WO2019064984A1

WO2019064984A1 - Phase difference observation device and cell treatment device

Info

Publication number: WO2019064984A1
Application number: PCT/JP2018/030464
Authority: WO
Inventors: 翔一本田; 松本　潤一; 忠夫森下
Original assignee: 株式会社片岡製作所
Priority date: 2017-09-28
Filing date: 2018-08-17
Publication date: 2019-04-04
Also published as: CN111164487B; DK3677945T3; CN111164487A

Abstract

The present invention provides a phase difference observation device of a reduced size that does not need an additional configuration for an imaging device and is capable of suppressing the degradation in a phase difference image due to a meniscus. A phase difference observation device according to the present invention comprises: a light source; an illumination optical system that guides illumination light from the light source to an observation target in a cell culture vessel; an imaging optical system for focusing the optical image of the observation target onto an imaging element; and a control unit. The illumination optical system includes a spatial modulation element that changes the intensity distribution of the illumination light. The control unit includes intensity distribution correction information that maps the position of the imaging optical system relative to the cell culture vessel to the intensity distribution of the illumination light at the position of the imaging optical system. The control unit acquires the imaging system position information that represents the position of the imaging optical system and changes the intensity distribution of the illumination light at the spatial modulation element on the basis of the imaging system position information and the intensity distribution correction information.

Description

Phase contrast observation device and cell processing device

The present invention relates to a phase contrast observation device and a cell processing device.

When observing the vicinity of the wall surface of the cell culture vessel, the liquid surface of the culture solution is curved due to surface tension (meniscus), light may be refracted, and light may be reflected in the phase difference image. In addition, when light is reflected in the phase difference image, there is a problem that the phase difference effect is lost such that the background of the phase difference image is dark and the observed object in the cell culture vessel looks bright ( Deterioration of the differential image).

As a method of preventing the deterioration of the phase difference image, a device corresponding to the deterioration of the phase difference image by moving the ring slit (opening ring) and the condenser lens of the phase difference observation device has been proposed (patent document 1).

International Publication 2014/091661

However, in the device of Patent Document 1, in order to sequentially adjust the positions of the ring slit and the condenser lens, an imaging unit capable of capturing an image of the aperture ring and an image of the phase plate, and an imaging unit for observation Are separately provided, and based on the obtained image of the aperture ring and the image of the phase plate, the amount of movement of the ring slit and the condenser lens is calculated. Then, the positions of the ring slit and the condenser lens are adjusted based on the obtained movement amount. For this reason, an additional configuration is required to adjust the position of the ring slit and the condenser lens, and there is a problem that the phase contrast observation device is enlarged.

Then, this invention aims at providing the phase contrast observation apparatus which can miniaturize an apparatus and can suppress deterioration of the phase contrast image by the said meniscus, for example by requiring the structure of an additional imaging part. I assume.

In order to achieve the above object, a phase contrast observation apparatus of the present invention comprises:
An illumination optical system for guiding illumination light from the light source to an object in a cell culture vessel;
An imaging optical system for forming an optical image of the object to be observed on an imaging device;
Including a control unit,
The illumination optical system includes a spatial modulation element that changes the intensity distribution of the illumination light,
The control unit
It includes intensity distribution correction information that associates the position of the imaging optical system with respect to the cell culture vessel and the intensity distribution of illumination light at the position of the imaging optical system,
Acquiring imaging system position information which is the position of the imaging optical system;
The intensity distribution of the illumination light in the spatial modulation element is changed based on the imaging system position information and the intensity distribution correction information.

The cell processing apparatus of the present invention comprises an observation unit capable of observing an object in a cell culture vessel.
A laser irradiation unit capable of irradiating a laser to the object to be observed;
A control unit that controls at least one of the observation unit and the laser irradiation unit;
The observation unit is the phase difference observation apparatus of the present invention.

According to the phase difference observation apparatus of the present invention, for example, since the configuration of the additional imaging unit is unnecessary, the apparatus can be miniaturized, and deterioration of the phase difference image due to the meniscus can be suppressed.

FIG. 1 is a schematic view showing an example of the phase difference observation apparatus in the first embodiment. FIG. 2 is a block diagram showing an example of a control unit in the phase difference observation apparatus of the first embodiment. FIG. 3 is a schematic view showing an example of a phase difference observation apparatus in the case of acquiring intensity distribution correction information in the first embodiment. FIG. 4 is a schematic view showing an example of an image of the phase plate 32 for correction and an image of illumination light captured by the imaging device in the first embodiment. FIG. 5 is a flowchart showing an example of a method of capturing a tiling image of a phase difference image using the phase difference observation device of the first embodiment. FIG. 6 is a flowchart showing another example of the method of acquiring intensity distribution correction information in the imaging method of the tiling image of the phase difference image using the phase difference observation apparatus of the first embodiment. FIG. 7 is a flowchart showing another example of the step S3 in the imaging method of the tiling image of the phase difference image using the phase difference observation apparatus of the first embodiment. FIG. 8 is a perspective view showing an example of a cell processing apparatus according to a second embodiment. FIG. 9 is a schematic view showing an example of the cell processing apparatus in the second embodiment. FIG. 10 is a perspective view showing an example of a first region in the cell processing device of the second embodiment. FIG. 11 is a cross-sectional view of the first region seen from the II direction in FIG. In FIG. 12, (a) is an exploded perspective view showing an example of the culture vessel placement portion in the cell processing apparatus of Embodiment 2, and (b) is a cross-sectional view seen from the III-III direction in FIG. It is. FIG. 13 is a perspective view showing an example of the first area and the circulating means when the outer wall of the first area is removed in the cell processing device of the second embodiment. FIG. 14 is a cross-sectional view of the upper portion of the first region and the circulating means, as viewed from the II-II direction in FIG. In FIG. 15, (a) is a perspective view showing an example of the configuration of the second area of the cell processing apparatus of Embodiment 2, and (b) is a perspective view showing another example of the configuration of the second area. It is. FIG. 16 is a block diagram showing an example of the configuration of a control unit of the cell processing apparatus of the second embodiment. FIG. 17 is a perspective view showing another example of the cell processing apparatus of the second embodiment. FIG. 18 is a schematic view showing a region in a cell culture vessel imaged in Example 1 using the phase contrast observation apparatus of the present invention. FIG. 19 is a photograph showing a phase difference image taken by the phase difference observation apparatus of the present invention in Example 1.

Hereinafter, in the present invention, the “Z-axis direction” refers to the direction perpendicular to the surface direction of the phase plate, and the “X-axis direction” refers to one direction in the plane (XY plane) direction of the phase plate, The “Y-axis direction” refers to a direction orthogonal to the X-axis direction in the surface direction of the phase plate.

In the present invention, “deterioration of phase difference image” means, for example, that the phase difference effect in the phase difference image is reduced, that is, the contrast is reduced.

Hereinafter, the phase contrast observation apparatus and the cell processing apparatus of the present invention will be described in detail with reference to the drawings. However, the present invention is not limited to the following description. In the following FIGS. 1 to 19, the same parts are denoted by the same reference numerals, and the description thereof may be omitted. Further, in the drawings, for convenience of explanation, the structure of each part may be appropriately simplified and shown, and the dimensional ratio of each part may be schematically shown differently from the actual one. Moreover, each embodiment can use the description for each other, unless it mentions in particular.

(Embodiment 1)
The present embodiment is an example of a phase difference observation apparatus. FIG. 1 is a schematic cross-sectional view showing the configuration of the phase difference observation apparatus 100 of the first embodiment. As shown in FIG. 1, the phase contrast observation apparatus 100 includes a light source 1, an illumination optical system 2, an imaging optical system 3, a stage 4, a control unit 5, a first moving unit 6, and a second moving unit 7. Including as a main configuration. The illumination optical system 2 includes a light source lens 21, a digital micro mirror device (DMD) 22, and a condenser lens 23 and is arranged in this order along the optical path of the illumination light emitted from the light source 1. The DMD 22, which is the spatial modulation element, is disposed as a configuration that exhibits the same function as a ring slit in a general phase contrast observation apparatus. The light source 1 and the light source lens 21 and the DMD 22 of the illumination optical system 2 are accommodated in a housing 86 having an opening. In addition, the condenser lens 23 of the illumination optical system 2 is provided at the opening of the housing 86 so that the illumination light (reflected light) guided from the DMD 22 can be guided to the observation object 42 in the cell culture vessel 41. It is arranged. The imaging optical system 3 includes an objective lens 31 including a phase plate 32, an imaging lens 33, and an imaging device 34, and is arranged in this order along the optical path of the illumination light. The imaging lens 33 and the imaging device 34 of the imaging optical system 3 are accommodated in a housing 87. The objective lens 31 is disposed at the opening of the housing 87 so that an optical image of the object to be observed 42 can be formed on the imaging device 34. At stage 4, a cell culture vessel 41 including an object to be observed 42 is disposed. The stage 4 is disposed between the illumination optical system 2 and the imaging optical system 3 in the optical path of the illumination light. The control unit 5 is electrically connected to the DMD 22, the first mobile unit 6, and the second mobile unit 7. The first moving unit 6 is disposed in contact with the housing 86 so as to move the light source 1 and the illumination optical system 2. The second moving unit 7 is disposed in contact with the housing 87 so as to move the imaging optical system 3. The phase contrast observation apparatus 100 of the present embodiment includes the stage 4, the cell culture vessel 41, the first mobile unit 6, the second mobile unit 7, the housing 86, and the housing 87, but any configuration is possible. And may or may not exist.

The light source 1 is, for example, a light source that generates illumination light to be irradiated to the observed object 42 cultured in the cell culture vessel 41. The light source 1 is not particularly limited, and for example, a known light source can be used, and specific examples thereof include a halogen lamp, a tungsten lamp, a white LED (Light Emitting Diode), a single color LED, a semiconductor laser and the like.

The illumination optical system 2 is, for example, an optical system that guides the illumination light from the light source 1 to the observation object 42 in the cell culture vessel 41. In the phase contrast observation apparatus 100 of the present embodiment, the illumination optical system 2 includes the light source lens 21, the DMD 22, and the condenser lens 23. However, the light source lens 21 and the condenser lens 23 have arbitrary configurations. Good or bad. The illumination optical system 2 includes, for example, a field stop, an aperture stop, a relay lens, a collimator lens, a lens such as a fly's eye lens and a diffuser, a prism such as an internal total reflection prism (TIR prism), and other configurations such as a mirror. May be.

The light source lens 21 is, for example, a lens that condenses the illumination light emitted from the light source 1. The light source lens 21 may be, for example, a known lens or lens system, and is preferably a lens or lens system serving as Kohler illumination capable of uniformly illuminating the surface on which the subject 42 is present.

The DMD 22 changes, for example, the intensity distribution of the illumination light emitted from the light source 1. Specifically, the DMD 22 guides the illumination light emitted from the light source 1 by, for example, reflecting it in the direction of the object 42. As described above, the DMD 22 exhibits the same function as a ring slit in a general phase contrast observation apparatus. For this reason, the DMD 22 is, for example, a position, a shape, and the like of the illumination light so that a phase difference effect can be obtained in a phase difference image (imaging data) imaged by the imaging device By shaping the size, the intensity distribution of the illumination light is arbitrarily changed. The position, shape, and size of the illumination light are controlled by the control unit 5, for example, as described later. As a specific example, when the liquid level of the culture solution in the cell culture vessel 41 is horizontal, the shape of the illumination light is, for example, a ring shape. The shape of the illumination light means, for example, the shape of the illumination light in a plane perpendicular to the optical axis direction between the DMD 22 and the objective lens 31. The DMD 22 is not particularly limited, and any known DMD element can be used. The DMD 22 includes, for example, a plurality of reflective surfaces that are pivotable about pivot axes parallel to each other. In this case, for example, the position, the shape, and the size of the illumination light are shaped by changing the angles of the plurality of reflecting surfaces.

The position of the DMD 22 in the phase contrast observation apparatus 100 is not particularly limited, but, for example, a larger contrast (phase contrast effect) can be obtained in the phase contrast image of the object 42 to be observed. It is preferable to be disposed at a position optically conjugate with the pupil position), more specifically, at a position optically conjugate with the phase plate 32.

The phase difference observation apparatus 100 of this embodiment includes the DMD 22 as the spatial modulation element, but the spatial modulation element can adopt any configuration capable of changing the intensity distribution of the illumination light. As a specific example, the spatial modulation element may be, for example, a liquid crystal panel, an array in which electrochromic elements are arranged in an array, an array in which light emitting elements such as organic electroluminescent (EL) elements are arranged in an array, . When the spatial modulation element is the liquid crystal panel or the electrochromic element, the light source 1, the illumination optical system 2, the stage 4, and the imaging optical system 3 are, for example, linearly arranged in this order. When the spatial modulation element is the light emitting element, the spatial modulation element may double as the light source 1, for example. The spatial modulation element is, for example, in the same manner as the DMD 22, in combination with the phase plate 32, in the image acquired by the imaging device 34, the position, shape, and size of the illumination light so as to obtain a phase difference effect. By shaping the intensity, the intensity distribution of the illumination light is arbitrarily changed. The position of the space modulation element is not particularly limited, and is, for example, the same as the position of the DMD 22.

The condenser lens 23 is, for example, a lens that condenses the illumination light onto the subject 42. As the condenser lens 23, for example, a known lens or lens system can be used.

