.

Hereinafter, the present invention will be described with reference to examples, but the scope of the present invention is not limited thereto. Hereinafter, the cell transport system produced in this example will be described with reference to a structural diagram, a usage mode diagram, and the like.

[Example 1] "Manufacturing of cell transport system"
As one aspect of the cell transport system according to the present invention, a cell transport system including the following constituent articles was manufactured.

(1) Three-dimensional automatic movement microscope sample stage A microscope sample stage (2) capable of automatic movement in the X-axis, Y-axis, and Z-axis directions constituting the cell transport system (1) according to this embodiment is manufactured. did. The structure of the microscope sample stage (2) according to this example is shown in FIGS. 1 to 3 and 5.
The microscope sample stage (2) in this embodiment is mainly composed of a sample stage surface (20), an X-axis moving actuator (22), a Y-axis moving actuator (23), and a Z-axis moving actuator (24). It becomes.
The upper surface of the sample stage surface (20) is provided with the sample container holding means (201), and is provided with an opening structure near the center where the sample container (5) is installed.
Each actuator moves along an axis by a support portion (221, 231 and 241), a power unit (222, 232, 242) embedded inside the support portion, and a force from the power unit. It is a structure composed of a driving unit (223, 233, 243) and the like as a main member. Each actuator is an actuator member provided with an electric drive means capable of freely moving in each axial direction. As the power unit and drive unit of the actuator according to this embodiment, a servomotor (RCP6 Robocylinder, manufactured by IAI) using DC (direct current power supply) drive was used.

The constituent articles of the microscope sample stage (2) are fixedly held by the microscope sample stage supporting means (25) in a state in which the articles are combined and configured. The microscope sample stage supporting means (25) is located at a position where the objective lens portion (62) is sandwiched from both the top view and the front view of the microscope device (6) when the microscope device (6) is arranged and used. It is a structure provided with a gantry (251) and a support member (252) horizontally bridged between the gantry.
The support portion (221) of the X-axis moving actuator is fixedly held on the bridged support (252) of the microscope sample stage supporting means: Y is attached to the driving portion (223) of the X-axis moving actuator. The support portion (231) of the axial movement actuator is fixedly held; the support portion (241) of the Z-axis movement actuator is fixedly held by the drive portion (233) of the Y-axis movement actuator; The stage surface (20) of the microscope sample stage is fixed to the drive unit (243) of the Z-axis moving actuator.

The movement control of the microscope sample stage (2) is performed by the control means (3), which is a semiconductor circuit arranged in a housing separate from the main body. The control means (3) can store the position coordinates in arbitrary three-dimensional coordinates, and by controlling the drive amounts of the three actuators in conjunction with each other based on the stored information, the microscope sample The stage is moved. The system according to this embodiment includes a touch panel controller (31) as an input means, and can input and store the position coordinates.

(2) Specially Shaped Fine Capillary Tube A fine capillary tube (4), which is a constituent article of the cell transport system (1) according to the present embodiment, was manufactured. The appearance of the fine capillary tube according to this embodiment is shown in FIG.
The fine capillary tube (4) is provided with a first bent shape (43) of 100 ° in a tip microstructure portion (41) located 1.25 mm from the tip, and the tip portion (42) has an inner diameter of φ0.05 mm. It is a straight tube shape. The Sugamoto side (44) of the first bent shape is formed of a straight tube shape having an outer diameter of φ1 mm, which is thicker than the tip microstructure (41), and is located 22 mm on the Sugamoto side of the first bent shape. It is provided with a second bent shape (45) that is bent by 105 ° in a direction opposite to the bending direction of the first bent shape.
The entire fine capillary tube (4) including the tip microstructure and the Sugamoto side structure is integrally molded with borosilicate glass.

The fine capillary pipe (4) is formed by connecting the Sugamoto side to the solution supply pipe (47) on the micro liquid amount pump (46) side. As the minute liquid amount pump (46), a TOPic pump (manufactured by Yodaka Giken Co., Ltd.), which is an oil-filled vibration-free pump, was used. The micro-liquid amount pump (46) is connected to and fixedly held by the micro-capillary pipe supporting means (48). In this embodiment, the microcapillary tube supporting means (48) is in a mode in which it can be directly connected to and fixed to the microscope device main body (6), and the tube tip position is finely adjusted by the operating means (49) using Joyce Tech. It can be adjusted. FIG. 8 shows a photographed image of the state in which the fine capillary tube (4) is arranged above the sample stage surface (20).