The imaging optical system 3 is, for example, an optical system for forming an optical image of the object to be observed 42 on the imaging device 34. In the phase difference observation apparatus 100 of the present embodiment, the imaging optical system 3 includes the objective lens 31 including the phase plate 32, the imaging lens 33, and the imaging device 34. However, the objective lens 31 including the phase plate 32 and the lens The image lens 33 may or may not have an arbitrary configuration. The imaging optical system 3 may include, for example, another configuration such as a mirror.

The objective lens 31 is, for example, a lens that magnifies the image of the object to be observed 42 to a target magnification. As the objective lens 31, for example, a known lens or lens system can be used, and can be appropriately selected according to the target magnification. In the phase contrast observation apparatus 100 of this embodiment, although the objective lens 31 is one, the objective lens 31 may be two or more. When two or more objective lenses 31 are provided, it is preferable that the respective objective lenses 31 can be enlarged to different magnifications. The objective lens 31 includes the phase plate 32, but as described above, the phase plate 32 may have any configuration, and may or may not have the configuration.

The phase plate 32 manipulates, for example, the phase of part of incident light. In the phase difference observation apparatus 100 of the present embodiment, the objective lens 31 includes the phase plate 32, that is, the objective lens 31 and the phase plate 32 are integrally configured, but the phase plate 32 31 may be configured independently. The phase plate 32 can be configured, for example, by forming a wavelength plate such as a 1⁄4 wavelength plate and a light absorbing filter such as a neutral density filter in a ring shape.

The imaging lens 33 is, for example, a lens for forming an optical image of the object to be observed 42 on the imaging device 34. As the imaging lens 33, for example, a known lens or lens system can be used.

The imaging element 34 is, for example, an element that captures an optical image (phase difference image) of the object 42 to be observed. As the image pickup device 34, for example, a known image pickup device can be used, and as a specific example, a device provided with a charge-coupled device (CCD, charge coupled device), a complementary metal oxide semiconductor (CMOS) or the like can be mentioned. The imaging surface of the imaging device 34 is preferably disposed at a position optically conjugate with the observation object 42 because, for example, a clearer phase difference image of the observation object 42 can be obtained. The imaging device 34 may output, for example, the phase difference image of the observed object 42 to the display device via the control unit 5. In this case, the imaging device 34 is electrically connected to the control unit 5.

Stage 4 supports, for example, cell culture vessel 41. The stage 4 has, for example, a recess in which the cell culture vessel 41 can be placed, and an opening at a position corresponding to the cell culture vessel 41.

The stage 4 may have a fixed position in the phase contrast observation apparatus 100, that is, not move but be movable. When the stage 4 is movable, the stage 4 is moved by, for example, a known moving means (driving means). The moving direction of the stage 4 is not particularly limited, and is, for example, any one direction, two directions or all directions among the X axis direction, the Y axis direction, and the Z axis direction. In the phase difference observation apparatus 100 of this embodiment, the position of the stage 4 is fixed. For this reason, the phase contrast observation apparatus 100 of the present embodiment can prevent, for example, the fluctuation of the liquid surface of the culture solution in the cell culture vessel 41 caused when the stage 4 moves in the phase contrast observation apparatus in which the stage 4 can move. . Moreover, since the phase contrast observation apparatus 100 of this embodiment can suppress the disorder of the phase contrast image of the to-be-observed body 42 by the fluctuation of the liquid surface of a culture solution, for example, it is suitable for automatic imaging of the cell culture container 41 is there. Moreover, according to the phase contrast observation apparatus 100 of the present embodiment, the waiting time until the fluctuation of the liquid surface of the culture solution can be reduced can be reduced. For example, the entire surface of the cell culture vessel 41 can be imaged in a shorter time. can do.

The phase contrast observation apparatus 100 of the present embodiment includes the stage 4 as the cell culture container arrangement unit, but the cell culture container arrangement unit can arrange the cell culture container 41, and Any configuration capable of observing the observation body 42 can be adopted. In the present embodiment, the stage 4 has an opening, but for example, a light transmitting material may be disposed on the side of the opening on the imaging optical system 3 side. Examples of the light transmitting material include a transparent glass plate and an acrylic plate. For example, the position in the phase contrast observation apparatus may be fixed or movable. For the movement of the cell culture vessel placement unit, for example, the description of the movement of the stage 4 described above can be used.

The cell culture vessel placement unit (stage 4) may further include, for example, temperature control means for adjusting the temperature of the cell culture vessel 41. By including the temperature adjusting means, culture conditions can be made constant while processing the cells in the cell culture vessel 41. For example, when the image of the object to be observed 42 is captured and the object to be observed 42 described later is processed. Damage can be reduced. Examples of the temperature adjusting means include heating means such as a heater.

The cell culture vessel placement unit (stage 4) may further include, for example, pH adjustment means for adjusting the pH of the culture solution in the cell culture vessel 41. By including the pH adjusting means, culture conditions can be made constant while processing cells in the cell culture vessel, and for example, damage to the cells during cell processing can be reduced. The pH adjusting means may be, for example, a carbon dioxide concentration adjusting means or the like, and as a specific example, a connection part connected to the carbon dioxide supplying means outside the phase difference observation apparatus 100 may be mentioned.

The cell culture vessel 41 is not particularly limited, and examples thereof include culture vessels such as known dishes and flasks used for cell culture. The forming material of the cell culture vessel 41 is not particularly limited, and examples thereof include a material that transmits a laser irradiated by a laser irradiation unit described later, and specific examples include a plastic that transmits the laser, glass, and the like. Examples of plastics include polystyrene polymers, acrylic polymers (polymethyl methacrylate (PMMA), etc.), polyvinylpyridine polymers (poly (4-vinylpyridine), 4-vinylpyridine-styrene copolymer, etc.), silicone resins Polymers (polydimethylsiloxane etc.), polyolefin polymers (polyethylene, polypropylene, polymethylpentene etc.), polyester polymers (polyethylene terephthalate (PET), polyethylene naphthalate (PEN etc.), polycarbonate polymers, epoxy polymers etc. Be

When subjecting the subject 42 in the cell culture vessel 41 to laser processing, for example, the cell culture vessel 41 has a pigment structure (coloring that absorbs the laser on the bottom of the cell culture vessel 41 (at the side of the It is preferable to include a laser absorbing layer formed of a polymer containing a group or a photoacid generator which absorbs a laser and generates an acidic substance. For the dye structure and the photoacid generator, for example, the description of Japanese Patent No. 6033980 can be used. The cell culture vessel 41 includes the laser absorption layer, for example, when the laser irradiation unit of the cell processing apparatus described later irradiates the laser, the energy of the laser is converted into heat, acid, etc., and the laser absorption The cells present at the top of the layer can be killed, released, etc.

The subject 42 is not particularly limited, and may be a cell, a cell mass composed of cells, a tissue, an organ or the like. The cells may be, for example, cultured cells or cells isolated from a living body. The cell mass, tissue or organ may be, for example, a cell mass, tissue or organ produced from the cell, or a cell mass, tissue or organ isolated from a living body.

The control unit 5 includes a configuration similar to a personal computer, a server computer, a work station and the like. FIG. 2 is a block diagram showing an example of the control unit 5 in the phase difference observation apparatus 100 of the present embodiment. As shown in FIG. 2, the control unit 5 includes a central processing unit (CPU) 51, a main memory 52, an auxiliary storage device 53, a video codec 54, an I / O (input-output) interface 55, etc. It is controlled by (a system controller, an I / O controller, etc.) 56 and operates in cooperation. The auxiliary storage device 53 may be a storage means such as a flash memory or a hard disk drive. The video codec 54 generates a screen to be displayed based on the drawing instruction received from the CPU 51, and transmits the screen signal to, for example, a display device or the like outside the phase difference observation apparatus 100. , A screen and a video memory for temporarily storing image data. The I / O interface 55 is a device for communicably connecting and controlling each member such as the DMD 22, the first mobile unit 6, and the second mobile unit 7. The I / O interface 55 may include a servo driver (servo controller). Further, the I / O interface 55 may be connected to, for example, input means outside the phase contrast observation apparatus 100. Examples of the display device include a monitor (for example, various image display devices such as a liquid crystal display (LCD) and a cathode-ray tube (CRT) display) which output as images, and the like. Examples of the input device include a touch panel that can be operated by the user with a finger, a track pad, a pointing device such as a mouse, a keyboard, and a push button.

The program executed by the control unit 5 and each information are stored in the auxiliary storage device 53. The program is read into the main memory 52 at the time of execution and is decoded by the CPU 51. And control unit 5 controls each member according to a program. Control of each member by the control unit 5 will be described later.

In the phase contrast observation apparatus 100 of the present embodiment, the control unit 5 is provided with a control unit individually for each member by providing the control function of the DMD 22, the first moving unit 6, and the second moving unit 7. Since the device does not have to be provided, the device can be miniaturized. However, the present invention is not limited to this. In the phase difference observation apparatus of the present invention, for example, a control unit is provided for each of the DMD 22, the first moving unit 6, and the second moving unit 7 as the control unit 5, and each member is controlled by the control unit of each member. You may control. Moreover, the phase difference observation apparatus of this invention may provide the control unit 5 and the control unit of each member, and may control each member jointly, for example. Further, the control unit 5 may be configured of one semiconductor element, may be a chip obtained by one package of a plurality of semiconductor elements, or may be configured to have a plurality of semiconductor elements provided on a substrate.

The first moving unit 6 is, for example, a moving unit capable of moving the light source 1 and the illumination optical system 2. The first moving unit 6 is, for example, a known moving means (driving means). The moving direction of the first moving unit 6 is not particularly limited, and is, for example, any one direction, two directions or all directions among the X axis direction, the Y axis direction, and the Z axis direction. The first moving unit 6 can move, for example, the light source 1 and the illumination optical system 2 such that the DMD 22 and the pupil (pupil position) of the imaging optical system 3 are in an optically conjugate position. In the 42 phase difference images, it is preferable to be movable in the Z-axis direction because a larger contrast (phase difference effect) can be obtained. In the phase contrast observation apparatus 100 of the present embodiment, the first moving unit 6 can move the light source 1 and the illumination optical system 2, but the first moving unit 6 includes, for example, the light source 1 and the illumination optical system 2. Only one of them may be movable. The first moving unit 6 may be, for example, movably disposed on the light source 1 and the illumination optical system 2, and can be appropriately disposed in accordance with the moving unit to be used. The movement by the first mobile unit 6 is controlled by the control unit 5, for example, as described later.

The second moving unit 7 is, for example, a moving unit capable of moving the imaging optical system 3. The second moving unit 7 includes known moving means (driving means). The moving direction of the second moving unit 7 is not particularly limited, and is, for example, any one direction, two directions, or all directions among the X axis direction, the Y axis direction, and the Z axis direction. The second moving unit 7 can move the imaging optical system 3 so that the imaging surface of the imaging device 34 and the observation object 42 are in an optically conjugate position, for example, and the observation object 42 is clearer It is preferable that it is movable in the Z-axis direction because a phase difference image of For example, the second moving unit 7 may be disposed so as to be able to move the imaging optical system 3, and can be appropriately disposed according to the moving unit to be used. The movement by the second moving unit 7 is controlled by the control unit 5, for example, as described later.

Next, an operation of the control unit 5 in the phase difference observation apparatus 100 of the present embodiment and a method of capturing a phase difference image using the phase difference observation apparatus 100 will be described.

The control unit 5 of the present embodiment includes intensity distribution correction information in which the position of the imaging optical system 3 with respect to the cell culture vessel 41 and the intensity distribution of the illumination light at the position of the imaging optical system 3 are associated. Therefore, first, the intensity distribution correction information is acquired and stored in the auxiliary storage device 53 of the control unit 5. The intensity distribution correction information may be, for example, information acquired in advance by another phase difference observation apparatus or the like, or may be information input by the user of the phase difference observation apparatus 100 of the present embodiment, or the position of the present embodiment. Although the information acquired by the phase difference observation apparatus 100 may be used, the information acquired by the phase difference observation apparatus 100 of the present embodiment is preferable because deterioration of the phase difference image due to the meniscus can be more effectively suppressed. When acquiring the said intensity distribution correction information using the phase difference observation apparatus 100 of this embodiment, the said intensity distribution correction information is specifically acquired as follows.

FIG. 3 is a schematic view showing the configuration of the phase difference observation apparatus 100 in the case of acquiring the intensity distribution correction information. In FIG. 3, the optical path of the illumination light is omitted. As shown in FIG. 3, a cell culture vessel 41 (cell culture vessel 41 for correction) not including the subject 42 is disposed on the stage 4 of the phase contrast observation apparatus 100. At this time, the liquid volume and type of the culture solution in the cell culture container 41 for correction are the same as the liquid volume and type in the cell culture container 41 including the object 42 to be observed. Further, a phase plate imaging lens 35 is inserted between the imaging lens 33 of the imaging optical system 3 of the phase contrast observation apparatus 100 and the imaging element 34. Thereby, the image of the phase plate 32 and the image of the illumination light (the image of the reflected light of the DMD 22) are imaged on the imaging device 34 as shown in FIG. Although not shown, the control unit 5 is electrically connected to the imaging device 34. As the phase plate imaging lens 35, for example, a known lens or lens system can be used.

Next, using the phase contrast observation apparatus 100 shown in FIG. 3, the cell culture vessel 41 for correction is divided into a plurality of sections, and each section is photographed (tiled photographing) to obtain a phase for correction. The image of the plate 32 and the image of the illumination light are acquired by the imaging device 34. At each imaging, the control unit 5 acquires the position of the imaging optical system 3 as imaging system position information. The imaging system position information (the position of the imaging optical system 3) is, for example, coordinates (three-dimensional coordinates) on the XYZ axes or coordinates (two-dimensional coordinates) on the XY axes.

FIG. 4 is a schematic view of the image of the phase plate 32 for correction and the image of the illumination light, which are imaged by the imaging device 34. As shown in FIG. The illumination optical system 2 and the imaging optical system 3 are disposed in the Z-axis direction of the horizontal plane of the culture solution in the cell culture vessel 41 for correction. At this time, when the DMD 22 and the phase plate 32 are optically conjugated and imaged by the imaging device 34, as shown in FIG. 4A, the phase difference image (image for correction) taken is An image 22 a is included in the image (phase plate image) 32 a of the phase plate 32. However, as described above, when observing the vicinity of the wall surface of the cell culture vessel 41, the liquid surface of the culture solution forms a meniscus, and a lens effect occurs. Therefore, the illumination light passing through the meniscus is refracted and the optical conjugate relationship between the DMD 22 and the phase plate 32 is broken. At this time, as shown in FIG. 4B, in the correction image, the image 22a of the illumination light is not included in the phase plate image 32a, or is deformed or blurred. To reduce the phase difference effect. Therefore, when the problem shown in FIG. 4B occurs, the control unit 5 corrects the relationship between the image 22a of the illumination light and the phase plate image 32a so as to be in the state of FIG. 4A. Information for acquiring the intensity distribution correction information. Specifically, with respect to the cell culture vessel 41 for correction, the control unit 5 generates an image 22a of the illumination light at each imaging position, that is, at the position of each imaging optical system 3 at the time of the tiling imaging. The intensity distribution of the illumination light included in 32a is determined. That is, the control unit 5 is configured to reflect the position information of the mirror of the DMD 22 that reflects the illumination light in the direction of the object 42 when the intensity distribution of the illumination light is corrected so that the image 22a of the illumination light is included in the phase plate image 32a. And position information of a mirror of the DMD 22 that does not reflect illumination light in the direction of the object 42 to be observed.

The control unit 5 detects whether or not the image 22a of the illumination light is included in the phase plate image 32a in the correction image at the time of the tiling imaging. When the image 22a of the illumination light is not included in the phase plate image 32a, that is, when the image 22a of the illumination light and the image 32a of the phase plate 32 deviate, the control unit 5 determines that the image 22a of the illumination light is The position and the size of the reflected light of the DMD 22 included in the phase plate image 32a are determined. Specifically, the control unit 5 detects a bright ring (image 22a of illumination light) and a dark ring (phase plate image 32a) in the correction image. Next, the control unit 5 determines the position and the size of the reflected light of the DMD 22 in which the bright ring and the dark ring are concentric and the image 22a of the illumination light is included in the phase plate image 32a. Specifically, the position of the reflected light of the DMD 22 is moved in at least one of the X-axis direction and the Y-axis direction within a predetermined range, and the size of the reflected light of the DMD 22 is also changed within the predetermined range. The position and the size of the reflected light of the DMD 22 where the bright ring and the dark ring are concentric are determined.

Next, the control unit 5 detects whether or not the image 22a of the illumination light is deformed in the correction image. Then, when the image 22a of the illumination light is deformed, the control unit 5 obtains the shape of the reflected light of the DMD 22 in which the image 22a of the illumination light has a substantially perfect circle on the outer periphery and the inner periphery of the ring. Specifically, the control unit 5 detects the shape of the bright ring in the correction image. Next, the shape of the reflected light of the DMD 22 is deformed within a predetermined range, and the shape of the reflected light of the DMD 22 in which the outer circumference and the inner circumference of the bright ring are substantially true circles is determined.

Then, the control unit 5 associates the information of the imaging position of the correction image, that is, the imaging system position information with the information of the position, the shape and the size of the reflected light of the DMD 22 which is the intensity distribution of the illumination light. Then, the information is stored in the auxiliary storage device 53 as the intensity distribution correction information. Each piece of information stored in the auxiliary storage device 53 depends on the conditions of the imaging target such as the type and size of the cell culture vessel 41, the type and volume of the culture fluid, and the like at the time of imaging using the phase difference observation apparatus 100 described later. Since it is possible to specify the intensity distribution correction information, it is preferable to store information in association with information on the type and size of the cell culture vessel 41, and the type and volume of the culture solution and the like.

Furthermore, the control unit 5 may detect whether a blur of the image 22a of the illumination light is present. Then, when the blur of the image 22a of the illumination light exists, the control unit 5 obtains the positions of the light source 1 and the illumination optical system 2 at which the blur of the image 22a of the illumination light is eliminated. Specifically, the control unit 5 moves the light source 1 and the illumination optical system 2 by moving the first moving unit 6 in the predetermined range in the Z axis direction, and the blur of the image 22a of the illumination light is eliminated. The positions of the light source 1 and the illumination optical system 2 are determined. Further, the control unit 5 acquires illumination system position information which is the position of the light source 1 and the illumination optical system 2 where the blur is eliminated. The illumination system position information (the positions of the light source 1 and the illumination optical system 2) is, for example, coordinates (three-dimensional coordinates) in the XYZ axes or coordinates in the Z axis. Then, the control unit 5 associates the imaging system position information with the illumination system position information which is the position of the light source 1 and the illumination optical system 2 where the blur is eliminated, to obtain the auxiliary storage device 53 as illumination system position correction information. Remember. The illumination system position correction information is preferably stored in association with the condition of the imaging target, as in the case of the intensity distribution correction information, for example.

In the phase difference observation apparatus 100 of the present embodiment, the control unit 5 acquires the intensity distribution correction information by correcting the entire intensity distribution of the illumination light, but the intensity distribution may be obtained by another method. Correction information may be acquired. Specifically, the control unit 5 operates, for example, one mirror of the DMD 22 at the position of one imaging optical system 3, and reflects illumination light toward the subject 42 by the operated mirror. At this time, for example, the control unit 5 picks up the correction image with the imaging device 34. Next, the control unit 5 specifies, for example, the position at which the reflected light from the mirror forms an image in the correction image, and generates positional information of the operated mirror and the image of the reflected light in the correction image Associate a position. Furthermore, for example, the control unit 5 performs this for all the mirrors of the DMD 22 and associates the position information of each mirror with the imaging position in the correction image of the reflected light. Then, the control unit 5 extracts an imaging position included in the phase plate image 32 a among imaging positions in the correction image of the reflected light of each mirror. The control unit 5 operates the mirror at the corresponding position of the DMD 22 based on, for example, the position information of the mirror corresponding to the extracted imaging position, and reflects the illumination light toward the object 42 by the operated mirror. The image 22a of the illumination light is included in the phase plate image 32a. Therefore, for example, the control unit 5 stores the imaging system position information and the position information of the mirror corresponding to the extracted imaging position in the auxiliary storage device 53 as intensity distribution correction information. . The control unit 5 performs the same process at, for example, the positions of the other imaging optical systems 3 and acquires intensity distribution correction information at the positions of the respective imaging optical systems 3.

Although the control unit 5 has described the case where the intensity distribution correction information is calculated as an example, the user of the phase difference observation apparatus 100 uses the intensity distribution correction information, for example, based on the correction image. , And may be input using the aforementioned input means or the like.

Thus, the intensity distribution correction information can be acquired using the phase difference observation apparatus 100 of the present embodiment.

FIG. 5 is a flowchart showing a method of capturing a tile image of a phase difference image using the phase difference observation apparatus 100 shown in FIG. As shown in FIG. 5, the imaging method of the phase difference image using the phase difference observation apparatus 100 of the present embodiment includes, for example, steps S1 to S4. The imaging method may further include steps S5 to S10. In the following description, although the case of imaging the entire surface of the cell culture vessel 41 will be described, the area imaged by the phase contrast observation apparatus 100 (imaging target area) is not limited to this. The phase difference observation apparatus 100 captures, for example, an imaging target area set by a user or the like. The imaging target area is, for example, part or all of the cell culture vessel 41. The image is, for example, an image including the subject 42 in the cell culture vessel 41.

First, the user of the phase contrast observation apparatus 100 selects the conditions of the imaging target such as the size of the cell culture container 41 to be imaged, the type of culture solution in the cell culture container 41, and the liquid volume (S1). Then, the intensity distribution correction information satisfying the condition of the selected imaging target is read from the auxiliary storage device 53 (S2). When the illumination system position correction information is stored in the auxiliary storage device 53, the illumination system position correction information may be read out from the auxiliary storage device 53.

Next, tiling imaging is performed on the cell culture vessel 41 including the subject 42 disposed in the phase contrast observation apparatus 100 (S3). The control unit 5 acquires imaging system position information which is the position of the imaging optical system 3 at each imaging, and based on the imaging system position information and the intensity distribution correction information, The intensity distribution of the illumination light is corrected to be the position, the shape, and the size of the reflected light of the corresponding DMD 22. Then, a phase difference image is taken by the image sensor 34 in the phase difference observation apparatus 100. In addition, when the illumination system position correction information is read, the control unit 5 further performs a first process corresponding to the position of the imaging optical system 3 based on the imaging system position information and the illumination system position correction information. The positions of the light source 1 and the illumination optical system 2 may be corrected by moving the first moving unit 6 to the position of the moving unit 6. Furthermore, the phase difference image may be captured by the image sensor 34 in the phase difference observation apparatus 100.

Then, the control unit 5 synthesizes the tile image based on the obtained phase difference image (S4). Thus, a tile image can be acquired using the phase contrast observation apparatus 100 of FIG. Note that although acquisition of tile images has been described as an example, it is also possible to acquire only phase-contrast images of the region where the object to be observed 42 exists in the cell culture vessel 41 using the phase contrast observation apparatus 100. .

When acquiring a tile image using the phase difference observation apparatus 100 of FIG. 1, the type and volume of the culture solution in the set intensity distribution correction information, the type and size of the cell culture vessel 41, etc., and the cell culture of the imaging target Since the control unit 5 can cope with the reduction of the phase difference effect caused by the difference in the type and volume of the culture solution in the container 41, the type and size of the cell culture container 41, etc., the control unit 5 further performs imaging used for synthesizing tile images. An imaging failure site in an image may be detected. In this case, the control unit 5 calculates the correction value of the imaging condition (correction value of the intensity distribution correction information) for the location (division) of the cell culture vessel 41 corresponding to the imaging failure site, and reimaging by the imaging device 34 Conduct.

Specifically, first, the control unit 5 detects whether there is an imaging failure site (S5). In the case of No, the control unit 5 ends the process. On the other hand, in the case of Yes, the process proceeds to S6. The imaging failure site means, for example, an area in which the contrast is lowered due to the increase in luminance. Specifically, the control unit 5 calculates a total value of luminance values in one phase difference image for each of the obtained phase difference images. And control unit 5 judges that it is an imaging defect part, when the sum total of the acquired luminosity value is more than a threshold. The threshold value may be any value, and specifically, a predetermined value or more (for example, 1 or more) from the total value of the brightness values of the phase difference image captured in the area where the culture fluid of the cell culture vessel 41 is horizontal. .15 times or more). The detection of the imaging failure site by the total value of the luminance values is performed, for example, on all or part of the obtained phase difference image. As a specific example, in the phase difference image, the stage 4 is, for example, a dark portion in the phase difference image. For this reason, when detecting an imaging failure part with the total value of luminance values, for example, the area of the tile image corresponding to the phase difference image including the stage 4 is excluded from the detection targets of the imaging failure part by the total value of luminance values. Processing such as may be performed. Whether or not the area of the tile image corresponding to the phase difference image subjected to the exclusion processing corresponds to an imaging failure site may be detected, for example, by another detection method of an imaging failure site.

Next, the control unit 5 calculates a correction value at the time of re-imaging of the phase difference image corresponding to the imaging failure site. The correction value may be, for example, an intensity distribution of the illumination light, that is, a correction value for correcting the position, size, and shape of the reflected light of the DMD 22 or a correction for correcting the positions of the light source 1 and the illumination optical system 2 It may be a value. The correction value is set, for example, such that the total value of the luminance values in the recaptured phase difference image is less than the threshold.

When the control unit 5 calculates the correction value regarding the position, the size, and the shape of the reflected light of the DMD 22, the correction value is implemented, for example, as follows. First, among the imaging failure parts, one place (one phase difference image) for calculating a correction value is extracted (S6). The correction value is calculated in the same manner as the position, size, and shape of the reflected light of the DMD 22 in the intensity distribution correction information (S7).

Then, the control unit 5 performs re-imaging based on the calculated correction value (S8). Specifically, the control unit 5 corrects the intensity distribution of the illumination light, the position, the size, and the shape of the reflected light of the DMD 22 based on the correction value, and re-images.

Next, the control unit 5 confirms whether re-imaging has been completed for all the imaging failure sites (S9). If No, return to S6. On the other hand, in the case of Yes, the process proceeds to S10. Then, the control unit 5 substitutes the phase difference image (re-captured image) re-captured in S10 with the original phase difference image, that is, the phase-difference image obtained after first capturing the phase-difference image. , Recompose tile images (S10). Then, the control unit 5 ends the process.