(3) Arrangement in a microscope device In this embodiment, a system is constructed in which the constituent articles of the cell transport system are arranged in an inverted optical microscope product (CKX41, manufactured by Olympus Corporation), which is a commercially available microscope device (6). did. In this embodiment, the manufactured microscope sample stage (2) is arranged at a position above the stage surface (61) originally provided by the main body of the microscope device (6) and below the illumination means (63). The mode to be used is shown. Further, in the present embodiment, the fine capillary tube supporting means (48) is connected and fixed to the side surface of the sample stage (61) originally provided in the main body of the microscope device (6), and the tube tip (42) is the present implementation. An embodiment in which the microscope sample stage surface (20) according to the example is arranged and used is shown.

The system (1) according to the present embodiment includes a touch panel controller (31) as a means for inputting position information of three-dimensional coordinates. Further, a monitor (32) for displaying a microscope image is provided.

(4) Device specifications The specifications of the cell transfer system (1) manufactured in this embodiment are shown below.

[Microscope sample stage]
X-axis direction movement range: 20 cm
Y-axis direction movement range: 10 cm
Z-axis direction movement range: 5 cm
Stage movement speed: 5 cm / sec Minimum fine movement unit: 5 μm
[Small liquid volume pump]
Type: Oil-filled vibration-free pump Set flow rate: 300 to 900 pL
[Fine capillary tube]
Tip microstructure: 100 ° bending shape at tip 1.25 mm Tube diameter: Tip inner diameter φ0.05 mm, Sugamoto outer diameter φ1 mm
Material: Made of transparent borosilicate

[Example 2] "Comparison with conventional cell transport system technology: automatic focusing operation"
When the cell transport system (Product 1 of the present invention) manufactured in Example 1 was used to perform an operation assuming one-cell analysis, the performance of the automatic focusing operation compared with the prior art was evaluated.

A sample container holding means (20) on a microscope sample stage surface (20) manufactured in Example 1 by placing an agarose fine particle suspension having a particle size of 30 μm on a slide glass which is a transport source container (51). It was arranged in 201). After placement, it was confirmed that the agarose fine particles were sunk on the bottom surface of the slide glass.
Further, 1 μL of distilled water was dispensed into the bottom surface of a polypropylene 8-unit PCR cap which is a transport destination container (53), and placed in a sample container holding means (201) different from the above. Here, it was confirmed that the liquid level of the trace solution on the bottom surface of the PCR cap was dropped due to surface tension.
Next, the three-dimensional coordinates of the upper surface (bottom surface of the container) of the slide glass and the three-dimensional coordinates of the bottom surface of one of the eight PCR caps are set, and the focus focus position is 4 times the objective lens and the working distance is 10 mm. Was set. Next, after confirming that the surface on the slide glass is in focus, one of the fine particles is sucked into the fine capillary tube, and the top surface of the bottom surface of one container of the 8-unit PCR cap is the tip of the fine capillary tube. The state of the microscope image was confirmed without automatically moving to the position and manually focusing.

As a result, by moving the microscope sample stage surface (20) and moving the bottom surface of the 8-unit PCR cap, which is the transport destination, to the position of the tube tip (42) of the fine capillary tube, the movement is automatically performed after the movement. It was confirmed that the lens was in focus. This is an operation of focusing by adjusting the position only on the container side while keeping the position (54) where the focus focus from the objective lens exists and the position (42) of the tip of the fine capillary tube fixed. It was recognized as an achievable effect (see FIG. 9).
On the other hand, as a comparative test, when an automatic focusing operation was performed on the trace solution at the bottom of the 8-unit PCR cap using a conventional microscope device equipped with a focus correction function by autofocus control, a high contrast phenomenon occurred. The autofocus correction did not work properly and a clear microscope image could not be obtained.