Although the correction value related to the position, size, and shape of the reflected light of the DMD 22 has been described as an example, the correction value for correcting the positions of the light source 1 and the illumination optical system 2 is described above. It may be. In this case, the calculation of the correction value can be performed, for example, as follows. First, one portion (one phase difference image) from which the correction value is calculated is extracted among the imaging failure parts. Next, correction values in the X axis direction, correction values in the Y axis direction, and correction values in the Z axis direction of the light source 1 and the illumination optical system 2 (illumination system) are calculated. Specifically, the control unit 5 causes the first moving unit 6 to move the positions of the light source 1 and the illumination optical system 2 in the X-axis direction, the Y-axis direction and / or the Z-axis direction within a predetermined range. At this time, the control unit 5 calculates the total value of the luminance values in the phase difference image captured by the imaging device 34 in parallel with the movement. Then, the control unit 5 calculates, as a correction value, a position where the total value of the luminance values in each phase difference image is equal to or less than the threshold value, preferably the position where the value is the minimum value. The position is, for example, coordinates (three-dimensional coordinates) on the XYZ axes.

In the imaging method using the phase difference observation apparatus 100, in the step S3, one of the images obtained by imaging using a plurality of illumination light intensity distributions for one section is suitable as a phase difference image The image of the part may be used to create an image of one section. That is, the intensity distribution correction information for one section may include the intensity distribution of a plurality of illumination lights. FIG. 6 is a flowchart showing another example of a method of acquiring intensity distribution correction information including the intensity distribution of one or more illumination lights using the phase difference observation apparatus 100 of FIG. 3. As shown in FIG. 6, the acquisition method includes steps S21 to S27 and S271. Acquisition of the intensity distribution correction information is performed, for example, prior to the step S1.

First, the imaging optical system 3 is moved to a section for acquiring intensity distribution correction information (S21). Next, in the section, the control unit 5 determines the intensity distribution of the illumination light (the intensity distribution of the first illumination light) contained in the phase plate image 32a by the image 22a of the illumination light (S22). That is, the control unit 5 is configured to reflect the position information of the mirror of the DMD 22 that reflects the illumination light in the direction of the object 42 when the intensity distribution of the illumination light is corrected so that the image 22a of the illumination light is included in the phase plate image 32a. And position information of a mirror of the DMD 22 that does not reflect illumination light in the direction of the object 42 to be observed. More specifically, as described above, the control unit 5 determines the position and the size of the reflected light of the DMD 22 in which the image 22a of the illumination light is included in the phase plate image 32a.

Next, the control unit 5 changes the intensity distribution of the illumination light in the DMD 22 to the intensity distribution of the illumination light obtained in the step S22, and images the section by the imaging element 34 (S23). It is determined whether the luminance value at the pixel of the obtained image is greater than or equal to the reference value of the luminance value (S24). The pixel may be one pixel or a plurality of pixels in the image, but the latter is preferable because it is easy to detect a change in luminance value and can suppress the influence of a meniscus. In the latter case, the pixels can also be referred to as pixel blocks. The pixel block can be produced, for example, by dividing an arbitrary number of adjacent pixels in the image into one unit and dividing the image into a plurality of units. As a specific example, the pixel block can be produced, for example, by dividing the image into squares. When the pixel is a pixel block, the luminance value of the pixel can be calculated, for example, as a total value of luminance values of each pixel of the pixel block. Further, the reference value of the luminance value can be an arbitrary value, and for example, in the obtained image, it is set so as to include pixels whose luminance value has reached the upper limit value. As a specific example, the reference value (B _s ) of the luminance value is, for example, 1.1 × B _L ≦ B _s ≦ 1.2 × B with reference to the lowest luminance value (B _L ) in the pixel of the image. _{L 1} , preferably B _s = 1.15 × B _L. Then, in the case of No, that is, when the luminance value in the image is less than the reference value of the luminance value, the process proceeds to step S26. On the other hand, in the case of Yes, that is, when the luminance value in the image is equal to or greater than the reference value of the luminance value, in the section, the control unit 5 detects the image 22a of the illumination light in a region equal to or more than the reference value of the luminance value of the image. To change the intensity distribution of the next illumination light (intensity distribution of the second illumination light) (S25). The change of the image 22a of the illumination light may be changed, for example, so that the image 22a of the illumination light is included in the phase plate image 32a, or a part or all thereof is not included in the phase plate image 32a. It may be changed to The change of the image 22a of the illumination light is preferably performed, for example, by moving the position of the image 22a of the illumination light. In this case, it is preferable not to change the size and shape of the illumination light image 22a. The phase difference observation apparatus 100 can suppress an increase in luminance value due to direct incidence of the illumination light, for example, by moving the position of the image 22a of the illumination light. Therefore, for example, the phase difference observation apparatus 100 may obtain the intensity distribution of the illumination light capable of acquiring the phase difference image of the object to be observed 42 with an appropriate contrast in the region of the luminance value or more in the image. it can.

Then, the intensity distribution of the first and second illumination light is associated with the position of the imaging optical system 3 and stored as the intensity distribution correction information (S26). In the step S26, for example, the intensity distribution of the illumination light each time may be stored as the intensity distribution correction information by associating the region with the reference value of the luminance value of the image or more. Thereby, the phase difference observation apparatus 100 can acquire the image of the said division simply, for example by unifying the area | region below the reference value of the luminance value of each image, after imaging with the image pick-up element 34. FIG. Further, in the description of the acquisition method in the imaging method of the present embodiment, after step S25, the process proceeds to step S26, but returns to step S23 again and replaces the intensity distribution of the first illumination light, and the intensity distribution of the second illumination light The steps from step S23 may be performed similarly using In this case, in the steps S24 and S25, when the intensity distribution of the first illumination light is applied and imaged among the images obtained by applying and imaging the intensity distribution of the second illumination light, It carries out by targeting the area (area before correction) equal to or more than the reference value of the luminance value. Then, the same process may be repeated until the luminance value at the pixel in the uncorrected area of the image obtained in each S23 process becomes less than the reference value of the luminance value. That is, the imaging method of the present embodiment may be implemented until an image of the entire section of the imaging target is obtained when the regions less than the reference value of the luminance value are integrated in the images captured each time.

In step S27, it is determined whether the acquisition of the intensity distribution correction information has been completed for all the segments. In the case of No, that is, when the acquisition of the intensity distribution correction information is not completed for all the divisions, the imaging optical system 3 is moved to the next division (S271), and the step S22 is performed again. On the other hand, in the case of Yes, that is, when the acquisition of the intensity distribution correction information has been completed for all the sections, the method of acquiring the intensity distribution correction information is ended.

Next, the intensity distribution correction information obtained by the acquisition method and an imaging method using the phase difference observation apparatus 100 will be described. FIG. 7 is a flowchart showing another example of the step S3 in the method of imaging a tile image of a phase difference image using the phase difference observation apparatus 100 shown in FIG. As shown in FIG. 7, the step S3 includes, for example, steps S31 to S37, S321, S351, and S371.

First, the imaging optical system 3 is moved to a section for imaging (S31). Next, the intensity distribution correction information corresponding to the section, that is, the intensity distribution correction information associated with the position of the imaging optical system 3 is acquired, and the intensity distribution correction information is the intensity distribution of a plurality of illumination lights. It is determined whether it contains (S32). In the case of No, that is, when the intensity distribution correction information includes the intensity distribution of one illumination light, the intensity distribution of the illumination light in the DMD 22 is changed to the intensity distribution of the illumination light obtained in the step S32, At 34, the section is imaged (S321), and the process proceeds to S37. On the other hand, if the determination is Yes, that is, if the intensity distribution correction information includes the intensity distribution of a plurality of illumination lights, the process proceeds to step S33. Hereinafter, the case where the intensity distribution of a plurality of illumination lights is N times will be described as an example. The N means an integer of 2 or more. In step S33, the number n of intensity distributions of illumination light applied at the time of imaging is set to 1 (S33). Next, the intensity distribution of the illumination light in the DMD 22 is changed to the intensity distribution of the first illumination light, and the image pickup device 34 picks up the section (S34).

After the imaging, it is determined whether there is an intensity distribution of the illumination light not applied among the intensity distributions of the Nth illumination light. That is, it is determined whether N = n. Then, in the case of No, that is, in the case of N ≠ n, n is set to n + 1 (in this case, n = 2, S351), and the process returns to the step S34. And the intensity distribution of illumination light of the 2nd time is applied, and the process from S34 process is implemented similarly. Then, in the imaging method, steps S34 and S35 are repeatedly performed until N = n. On the other hand, in the case of Yes, that is, in the case of N = n, an image (a region to be corrected) in which luminance values at pixels of the obtained N images are less than the reference value of the luminance value Integrate. As a result, one image obtained by imaging the section is acquired (S36).

Then, in step S37, it is determined whether imaging has been completed for all the segments. In the case of No, the process moves to the next section (S371), and the processes from S32 are performed in the same manner. On the other hand, in the case of Yes, the step S3 ends.

In the S3 step, by applying intensity distribution correction information including the intensity distribution of one or more illumination lights and imaging, in only one section, only correction based on the intensity distribution correction information including the intensity distribution of one illumination light Even in the case where it is difficult, it is possible to acquire an image in which the deterioration of the phase difference image is suppressed.

According to the phase difference observation apparatus 100 of the present embodiment, the configuration of the additional imaging unit for suppressing the deterioration of the phase difference image due to the meniscus as in Patent Document 1 is unnecessary. Therefore, according to the phase difference observation apparatus 100 of the present embodiment, the apparatus can be miniaturized. In addition, the phase contrast observation apparatus 100 according to the present embodiment corrects the intensity distribution of the illumination light at the time of imaging based on the intensity distribution correction information acquired using, for example, the cell culture container 41 not including the object 42 to be observed. For example, the correction value is calculated at the time of imaging, and imaging can be performed in a shorter time as compared with the device of Patent Document 1 that performs the correction. Further, the phase difference observation apparatus 100 according to the present embodiment corrects the intensity distribution of the illumination light at the time of imaging based on the intensity distribution correction information, and therefore, the deterioration of the phase difference image due to the meniscus can be suppressed. For this reason, according to the phase difference observation apparatus 100 of the present embodiment, it is possible to capture a phase difference image with a large contrast. These effects are the same as in the cell processing apparatus described later.

Second Embodiment
The present embodiment is an example of a cell processing apparatus. 8 to 17 show an example of the configuration of the cell processing apparatus of the present embodiment. FIG. 8 is a perspective view showing an example of the configuration of the cell processing apparatus of the present embodiment, and FIG. 9 shows the configurations of the first area, the second area, and the third area in the cell processing apparatus of the present embodiment. FIG. 10 is a schematic cross-sectional view, FIG. 10 is a perspective view showing an example of the configuration of a first region of the cell processing apparatus of the present embodiment, and FIG. 11 is a cross-sectional view of the first region viewed from II in FIG. It is a figure, and in Drawing 12, (a) is an exploded perspective view showing an example of a culture vessel placement part in a cell processing device of this embodiment, (b) is a III-III direction in Drawing 12 (a). 13 is a perspective view of the first region and the circulating means when the outer wall of the first region is removed, and FIG. 14 is a cross-sectional view of the first region viewed from the II-II direction in FIG. 15 is a cross-sectional view of the top of the area and the circulating means, and in FIG. FIG. 16 is a perspective view showing an example of the configuration of the second area of the cell processing apparatus of the present embodiment, (b) is a perspective view showing another example of the configuration of the second area, and FIG. It is a block diagram which shows an example of the control unit in the cell processing apparatus of this embodiment, FIG. 17 is a perspective view which shows the other example of a structure of the cell processing apparatus of this embodiment.

As shown in FIG. 8, the cell processing apparatus 200 of this embodiment includes a first chamber 81 which is a first region, a second chamber 82 which is a second region, and a third chamber 83 which is a third region. A first chamber 81, a second chamber 82, and a third chamber 83, including a circulating means 84, are arranged in this order sequentially from the top to the bottom. The cell processing apparatus 200 of the present embodiment includes the circulating means 84, but the circulating means 84 may have any configuration, and may or may not be. Further, the positional relationship between the first chamber 81, the second chamber 82, and the third chamber 83 may be such that the first chamber 81 and the second chamber 82 are disposed continuously (adjacent) to each other, and the third chamber 83 is , Can be placed at any position. For example, as shown in FIG. 17, the third chamber 83 may be disposed separately from the first chamber 81 and the second chamber 82. As shown in FIG. 17, when the third chamber 83 is disposed separately from the first chamber 81 and the second chamber 82, the cell processing apparatus 200 can also be referred to as, for example, a cell processing system. The cell processing system may be, for example, a desktop system. It is preferable that the first chamber 81 be disposed at the top of the second chamber 82. When the laser is irradiated by the laser irradiation unit 85 described later from the upper part of the cell culture vessel 41, in order to stabilize the focal position of the laser irradiation unit 85, the medium inside the cell culture vessel 41 contains the emission of the laser emitting unit 85c. Need to put the mouth. However, when the laser irradiation is performed in this state, the components of the culture medium adhere to the emission port of the laser emission unit 85c, burn-in and the like occur, and the emission port of the laser emission unit 85c becomes dirty. Therefore, by arranging the cell processing apparatus 200 according to the present embodiment, for example, when irradiating a cell in the cell culture vessel 41 with the laser irradiation unit 85 described later, the laser emission port of the laser irradiation unit 85 It can control the dirt of Therefore, according to the cell processing apparatus 200 of the present invention, for example, the output of the laser emitted from the laser irradiation unit 85 can be stabilized, and the cells can be processed efficiently. The forming material of each region (each chamber) is not particularly limited, and examples thereof include a stainless steel plate, an iron plate subjected to rustproofing, and a resin plate that can be molded by vacuum molding, injection molding, pressure molding and the like. The formation material of each region is preferably a non-transparent material because the cells in the cell culture vessel 41 can be imaged more clearly by the observation unit described later. The “non-translucent” means, for example, suppressing the transmission of light of a wavelength that affects the imaging by the observation unit. When the observation unit can observe fluorescence, the wavelength of the light may be, for example, a wavelength corresponding to the fluorescence to be detected. As a specific example, as the non-light transmitting material, for example, a forming material of each of the above-mentioned regions and the like can be mentioned. The size and shape of each region are not particularly limited, and can be appropriately set according to the size and shape of members (means) disposed in each region. In the cell processing apparatus 200 of the present embodiment, the first chamber 81 and the second chamber 82 are configured as separate housings, and the housing configuring the first chamber 81 and the housing configuring the second chamber 82 Although arranged adjacent to each other, the present invention is not limited to this, and the first chamber 81 and the second chamber 82 are configured in one case, and the first chamber 81 and the second chamber 82 are formed in one case. You may constitute by dividing. The cell processing apparatus 200 of the present embodiment can easily perform maintenance of each member in the cell processing apparatus 200, for example, by configuring the first chamber 81 and the second chamber 82 in separate housings, and Assembly of the cell processing apparatus 200 is facilitated.

Next, as shown in FIG. 9, the cell processing apparatus 200 of this embodiment includes a light source 1, an illumination optical system 2, an imaging optical system 3, a cell culture vessel placement unit 4, a control unit 5, and An observation unit mainly including one moving unit 6 and a second moving unit 7 and a laser irradiation unit 85 are mainly included. The configuration other than the cell culture vessel placement unit 4 of the observation unit is the same as that of the phase contrast observation apparatus 100 of the first embodiment, and the description thereof can be used. In FIG. 9, the outer wall of the double wall of the first chamber 81 described later is omitted. The laser irradiation unit 85 includes a laser light source 85a, an optical fiber 85b, and a laser emitting unit 85c. The laser light source 85a and the laser emitting unit 85c are optically connected by an optical fiber 85b. In the first chamber 81, the light source 1 and the illumination optical system 2 accommodated in the housing 86, and the first moving unit 6 are disposed. The housing 86 accommodating the light source 1 and the illumination optical system 2 is movable by the first moving unit 6. In the second chamber 82, the imaging optical system 3 accommodated in the housing 87, the second moving unit 7, and the laser irradiation unit 85 are disposed. The imaging optical system 3 and the laser emitting unit 85 c in the laser irradiation unit 85 are movable by the second moving unit 7. The control unit 5 and the power supply unit 57 are disposed in the third chamber 83. The cell culture vessel placement unit 4 is formed as part of a partition between the first chamber 81 and the second chamber 82. The cell processing apparatus 200 of the present embodiment includes a cell culture vessel placement unit 4, a cell culture vessel 41, a first mobile unit 6, a second mobile unit 7, a housing 86, a housing 87, a first chamber 81, Although the two chambers 82 and the third chamber 83 are included, each may have any configuration, and may or may not be present.

The first chamber 81 includes a working opening 811 a on the front surface (the near side in FIG. 8) and an opening 811 b on the side of which maintenance can be performed. The opening 811 a is an opening for performing an operation related to the processing of the object in the object processing chamber which is the first chamber 81. The opening 811 b is an opening capable of maintaining the object processing chamber. The opening area of the opening 811a is preferably smaller than the opening area of the opening 811b, for example, in order to facilitate maintenance work. The size and number of the openings 811a and the openings 811b are not particularly limited, and for example, the sizes and numbers of working openings and openings that can be maintained in the safety cabinet can be referred to. As a specific example, the sizes and the numbers of the openings 811 a and the openings 811 b can be referred to, for example, the standard of the safety cabinet specified by EN 12469: 2000 which is the EN standard. The number of the openings 811 b is not particularly limited and may be any number, but for example, two or more is preferable because maintenance becomes easier. The arrangement location of the opening 811 a and the opening 811 b in the first chamber 81 is not particularly limited and may be any place, but the opening 811 a and the opening 811 b may be different locations (for example, different) of the first chamber 81 It is preferable to arrange on the side surface). In the present embodiment, the opening 811 b is mainly intended to easily perform maintenance in the cell processing apparatus 200, but may be used for other purposes. The cell processing apparatus 200 according to the present embodiment can observe, for example, the defective portion directly when a trouble occurs in the cell processing apparatus 200 by making it possible to observe the movement and the like of the internal members from the opening 811b. It is possible to consider measures.

The front wall of the first chamber 81 is a double wall having an outer wall and an inner wall, and the door 812a is an opening by raising and lowering a rail disposed in a space between the outer wall and the inner wall Open and close the opening of 811a. The opening 811 b can be opened and closed by attaching and detaching a door 812 b covering the opening. The opening 811 b is preferably sealed in the door 812 b, for example, when processing cells in the observation object processing chamber. Thus, for example, the gas outside the cell processing apparatus 200 and the dust contained therein can be prevented from flowing into the object processing chamber. In the cell processing apparatus 200 of the present embodiment, the opening 811a and its door 812a, and the opening 811b and its door 812b have any configuration, and may or may not exist, and any opening It may include only the department and its door. The wall of the first chamber 81 may be double-walled or single-walled, but the former is preferable because the size of the cell processing apparatus 200 can be reduced by arranging other members inside. When the wall of the first chamber 81 is a single wall, the door 812a is disposed outside the first chamber 81, for example, as a door 812b. The type of opening and closing of the door is not particularly limited, and may be, for example, a lifting type like the door 812a, an external type like the door 812b, or any other type. Examples of the other types include a double-door type, an accordion type, and a sliding door type. The material for forming the door is not particularly limited, and, for example, the materials for forming the above-mentioned regions can be incorporated, and a non-light-transmitting material is preferable.

As shown in FIG. 10, the inside of the first chamber 81 of the cell processing apparatus 200 of this embodiment is an object processing chamber for processing an object to be observed, and can be closed by closing the

doors

812a and 812b. That is, it can be opened and closed. The to-be-observed object processing chamber includes a first moving unit 6 including an XY stage 61 and an arm 62, a suction / discharge unit 813, a housing 86 for housing the illumination optical system 2, a drainage container placement portion 814a, and a storage container A placement unit 815a, a cell culture vessel placement unit 4, and a collection vessel placement unit 816a are included. In the present embodiment, the object processing chamber includes a first moving unit 6 including an XY stage 61 and an arm 62, a suction / discharge unit 813, a drainage container placement portion 814a, a storage container placement portion 815a, and a recovery container placement portion. Although 816 a is included, any configuration may be or may not be present, and any may be included or any two or more may be included. The XY stage 61 is disposed on the bottom surface of the object processing chamber, and is disposed so as to be movable in the X-axis direction and the Y-axis direction. At the top of the XY stage 61, an arm 62 including a pair of arms is disposed. A suction / discharge unit 813 is disposed at the tip of one arm of the arm 62 with its suction / discharge port directed downward. Further, at the tip of the other arm of the arm 62, a housing 86 including the illumination optical system 2 is disposed so as to be capable of guiding (illuminating) illumination light in the direction of the object 42. The drainage container placement unit 814a, the storage container placement unit 815a, the cell culture container placement unit 4, and the collection container placement unit 816a are along the moving direction of the XY stage 61 in the X axis direction on the bottom surface of the object processing chamber. It is arranged in this order. A drainage container 814b having a tip member detachment means 814c is disposed in the drainage container placement portion 814a, a storage container 815b is disposed in the storage container placement portion 815a, and a collection container is disposed in the collection container placement portion 816a. 816b is arranged.

The cell processing apparatus 200 of this embodiment can move the illumination optical system 2 and the suction / discharge unit 813 by the XY stage 61 and the arm 62 which are the first moving unit 6, but the suction / discharge unit 813 It may be movable by drive means other than moving unit 6 of the above. In this case, the moving direction of the drive unit capable of moving the suction and discharge unit 813 is not particularly limited. For example, any one, two, or all directions among the X axis direction, the Y axis direction, and the Z axis direction It is. In the present embodiment, the XY stage 61 is a known one that can move an object at high speed and precisely along the X-axis direction and the Y-axis direction via, for example, a linear motor carriage or the like. The arm 62 can extend and contract in the vertical direction (Z-axis direction), but the arm 62 may be fixed. In the latter case, the first moving unit 6 can move the suction and discharge unit 813 only on the XY plane, that is, only in the X-axis direction and the Y-axis direction in FIG.

The suction and discharge unit 813 sucks and discharges, for example, the culture medium, cells, and the like in the cell culture vessel 41. For example, the suction and discharge unit 813 mounts and uses a tip member described later on the side of the suction and discharge port. The suction and discharge unit 813 is not particularly limited, and, for example, a known suction and discharge unit can be used, and specific examples include an electric pipettor, an electric syringe pump, and the like.

The drainage container placement portion 814 a is a region where a drainage container 814 b for draining the suctioned liquid suctioned by the suction and discharge unit 813 can be arranged. In the present embodiment, the drainage container 814 b is disposed in the drainage container placement portion 814 a, but the drainage container 814 b may have any configuration, and may or may not be provided. In the present embodiment, the drainage container 814b is a box with an upper opening, the wall on the storage container placement portion 815a side extends upward, and is formed as a semicircular recess (notch) at the upper end thereof It has a wall (upper surface) in a direction substantially parallel to the bottom surface of the observation object processing chamber, including the tip member detachment means 814c. The drainage container 814b can recover the tip member detached from the suction and discharge unit 813, so it can be called, for example, a tip member collection container, and the drainage container placement portion 814a has a tip member collection container arrangement It can also be called a department. The tip member detachment means 814c is formed in the drainage container 814b, but may be separately disposed. Further, the tip end member detachment unit 814c may be disposed in the vicinity of the suction and discharge unit 813, specifically, the arm 62 of the first moving unit 6 in which the suction and discharge unit 813 is disposed.

The storage container placement portion 815a is a region where the storage container 815b in which the tip member which can be attached and detached is accommodated in the suction and discharge unit 813 can be disposed. In the present embodiment, the storage container 815 b is disposed in the storage container disposition portion 815 a, but the storage container 815 b may have any configuration, and may or may not have any configuration. The tip member is not particularly limited as long as it is a member capable of internally storing the liquid sucked by the suction and discharge unit 813. For example, when the suction and discharge unit 813 is a pipettor, a tip can be mentioned. The storage container 815b is, for example, a rack in which the tip is stored. The cell processing apparatus 200 according to the present embodiment includes the tip member detaching means 814c and the storage container placement portion 815a to simplify movement when sucking and discharging the medium, cells and the like in the cell culture container 41 (shortly )it can.

The collection container placement part 816 a is a region where a collection container 816 b for collecting a suction liquid containing cells collected by the suction and discharge unit 813 can be placed. In the present embodiment, the recovery container 816b is disposed in the recovery container placement part 816a, but the recovery container 816b may have any configuration, and may or may not have any configuration. Examples of the collection container 816b include known culture containers such as dishes and flasks.

In the present embodiment, on the bottom surface of the observation object processing chamber, along the movement direction of the XY stage 61 in the long axis direction (X axis direction) on the arrangement surface of the cell culture vessel placement unit 4, ie, the XY plane. The drainage container placement portion 814a, the storage container placement portion 815a, the cell culture container placement unit 4, and the collection container placement portion 816a are disposed in this order, but each placement portion is along the long axis direction. It may not be arranged, and may not be arranged in this order. In the present embodiment, by disposing the drainage container placement portion 814a, the storage container placement portion 815a, the cell culture container placement unit 4, and the collection container placement portion 816a in the above-described order, for example, The movement of the medium can be made linear, and the movement when sucking and discharging the medium, cells and the like in the cell culture vessel 41 can be simplified (shortened).

In addition, as shown in FIG. 11, a camera 817,

illumination lights

818a and 818b, and a germicidal lamp are provided above the opening 811a on the front wall of the subject processing chamber of the cell processing apparatus 200 of this embodiment Including 819.

Illumination lights

818 a and 818 b are disposed on both sides of the camera 817 in the X-axis direction, and a germicidal lamp 819 is disposed on the top.