From the above results, in the cell transport system according to the present invention, the focusing operation can be performed without any problem even when the solution at the cell transport destination is a trace amount solution having high contrast, which is difficult to control autofocus. Was shown.
From this, in the cell transport system according to the present invention, the work assuming one-cell analysis (that is, the bottom structures of the cell transport source container and the transport destination container are different, and the solution in the transport destination container has high contrast. It was shown that the focusing operation can be performed simultaneously by performing only one movement operation of the microscope sample stage (under the situation).

[Example 3] "Comparison with conventional cell transport system technology: evaluation of operational efficiency"
When the cell transport operation assuming 1-cell analysis was performed using the cell transport system (Product 1 of the present invention) manufactured in Example 1, the efficiency of the cell transport operation compared with the prior art was evaluated.

In the cell transport system (Product 1 of the present invention) produced in Example 1, a slide glass in which agarose fine particles were sunk on the bottom surface and 1 μL of distilled water were dispensed in the same manner as in Example 2. An 8-strand PCR cap made of agarose was placed on the sample container holding means (201) on the microscope sample stage surface (20) manufactured in Example 1. Here, the slide glass which is the transport source container (51) and the PCR cap which is the transport destination container (53) are containers having different shapes on the bottom surface (upper side surface in contact with the liquid).
Next, set the 3D coordinates of the upper surface (bottom surface of the container) of the slide glass and the 3D coordinates of the bottom surface of each of the 8-unit PCR caps, and set the focus focus position to be 4 times the objective lens and a working distance of 10 mm. did. An operation was performed in which fine particles were transferred one by one from the slide glass to each hole of the 8-unit PCR cap.

On the other hand, as a comparative test, a system (comparative product 1) in which a support means equipped with a fine capillary tube manufactured in Example 1 is connected and fixed to a sample stage of an XY planar moving microscope, which is a conventional technique, is constructed and similarly fine. The operation of transporting the particles one by one was performed.

As a result, the amount of transmitted light and the thickness of the bottom surface (the surface in contact with the liquid) of the slide glass and the PCR cap used in this example are different. It was shown that it can be easily executed only by the operation.
Specifically, in the cell transport system according to the present invention (product 1 of the present invention), the position (54) where the focus focus from the objective lens exists and the position (42) of the tip of the fine capillary tube remain fixed. , It was shown that the cell transport operation is possible. Due to the characteristics of this operation, in the product 1 of the present invention, it was shown that the operation step required for one operation from cell suction to cell ejection can be performed only by the four operation steps shown in Table 2 (Fig.). 9). Moreover, these operation steps could be executed only by a simple button operation.

On the other hand, when the XY plane moving microscope sample stage (comparative product 1), which is a conventional technique, is used, a manual focusing operation is performed to compensate for the inability of autofocus each time cell suction and cell ejection are performed. It took. In addition to this, in the operation using Comparative Product 1, in order to avoid contact between the fine capillary tube and the container wall, it was necessary to separately move the fine capillary tube up and down each time the microscope sample stage was moved in a plane. ..
Finally, in order to perform one operation from cell aspiration to cell ejection using Comparative Product 1, nine operation steps shown in Table 2 are required, and the process is repeated for each of the eight PCR caps. The effort required to perform the operation was great.

As shown in the above results, when the cell transport operation is performed using the system (1) according to the present invention, the work assuming one cell analysis (that is, the bottom structures of the cell transport source container and the transport destination container are different). In addition, even under the condition that the solution in the transport destination container has high contrast), the moving operation and the focusing operation of the microscope sample stage surface (20) can be performed simultaneously, and continuous cell transport can be performed. It was shown that the operation can be performed efficiently.
As a result, in the cell transport system (1) according to the present invention, the more the multi-sample analysis requires many repetitive steps of the cell transport operation, the more the system has structural features that can exert an advantage in terms of work efficiency. It was shown to be.