In the present embodiment, the camera 817 is provided as the imaging unit of the first chamber 81, but the imaging unit of the first chamber 81 may have any configuration, and may or may not be. Further, the imaging means of the first chamber 81 is not limited to a camera, and may be capable of imaging in the first chamber 81, that is, the object processing chamber. The imaging means in the first chamber 81 is not particularly limited, and a known imaging means such as a microscope or a camera can be used, and a known imaging means and a solid-state imaging device (image sensor) such as a CCD or CMOS (Complementary MOS) And a combination of In the present embodiment, the camera 817 is disposed on the front wall in the object processing chamber, but the position of the camera 817 is not particularly limited and can be any position. It is preferable to arrange so that a wide range can be imaged. Specifically, as in the cell processing apparatus 200 of the present embodiment, the first mobile unit 6 is located on the back side (upper left side in FIG. 10) of the cell culture vessel placement unit 4 in the subject processing chamber. In the case where a certain XY stage 61, arm 62 and suction / discharge unit 813 are arranged, a wide range in the object processing chamber can be imaged, so that the front side of the object processing chamber (FIG. 10) It is preferable to arrange in the lower right side). The first imaging means is preferably capable of imaging at a plurality of magnifications (for example, different magnifications), but may be capable of imaging at one magnification. The magnification means, for example, an imaging magnification. As a specific example, the camera 817 includes, for example, lenses of multiple magnifications (eg, different magnifications). The imaging means of the first chamber 81 may be capable of optical zoom, digital zoom, etc., for example. By including the camera 817, the cell processing apparatus 200 of the present embodiment can confirm, for example, the operation in the observation object processing chamber, and the reliability of the operation is improved. The number of imaging means in the first chamber 81 disposed in the object processing chamber is not particularly limited, and may be one or more.

In the present embodiment, the

illumination lights

818a and 818b are provided as the illumination means, but the illumination means may have any configuration, and may or may not be present. Further, the illumination means is not limited to a lamp, and it may be capable of emitting light (illumination) into the observation object processing chamber. The illumination means is not particularly limited, and for example, known illuminations such as fluorescent lamps and LED lamps can be used. In the present embodiment, the

illumination lights

818a and 818b are disposed on the front wall in the object processing chamber, but the positions of the

illumination lights

818a and 818b are not particularly limited, and can be arbitrary positions. It is preferable to arrange so that light can be projected to a wide range in the object processing chamber, that is, shadows are not easily generated in the object processing chamber. Specifically, as in the cell processing apparatus 200 of the present embodiment, the first mobile unit 6 is located on the back side (upper left side in FIG. 10) of the cell culture vessel placement unit 4 in the subject processing chamber. When a certain XY stage 61, an arm 62 and a suction / discharge unit 813 are disposed, light can be projected to a wide range in the observation object processing chamber, so the front side of the observation object processing chamber (FIG. 10) In the lower right side). The cell processing apparatus 200 of the present embodiment includes the

illumination lights

818a and 818b, so that, for example, the work in the object processing chamber can be confirmed, and the reliability of the work is improved. The number of illumination units disposed in the object processing chamber is not particularly limited, and may be one or more.

In the present embodiment, a sterilizing lamp 819 is provided as a sterilizing means, but the sterilizing means may have any configuration, and may or may not be provided. Further, the sterilizing means is not limited to the germicidal lamp, and it may be capable of sterilizing the observation object processing chamber, particularly, around the cell culture vessel placement unit 4. The sterilization means is not particularly limited, and for example, known sterilization means such as a germicidal lamp and an ultraviolet LED lamp can be used. In the present embodiment, the germicidal lamp 819 is disposed on the front wall in the treatment subject treatment chamber, but the position of the germicidal lamp 819 is not particularly limited, and may be any position. As for the position of the germicidal lamp 819, for example, dust and the like outside the cell processing apparatus 200 preferably flows in from the

openings

811a and 811b, so that it is preferable to sterilize the vicinity of the

openings

811a and 811b. Specifically, as in the cell processing apparatus 200 of the present embodiment, in the case where an opening 811 a is provided on the front wall of the subject processing chamber, the front side of the subject processing chamber Preferably, the sterilizing means is disposed on the wall above the opening 811a. As in the cell processing apparatus 200 of the present embodiment, when the opening 811 b is provided on the side wall of the object processing chamber, the opening on the side wall of the object processing chamber It is preferable to arrange the sterilizing means on top of 811 b. Moreover, when the cell processing apparatus 200 includes the illumination means and the sterilizing means, it is preferable to dispose both on the same wall of the object processing chamber, for example, the wall provided with the opening 811a. In this case, the sterilizing means is preferably provided above the lighting means. The cell processing apparatus 200 according to the present embodiment includes the germicidal lamp 819 to improve, for example, the cleanliness in the object processing chamber. The number of sterilization means disposed in the object treatment chamber is not particularly limited, and may be one or more.

In the present embodiment, the size, shape, structure, etc. of the object processing chamber which is the first chamber 81 can be referred to, for example, the size, shape, structure, etc. of the safety cabinet. The standard of the safety cabinet specified in EN12469: 2000 can be referred to.

As shown in FIG. 12, the cell culture vessel disposition unit 4 of the cell processing apparatus 200 of the present embodiment includes an upper lid 43 and a bottom 44, and the upper lid 43 is detachably attached to the bottom 44. In the present embodiment, the cell culture vessel placement unit 4 is a box including the upper lid 43 and the bottom 44, and the cell culture vessel 41 is placed inside thereof, but the cell culture vessel placement unit 4 is limited to this. In addition, the cell culture vessel 41 can be disposed, and is disposed adjacent to the second chamber 82 in the observation object processing chamber, and a portion adjacent to the second chamber 82 in the cell culture vessel disposition unit 4 (FIG. 12). It is sufficient if the bottom plate 47) can transmit light. The “light transmission” means, for example, that the laser irradiated from the laser irradiation unit 85 of the second chamber 82 is transmitted. Also, it means that the imaging device 34 of the observation unit can be imaged via the bottom plate 47. The upper lid 43 is provided with a translucent region 45 so that illumination light can be emitted from the light source 1 to the cell culture vessel 41. The translucent region 45 is formed of, for example, a transparent glass plate, an acrylic plate, or the like. The bottom portion 44 includes a bottom wall 46 and a translucent bottom plate 47. The translucent bottom plate 47 is formed of, for example, a transparent glass plate, an acrylic plate or the like. The bottom plate 47 is adjacent to the second chamber 82. Therefore, it can be said that the adjacent part of the cell culture vessel placement unit 4 to the second chamber 82, that is, the bottom plate 47 is formed as a part of the wall of the object processing chamber. Preferably, the contact portion between the bottom plate 47 and the wall of the object processing chamber is sealed by a sealing member such as a packing or a sealing material, for example. This can prevent, for example, the gas in the second chamber 82 and dust contained therein from flowing into the cell culture vessel placement unit 4 and the object processing chamber. The bottom wall 46 includes four recesses 48 in which four cell culture vessels 41 can be arranged, and the side surface of each recess 48 extends from the inside of the object processing chamber to the outside of the object processing chamber ( In FIG. 12 (b), it has a reverse taper shape which narrows from top to bottom). Each recess 48 includes, on the bottom plate 47 end side, a projecting portion 49 projecting in the inward direction of the recess 48. The bottom end of the cell culture vessel 41 is in contact with the protrusion 49. In the cell processing apparatus 200 of the present embodiment, the bottom wall 46 has four recesses 48, but the number of the recesses 48 included in the bottom wall 46 is not limited to this, and the number corresponds to the number of cell culture vessels 41 to be disposed. Can be set appropriately. The size of the recess 48 can be appropriately set according to the size of the cell culture vessel 41 to be disposed. In the cell culture vessel arranging unit 4 of the present embodiment, the recess 48 has the above-described structure, so that the cell culture vessel arranging unit 4 is arranged with the cell culture vessel arranging unit 4 regardless of the shape of the side of the cell culture vessel 41 It becomes possible. In the cell processing apparatus 200 of the present embodiment, the bottom wall 46 is integrally formed with the bottom wall and the side wall thereof, but the bottom wall 46 is not limited to this, and each is used as a separate member It is also good. By forming the bottom wall 46 as a separate member, for example, members of the bottom wall of the plurality of bottom walls 46 having recesses 48 of different numbers and sizes can be prepared. Thus, for example, depending on the size and number of cell culture vessels 41, it can be replaced with a member of the bottom wall of bottom wall 46 having a size and number suitable for the arrangement of cell culture vessels 41. The container 41 can be suitably arranged.

As shown in FIGS. 13 and 14, in the cell processing apparatus 200 of the present embodiment, the circulation means 84 includes an intake part 84a, a circulation channel 84b, a gas supply part 84c, and an exhaust part 84d. Thus, the circulation means 84 circulates the gas in the object processing chamber.

The suction unit 84 a sucks in the gas in the observation object processing chamber. The air suction unit 84 a may suck in the gas outside the cell processing apparatus 200 instead of or in addition to the gas in the observation object processing chamber. In the present embodiment, the intake portion 84 a is disposed in the vicinity (for example, immediately below) of the opening 811 a of the object processing chamber. Specifically, the intake portion 84a has a plurality of openings (for example, slits) formed on the upper surface thereof (not shown), and the lower side of the opening 811a so that the opening communicates with the opening 811a. Is located in Thus, by disposing the intake part 84a in the vicinity of the opening 811a of the object processing chamber, for example, when the door 812a is opened and the worker works in the object processing chamber, the cells are removed. It is possible to prevent the gas outside the processing apparatus 200 and the dust contained therein from flowing into the observation object processing chamber. The intake portion 84a may be disposed in the vicinity of the opening 811b instead of or in addition to the opening 811a. For example, the air suction unit 84a may suck in the gas in the observation object processing chamber by a blowing unit such as a fan.

The circulation flow path 84 b connects the intake portion 84 a to the gas supply portion 84 c and the exhaust portion 84 d. In the present embodiment, the circulation flow passage 84 b is disposed in the space between the outer wall and the inner wall and in the upper portion of the first chamber 81. The circulation channel 84 b is, for example, a hollow cylinder. Further, one end of the circulation flow passage 84b communicates with the intake portion 84a, and the other end communicates with the gas supply portion 84c and the exhaust portion 84d. As in the cell processing apparatus 200 of the present embodiment, by arranging the circulation flow path 84b in the space between the outer wall and the inner wall, for example, the size of the cell processing apparatus 200 can be reduced. Further, in the present embodiment, the circulation means 84 includes the circulation flow passage 84b, but the circulation flow passage 84b may or may not be present. In the latter case, the intake part 84a is directly connected to, for example, the gas supply part 84c and the exhaust part 84d. For example, the circulation flow path 84b may blow the gas sucked by the suction portion 84a to the gas supply portion 84c and the exhaust portion 84d by a blowing unit such as a fan.

When the circulation flow path 84b includes the air blowing means, the air blowing means may be disposed in the vicinity of the air intake portion 84a, the gas supply portion 84c, or the exhaust portion 84d, or at other positions such as the central portion thereof. Although it may be arranged, air intake from the air intake portion 84a is improved, and for example, dust and the like are more effective to flow into the object processing chamber in comparison with the downflow caused by the gas supply portion 84c described later. It is preferable to dispose it in the vicinity of the intake part 84a because it can be prevented. When the air blowing means is disposed in the vicinity of the intake portion 84 a, the air blowing means is preferably disposed, for example, in the second chamber 82 or the third chamber 83. As a specific example, in the cell processing apparatus 200 of the present embodiment, when the circulation flow path 84b further includes the air blowing means, the air blowing means is the near side in the second chamber 82 or the third chamber 83 (FIG. 8). And the lower side of the intake part 84a). And, in this case, the circulation flow path 84b connects the suction part 84a and the suction side of the blowing means, and connects the blowing side of the blowing means to the gas supply part 84c and the exhaust part 84d. That is, the circulation flow passage 84 b is disposed in the second chamber 82 or the second and

third chambers

82 and 83, the space between the outer wall and the inner wall, and the upper portion of the first chamber 81.

The gas supply unit 84c supplies a part of the gas sucked by the suction unit 84a into the observation object processing chamber. In the present embodiment, the gas supply unit 84c is in communication with the upper end of the first chamber 81 so as to be able to supply the gas sucked from the suction unit 84a into the observation object processing chamber. The gas supply unit 84c may supply the gas into the observation object processing chamber by, for example, a blowing unit such as a fan. In addition, the gas supply unit 84c may include, for example, a gas purification unit. In this case, the gas supplied from the gas supply unit 84c into the object processing chamber passes through the gas cleaning means. By including the gas cleaning means, for example, dust and the like can be prevented from flowing into the observation object processing chamber. Examples of the gas cleaning means include particulate collection filters such as HEPA filters (High Efficiency Particulate Air Filter) and ULPA filters (Ultra Low Penetration Air Filter). The cell processing apparatus 200 of the present embodiment is connected to the gas supply unit 84c in the upper part of the observation object processing chamber, so that, for example, a downflow occurs due to air flow from the gas supply unit 84c, thereby opening the opening Dust and the like can be more effectively prevented from flowing into the observation object processing chamber from 811a.

The exhaust unit 84 d exhausts the remaining portion of the gas taken by the intake unit 84 a to the outside of the object-to-be-observed body processing, specifically to the outside of the cell processing apparatus 200. In the present embodiment, the exhaust unit 84 d is disposed at the upper end (uppermost portion) of the cell processing device 200 so that the gas sucked from the intake unit 84 a can be exhausted to the outside of the cell processing device 200. Thus, by providing the exhaust part 84d at the top of the cell processing apparatus 200, for example, the size of the cell processing apparatus 200 can be reduced, and dust that has been blown up by the exhaust flows into the observation object processing chamber. Can be prevented. The exhaust unit 84 d may exhaust the gas to the outside of the cell processing apparatus 200 by, for example, a blowing unit such as a fan. In addition, the exhaust unit 84d may include, for example, the gas purification unit. In this case, the gas exhausted from the exhaust unit 84 d to the outside of the cell processing apparatus 200 passes through the gas purification unit. By including the gas cleaning means, for example, it is possible to prevent the outflow of the particles and the like generated in the observation object processing chamber to the outside of the cell processing apparatus 200.

In the circulation means 84, the size, shape, structure, etc. of each part can refer to, for example, the size, shape, structure, etc. of the safety cabinet, and as a specific example, the standard of the safety cabinet specified in the above-mentioned EN 12469: 2000 You can refer to

As shown in FIG. 15A, in the cell processing apparatus 200 of the present embodiment, the second chamber 82 includes a second moving unit 7, a housing 87 in which the imaging optical system 3 is housed, and a laser irradiation unit 85. including. The cell processing apparatus 200 of the present embodiment includes the second mobile unit 7. However, as described above, the second mobile unit 7 may or may not have any configuration. Also, either one may be included. The second moving unit 7 includes an XY stage 71 and

carriages

711a and 711b. The XY stage 71 is arranged on the arrangement surface of the cell culture vessel arrangement unit 4, that is, on the bottom of the second chamber 82 substantially parallel to the XY plane. In the XY stage 71, on the common rail (moving path) in the Y-axis direction, two rails in the X-axis direction are disposed movably on the common rail.

Bogies

711a and 711b are disposed on the two rails in the X-axis direction so as to be able to move the rails. The laser irradiation unit 85 includes a laser light source 85a, an optical fiber 85b, and a laser emission unit 85c. In the upper part of the XY stage 71, a casing 87 in which the imaging optical system 3 is accommodated directs the objective lens 31 of the imaging optical system 3 upward (Z-axis direction) to the carriage 711b, and laser irradiation It is disposed on the carriage 711a with the laser emission port of the laser emission unit 85c of the unit 85 directed upward (in the Z-axis direction). The carriage 711a can move up and down in the vertical direction (Z-axis direction). The laser light source 85 a is disposed on the bottom surface of the second chamber 82 in a region not overlapping the movable range of the XY stage 71 in the second chamber 82. One end of the optical fiber 85b is optically connected to the laser light source 85a, and the other end is optically connected to the laser emission portion 85c.

The cell processing apparatus 200 of the present embodiment can move the imaging optical system 3 and the laser irradiation unit 85 by the XY stage 71 which is the second moving unit 7, but the laser irradiation unit 85 is a second moving unit. It may be movable by drive means other than seven. In this case, the moving direction of the driving unit capable of moving the laser irradiation unit 85 is not particularly limited, and, for example, any one, two or all directions among the X-axis direction, the Y-axis direction and the Z-axis direction It is. Further, in the present embodiment, the driving means (laser moving means) capable of moving the laser irradiation unit 85 and the second moving unit 7 share a rail in the Y-axis direction (first direction). The moving means and the second mobile unit 7 may be independent. As a specific example, as shown in FIG. 15B, the laser moving unit at the bottom of the second chamber 82 is disposed, for example, as an XY stage 71a, and the second moving unit 7 is disposed as an XY stage 71b. May be The moving direction of the laser moving unit and the second moving unit 7 is not particularly limited, and is, for example, any one, two, or all directions among the X-axis direction, the Y-axis direction, and the Z-axis direction. . If the laser moving means can move the laser irradiation unit 85 in a direction substantially orthogonal to, for example, the arrangement surface of the cell culture vessel placement unit 4, that is, the bottom of the cell culture vessel 41, the laser moving means The spot diameter described later can be adjusted. In this case, the laser moving means doubles as, for example, a spot diameter adjusting means described later. In the present embodiment, the XY stage 71 is a known one that can move the object at high speed and precisely along the X-axis direction and the Y-axis direction via, for example, a linear motor carriage or the like.

As in the XY stage 71 of the present embodiment, the laser moving means and the second moving unit 7 are each arranged in a first direction (for example, as shown in FIG. The laser irradiation unit 85 and the second imaging means can be moved in the direction of the arrow Y in 15 (a), and the movement of the laser irradiation unit 85 in the first direction by the laser moving means and the second movement The movement of the imaging optical system 3 in the first direction by the unit 7 is preferably on the same straight line. Thus, by moving the laser irradiation unit 85 and the imaging optical system 3 on the same straight line, for example, the cells in the cell culture vessel 41 are imaged by the imaging optical system 3 and then processed by the laser irradiation unit 85. When performing cell processing such as, the number of movements of each means can be reduced, and processing time can be reduced. Further, as in the case of the XY stage 71 of the present embodiment, the laser moving unit moves the carriage 711a for arranging the laser irradiation unit 85 and the movement path along which the carriage 711a is moved and arranged along The second moving unit 7 includes a carriage 711b for disposing the imaging optical system 3, and a movement path (rail) along which the carriage 711b is moved and arranged along the first direction, It is preferable that the moving path of the laser moving means and the moving path of the imaging optical system 3 be the same. By configuring in this way, when performing cell processing such as processing by the laser irradiation unit 85 after imaging by the imaging optical system 3, the number of movements of each means can be further reduced, and processing time can be further reduced. It is preferable that the second mobile unit 7 is configured to be movable independently of the first mobile unit 6 as in the cell processing device 200 of the present embodiment.

In the cell processing apparatus 200 of the present embodiment, a housing 87 for housing the imaging optical system 3 having the objective lens 31 of one type of magnification as the imaging optical system 3 is disposed, but the invention is not limited thereto. A housing 87 that houses the imaging optical system 3 having a plurality of types of objective lenses 31 may be disposed. In this case, the magnifications of the plurality of types of objective lenses 31 are preferably different magnifications, such as 2 ×, 4 ×, and 8 ×, for example. Further, as in the cell processing apparatus 200 of the present embodiment, when the imaging optical system 3 having the imaging means in the first chamber 81 and the objective lens 31 is included, the cells in the cell culture vessel 41 can be imaged more clearly. The magnification of the objective lens 31 of the imaging optical system 3 is preferably higher than the magnification of the imaging means of the first chamber 81.

In the cell processing apparatus 200 of the present embodiment, the laser irradiation unit 85 includes the laser light source 85a, the laser emitting unit 85c, and the optical fiber 85b, but the laser irradiation unit 85 is not limited thereto. It is sufficient if the cell culture vessel 41 disposed in the above can be irradiated with a laser. The laser irradiation unit 85 may include, for example, a laser light source 85a, and may irradiate the cell culture vessel 41 with laser directly from the laser light source 85a. When the laser of the laser light source 85a is guided to the laser emitting unit 85c, the light may be guided using a light guiding means such as a mirror or MEMS (Micro Electro Mechanical Systems) instead of the optical fiber 85b. The arrangement of the laser light source 85a in the second chamber 82 can be freely set. For example, in the second chamber 82, other means such as the laser moving means, the imaging optical system 3, and the second moving unit 7 By arranging the laser light source 85a in a region which is not disposed and does not overlap with the movable range of other means, the size of the cell processing apparatus 200 can be reduced, and the cell processing compared with other light guiding means The optical fiber 85 b is preferable because the weight of the device 200 can be reduced.

The laser light source 85 a is, for example, a device that oscillates a continuous wave laser or a pulse laser. The laser light source 85a may be, for example, a high frequency laser having a long pulse width close to a continuous wave. The output of the laser oscillated from the laser light source 85a is not particularly limited, and can be determined appropriately according to, for example, the treatment and the cells. The wavelength of the laser emitted by the laser light source 85a is not particularly limited, and examples thereof include visible light lasers such as 405 nm, 450 nm, 520 nm, 532 nm, and 808 nm, infrared lasers, and the like. As described above, when the cell culture vessel 41 is provided with a laser absorption layer, the laser light source 85 a oscillates, for example, a wavelength that can be absorbed by the laser absorption layer. The laser light source 85a preferably oscillates a laser having a wavelength longer than 380 nm because it can suppress the influence on cells. A specific example of the laser light source 85a is a continuous wave diode laser having a wavelength of about 405 nm and a maximum output of 5 W.

When the laser irradiation unit 85 includes the laser emitting unit 85c, the laser moving unit preferably moves the laser emitting unit 85c. When the laser moving means moves the laser emitting unit 85c in the vertical direction (in the direction of arrow Z in FIG. 15), the laser emitting port of the laser emitting unit 85c is the bottom surface of the object processing chamber, preferably It is preferable to move so as not to contact the bottom of the cell culture vessel placement unit 4. As a specific example, it is preferable that the laser moving means move the laser emitting port of the laser emitting unit 85 c so as not to approach within 1 mm with reference to the bottom surface of the cell culture vessel placement unit 4. In such a range, when the laser moving unit moves the laser emitting unit 85c, for example, it is arranged in the cell culture vessel arranging unit 4 generated by the contact between the laser emitting unit 85c and the bottom of the cell culture vessel arranging unit The shaking of the culture medium in the cell culture vessel 41 can be prevented.

In the present embodiment, the imaging optical system 3 is disposed on the near side (the lower left side in FIG. 15), and the laser irradiation unit 85 is disposed on the far side (the upper right side in FIG. 15). However, the positional relationship between the imaging optical system 3 and the laser irradiation unit 85 is not limited to this. For example, even if the imaging optical system 3 is disposed on the back side and the laser irradiation unit 85 is disposed on the front side Good. Generally, the imaging optical system 3 of the observation unit has a large volume compared to the laser irradiation unit 85. Therefore, when the cell culture vessel placement unit 4 is disposed on the front side in the first chamber 81, the imaging optical system 3 is disposed on the back side, and the laser irradiation unit 85 is disposed on the front side. The size of the cell processing apparatus 200 can be reduced.

The cell processing apparatus 200 of the present embodiment may further include spot diameter adjusting means for adjusting the diameter of the spot that the laser forms on the irradiated portion of the object to be irradiated. The spot diameter means the beam diameter of the laser at the contact portion between the laser and the object to be irradiated. The spot diameter can be adjusted, for example, by switching at least one of the laser condenser lens and the collimator lens (collimation lens) of the laser irradiation unit 85 or changing the distance between the laser irradiation unit 85 and the object to be irradiated. . In the former case, it is preferable that the laser irradiation unit 85 includes, for example, a plurality of lenses, and the spot diameter adjusting unit adjusts the diameter of the spot by changing the lens. The plurality of lenses may be, for example, a plurality of condenser lenses, a plurality of collimator lenses, or a combination of one or more condenser lenses and one or more collimator lenses. The plurality of focusing lenses have, for example, different focal lengths. The plurality of collimator lenses have, for example, different focal lengths. The change of the lens may be performed manually, for example, or may be changed by the control unit 5 described later. In the latter case, for example, a lens changing means is included, and the changing means changes the lens. Moreover, when the spot diameter adjusting means changes the distance, it is preferable that the spot diameter adjusting means adjust the diameter of the spot by adjusting the distance between the laser irradiation unit 85 and the object to be irradiated. . The distance between the laser irradiation unit 85 and the object to be irradiated means, for example, a distance in a direction substantially orthogonal to the arrangement surface of the cell culture container placement unit 4, that is, the bottom surface of the cell culture container 41. When the laser irradiation unit 85 includes the laser emitting unit 85c, the distance between the laser irradiation unit 85 and the object to be irradiated means the distance between the laser emitting unit 85c and the object to be irradiated. The distance between the laser irradiation unit 85 and the object to be irradiated can be adjusted, for example, by the laser moving unit. As a specific example, it is possible to adjust the distance from the bottom of the cell culture vessel 41, which is the object to be irradiated, by the movement of the laser moving means in the direction of the arrow Z. In the cell processing apparatus 200 of the present embodiment, the carriage 711a of the XY stage 71, which also serves as the laser moving means, can move up and down in the vertical direction (arrow Z direction). For this reason, the laser moving means in the present embodiment can also be called, for example, a spot diameter adjusting means. The spot diameter adjusting means adjusts the spot diameter to a small size, for example, when performing cell processing in which a small spot diameter is preferable, for example, dividing a cell mass, or cutting out a cell or cell mass in a specific region. Further, the spot diameter adjusting means adjusts the spot diameter to a large size, for example, in the case of performing cell treatment in which a large spot diameter is preferable, for example, the killing of cells in a specific region. The size of the spot diameter is not particularly limited, and can be appropriately set according to, for example, the type of cell treatment, the size of cells, and the like. The cell processing apparatus 200 according to the present embodiment includes the spot diameter adjusting means, so that the spot diameter can be adjusted to an appropriate size, for example, by the processing performed on the cells, and the cell processing can be performed rapidly. In addition, since the diameter of the spot can be adjusted to an appropriate size, for example, the influence on the non-treated cells can be reduced.

When the cell processing apparatus 200 of the present embodiment includes the spot diameter adjusting means, the control unit 5 described later preferably controls the adjustment of the diameter of the spot by the spot diameter adjusting means.

In the cell processing apparatus 200 of the present embodiment, it is preferable that the movement of gas be suppressed between the observation object processing chamber and the second chamber 82. The movement of the gas can be suppressed, for example, by sealing the portion adjacent to the second chamber 82 in the object processing chamber with a sealing member such as the packing and the sealing material described above. By thus suppressing the movement of the gas, for example, the inflow of dust contained in the gas into the observation object processing chamber can be prevented.