1. 1. Cell transport system 2. Microscope sample stage (three-dimensional space moving sample stage)

20. Sample stage surface 201. Sample container holding means

21. Drive means

22. Actuator for X-axis movement 221. Support part 222. Power unit 223. Drive 225. cable

23. Actuator for Y-axis movement 231. Support part 232. Power unit 233. Drive unit 235. cable

24. Actuator for Z-axis movement 241. Support part 242. Power unit 243. Drive 244. cable

25. Microscope sample stage support means 251. Stand 252. Support member

3. 3. Control means 31. Input means (touch panel controller)
32. Monitor (microscope image output means)

4. Fine capillary tube 41. Tip microstructure 42. Pipe tip 43. First bent or curved shape 44. Sugamoto side pipe structure 45. Second bent or curved shape 46. Micro liquid volume pump 47. Solution supply tube 48. Fine capillary tube support means 49. Fine capillary tube operating means

5. Sample container 51. Transport source container 52. Sample 53. Destination container 54. The position where the focus is located

6. Microscope device 61. Original microscope sample stage 62. Objective lens 63. Lighting means 64. Eyepiece

The present invention is expected to be effectively used in various industrial fields that require a cell transport operation as a physics and chemistry device that enables cell transport of a minute amount of liquid. For example, it is expected to be effectively used in various industrial fields such as life science field, medical field, pharmacy field, food field, bio-related product development field, agriculture field, and livestock field.
In particular, the present invention is effectively used as a basic technology for significantly improving the efficiency of single cell analysis, and contributes to research progress in cancer research, stem cell research, iPS cell research, biopharmacy research, microbial substance production, and the like. It is expected.

Claims (3)

Using a cell transfer system that holds a transfer source container and a transfer destination container, which are sample containers, on the sample stage surface, the transfer source of the sample stage surface is at the tip of the microstructure of the tip of the fine capillary tube provided in the cell transfer system. It is a method of transporting the target cells from the container to the solution in the destination container in units of one cell;
The cell transport system is a cell transport system capable of transporting target cells to a desired transport destination container in units of one cell, and (Constituent Article A) is provided with a sample container holding means on the sample stage surface and X. Microscope sample stage with drive means capable of automatic movement in the axis, Y-axis, and Z-axis directions, (Constituent B) Three-dimensional coordinates with respect to the sample stage surface are memorable and the stored relatives. A control means for automatically moving the sample stage surface to an arbitrary set three-dimensional coordinate based on position information (Constituent Article C) A fine capillary tube arranged above the sample stage surface for cell suction. And a cell transport system comprising a microcapillary tube comprising a bent or curved shape having a tip microstructure in the range of 100-120 ° for cell ejection, and (Constituent D) an optical microscope device. Yes;
(Solution in the transport destination container) The solution in the transport destination container is a solution in which the liquid level is dropped due to surface tension and the microscopic image is highly contrasted.
(Step A) The position where the focus focus from the objective lens of the optical microscope device exists is set to the position that matches the bottom surface of the transport source container, and the tip of the fine structure at the tip of the fine capillary tube is placed in the tube with the focus focus. The position where cells can be aspirated is set, and the position of the focus focus and the position of the tip of the fine structure at the tip of the fine capillary tube are fixed.
(Step B) The bottom surface of the transport destination container held on the sample stage surface is moved downward in the Z-axis direction to three-dimensional coordinates set based on the relative position information stored in advance. , The movement in the XY plane direction, and the upward movement in the Z-axis direction are automatically performed, and the movement fixes the position where the focus focus from the objective lens exists and the tip of the tip microstructure. Focusing on the trace amount of solution in the destination container in that state;
A method for transporting the target cells in units of one cell, which comprises a step . The tip microstructure of the fine capillary tube is a tube structure having an inner diameter of 5 to 500 μm, and is a tip microstructure including a bent or curved shape portion within a range of 5 mm or less from the tube tip. A method for transporting the described target cells in units of one cell. The driving means provided in the microscope sample stage has a driving unit capable of moving in units of 0.1 to 100 μm, and the first to third actuators have different operating axes in three-dimensional directions. Including a structure, which is arranged such that;
The support portion of the first actuator is fixedly held by the microscope sample stage support means, the support portion of the second actuator is fixedly held by the drive portion of the first actuator, and the drive portion of the second actuator is fixedly held. The support portion of the third actuator is fixedly held, and the stage surface of the microscope sample stage is fixed to the driving portion of the third actuator;
The method for transporting the target cells according to claim 1 or 2 in units of one cell.

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