In the cell processing apparatus 200 of the present embodiment, the third chamber 83 includes the control unit 5 and the power supply unit 57. As shown in FIG. 16, in the control unit 5 of this embodiment, the I / O interface 55 includes the DMD 22, the first moving unit 6, the second moving unit 7, the suction / discharge unit 813, the camera 817, the observation unit , And a device for communicably connecting to and controlling each member such as the laser irradiation unit 85. Except for this point, the control unit 5 of the present embodiment has the same configuration as the control unit 5 of the first embodiment, and the description thereof can be used.

The cell processing apparatus 200 of this embodiment controls the DMD 22, the first mobile unit 6, the second mobile unit 7, the suction / discharge unit 813, the camera 817, the observation unit, and the laser irradiation unit 85 in the control unit 5. By providing the function, it is not necessary to individually provide the control unit to each member, so the device can be miniaturized. However, the present invention is not limited to this. The cell processing apparatus of the present invention includes, for example, the DMD 22, the first moving unit 6, the second moving unit 7, the suction / discharge unit 813, the camera 817, the observation unit, and the laser irradiation unit 85 as the control unit 5. A control unit may be provided to control each member by the control unit of each member. Moreover, the cell processing apparatus of this invention may provide control unit 5 and the control unit of each member, and may control each member jointly, for example.

In the present embodiment, the control unit 5 controls the observation unit and the laser irradiation unit 85, but the control unit 5 may control either one.

In the present embodiment, the control unit 5 controls the laser irradiation by the laser irradiation unit 85 and the movement of the laser emitting unit 85 c of the laser irradiation unit 85 by the XY stage 71 and the carriage 711 a which also serve as the laser moving unit. May control one or the other.

In the present embodiment, the control unit 5 controls the suction and discharge by the suction and discharge unit 813 and the movement of the suction and discharge unit 813 by the XY stage 61 and the arm 62 which are the first moving unit 6. One of them may be controlled.

In the present embodiment, the control unit 5 controls imaging in the object processing chamber by the camera 817 which is an imaging unit of the first chamber 81.

In the present embodiment, the control unit 5 turns on / off the light source 1, moves the illumination optical system 2 by the XY stage 61 and the arm 62 as the first moving unit 6, and observes the image pickup element 34 of the imaging optical system 3. Although the imaging of the body and the movement of the imaging optical system 3 by the XY stage 71 and the carriage 711b which are the second moving unit 7 are controlled, the control unit 5, one or more of them may be controlled .

The power supply unit 57 is not particularly limited, and a known power supply can be used. The power supply unit 57 includes, for example, a laser irradiation unit 85, the observation unit, the first moving unit 6, the second moving unit 7, a suction / discharge unit 813, a circulating unit 84, the illumination unit, the sterilizing unit, the control unit 5, etc. Power is supplied to a member (means) operated by the power of For this reason, the power supply unit 57 is electrically connected to, for example, a member (means) operated by the power. The power supply unit 57 supplies power at a voltage of 100 V, for example. Thereby, for example, the cell processing apparatus 200 can be used also in a general power environment. In the cell processing apparatus 200 of the present embodiment, the power supply unit 57 carries the entire power supply, so that it is not necessary to individually provide the power supply unit to each member. And weight reduction can be realized. However, the present invention is not limited to this, and for example, at least one of the respective units may be provided with a dedicated power supply unit.

In the cell processing apparatus 200 of the present embodiment, a communication unit (not shown) may be further provided in the third chamber 83. The communication unit has, for example, a function of transmitting / receiving data to / from an external device such as a personal computer or a mobile communication device or the Internet by wire or wirelessly. The communication unit may be, for example, an existing communication module. By providing the communication unit in this manner, the cell processing apparatus 200 can be connected to the outside, so, for example, the cell processing apparatus 200 is operated from the outside, or the cell processing apparatus 200 receives data from the outside. be able to. Further, data in the cell processing apparatus 200 can be browsed by, for example, connecting from outside.

Next, processing of cells using the cell processing apparatus 200 of the present embodiment and recovery of the treated cells will be described by way of examples.

First, the germicidal lamp 819 is turned off and the

lamp

818a, 818b are turned on. Further, the control unit 5 activates the camera 817 to start imaging in the observation object processing chamber. The image in the to-be-observed object processing chamber captured by the camera 817 is output to the display device via the control unit 5, for example. Next, the circulation means 84 is operated to circulate the gas in the object processing chamber. Further, the worker opens the door 812a of the opening 811a, arranges the cell culture vessel 41 in the cell culture vessel arrangement unit 4, and arranges the collection vessel 816b in the collection vessel arrangement part 816a. The laser absorption layer is formed on the bottom of the cell culture vessel 41. After the placement, the worker closes the door 812a of the opening 811a.

Next, the control unit 5 controls the XY stage 71 and the carriage 711b to move, and the housing 87 accommodating the imaging optical system 3 moves to the lower side of the bottom surface of the cell culture vessel 41. In addition, the control unit 5 controls the XY stage 61 to move, and the housing 86 accommodating the illumination optical system 2 is placed on the upper surface of the cell culture container 41, ie, on the cell culture container arrangement unit 4. Moving. Then, the tile image of the cell culture vessel 41 is obtained in the same manner as the imaging method of the phase contrast observation apparatus 100 of the first embodiment. The tile image may be acquired, for example, using an objective lens 31 of different magnification depending on the size of the object 42 to be processed. An image captured by the imaging element 34 may be, for example, a phase difference image captured by a phase contrast microscope. When the imaging optical system 3 can perform fluorescence observation, the image captured by the imaging device 34 may be a fluorescence image. The captured image is output to the display device via, for example, the control unit 5.

When, for example, a worker designates a processing target area (for example, a cell area to be collected) by the input device based on the captured tile image, the control unit 5 moves the XY stage 71 and the carriage 711a. The laser emission unit 85 c is controlled to move to a position where the target such as cells around the processing target area can be irradiated with the laser on the lower side of the bottom surface of the cell culture vessel 41. Then, the control unit 5 controls the laser light source 85 a to oscillate a laser. The oscillated laser is guided by the optical fiber 85 b and emitted from the laser emission unit 85 c. Further, together with the laser irradiation, the control unit 5 moves the XY stage 71 and the carriage 711a around the processing target area. At this time, the size of the spot diameter is adjusted to an appropriate size by raising and lowering the carriage 711 a according to the size of the object around the processing target area, and the target within the processing target area is adjusted. The observation object 42 is not affected. The irradiated laser is absorbed by the laser absorption layer formed on the bottom surface of the cell culture vessel 41, and the target area around the processing target area is killed by heat or the like generated from the laser absorption layer. Thereby, the processing target area can be cut out.

Next, the XY stage 61 is controlled to move by the control unit 5, and the suction and discharge unit 813 moves to the upper part of the storage container 815b. The control unit 5 controls the arm 62 to be lowered and raised, and the tip as the tip member is mounted on the suction / discharge port side of the suction / discharge unit 813. Furthermore, the XY stage 61 is controlled to move by the control unit 5, and the suction and discharge unit 813 is moved to the upper part of the processing target region in the upper part of the cell culture vessel 41. The control unit 5 controls the arm 62 to descend, and places the opening of the tip near the processing target area. In this state, the control unit 5 controls the suction and discharge unit 813 to suction so as to suction the observation object 42 in the processing target area into the chip together with the surrounding medium.

Further, the control unit 5 controls the arm 62 to move upward and the XY stage 61 to move, and the suction and discharge unit 813 moves to the upper part of the collection container 816b. Further, the control unit 5 controls the arm 62 to be lowered, and the opening of the tip moves inside the collection container 816b. In this state, the control unit 5 controls the suction and discharge unit 813 to discharge, and discharges the culture medium including the object 42 to be observed in the processing target area in the chip into the collection container 816b.

After the discharge, the control unit 5 controls the arm 62 to move up and the XY stage 61 to move, and the suction and discharge unit 813 moves to the upper part of the drainage container 814 b. Further, the control unit 5 controls the arm 62 to be lowered and the XY stage 61 to be moved, and the upper end of the tip is a tip member detachment which is a concave portion of the upper surface provided in the drainage container 814b. Hook on means 814c. In this state, the control unit 5 controls the arm 62 to ascend, and the tip is detached from the suction and discharge unit 813.

Then, the worker opens the door 812a of the opening 811a, recovers the cell culture vessel 41 from the cell culture vessel placement unit 4, and recovers the recovery vessel 816b from the recovery vessel placement portion 816a. By doing so, the cell processing apparatus 200 according to the embodiment can process the object to be observed 42 such as cells and collect the processed object to be observed 42.

According to the cell processing apparatus 200 of the present embodiment, since the deterioration of the phase difference image due to the meniscus can be suppressed, it is possible to capture the phase difference image with large contrast by the observation unit. Since the to-be-observed object 42 which implements a laser processing, area | region, etc. can be clarified if it is based on the phase contrast image with large said contrast, a laser processing can be implemented with high precision. For this reason, according to the cell processing apparatus 200 of the present embodiment, for example, damage at the time of laser processing can be reduced.

According to the cell processing apparatus 200 of the present embodiment, for example, processing such as sorting and recovery can be easily performed on the cells of the cell culture vessel 41. Moreover, since the cell processing apparatus 200 of this embodiment processes a cell not with a worker itself but with the laser irradiation unit 85, for example, it is not influenced by the skill level of the worker. Thus, for example, the quality of cells obtained after treatment is stabilized.

Next, examples of the present invention will be described. However, the present invention is not limited by the following examples. Commercially available reagents were used based on their protocol unless otherwise indicated.

Example 1
By imaging the cells in the cell culture vessel using the phase contrast observation device of the present invention, it was confirmed that deterioration of the phase contrast image due to the meniscus can be suppressed.

When 3 mL of culture solution is introduced into a 35 mm (diameter) dish (manufactured by IWAKI), deterioration of the phase difference image in a range of about 7 mm from the wall surface (region between dashed line and solid line) by meniscus as shown by arrows in FIG. Will occur. Therefore, in Example 1, regions a to d shown in FIG. 18 of the dish into which the culture solution was introduced were imaged using the phase contrast observation apparatus 100. Specifically, for the regions a to d, after the intensity distribution correction information was acquired by the acquisition method shown in FIG. 6, imaging was performed by the imaging method shown in FIG. In addition, each process in the acquisition method shown in FIG. 6 was implemented by the user. In addition, the intensity distribution of the next illumination light was set by moving the illumination image. In the regions a to d, the regions b to d are regions where deterioration of the phase difference image occurs due to the meniscus. Therefore, the region b is corrected using intensity distribution correction information including the intensity distribution of one illumination light to obtain an image (corrected image 1). The area c is corrected using intensity distribution correction information including the intensity distribution of two illumination lights, and after obtaining two images (corrected images 2 to 3), one image is integrated by integrating the correction target areas. I got an image of. The area d is corrected using intensity distribution correction information including the intensity distribution of three illumination lights, and after acquiring three images (corrected images 4 to 6), one image is integrated by integrating the correction target areas. I got an image of. The results are shown in FIG.

FIG. 19 is a photograph showing a phase difference image taken by the phase difference observation apparatus 100. As shown in FIG. 19, in the region a where no meniscus is generated, deterioration of the phase difference image was not seen even in a state without correction. On the other hand, deterioration of the phase difference image has occurred in the regions b to d where the meniscus is generated, but by adopting or integrating the images (corrected images 2 to 6) obtained by the correction with the intensity distribution correction information. The deterioration of the phase difference image in these regions could be further suppressed.

From these results, it was found that deterioration of the phase contrast image due to the meniscus can be suppressed by imaging the cells in the cell culture vessel using the phase contrast observation device of the present invention.

Example 2
It was confirmed by the phase contrast observation device of the present invention that the area in which an image in which the deterioration of the phase contrast image due to the meniscus is suppressed can be captured is expanded.

A phase difference image was taken in the same manner as in Example 1 for the entire surface of a 35 mm dish into which 3 mL of culture solution was introduced. Moreover, the control imaged the phase difference image in the same manner except that the correction based on the intensity distribution correction information and the correction value of the intensity distribution correction information were not used. Then, in each phase difference image, the area of the area where the meniscus was suppressed, that is, the area in which the cells in the dish could be observed was calculated. Moreover, the average value ( _RA ) of the distance from the wall surface of a 35 mm dish to the said observable area was calculated about the image before and behind correction | amendment by the correction value of the said intensity distribution correction information.

Next, for a 60 mm (diameter) dish (φ 60, manufactured by IWAKI) and a 100 mm (diameter) dish (φ 100, manufactured by IWAKI), the average value of the distance from the wall surface to the observable region in a 35 mm dish ( _RA Based on the above, the area of the area in which cells in the dish can be observed before and after correction with the correction value of the intensity distribution correction information was calculated. The results are shown in Table 1 below.

As shown in Table 1, according to the phase difference observation apparatus 100, the observable region of the dish is greatly expanded, and the effect is remarkable particularly when the diameter of the dish is small.

From these results, it was found that the phase contrast observation device of the present invention enlarges the area in which the image in which the deterioration of the phase contrast image due to the meniscus is suppressed can be captured.

Although the present invention has been described above with reference to the embodiments, the present invention is not limited to the above embodiments. Various changes that can be understood by those skilled in the art can be made to the configuration and details of the present invention within the scope of the present invention.

This application claims priority based on Japanese Patent Application No. 2017-189170 filed on September 28, 2017, and Japanese Patent Application No. 2081-10020 filed on June 8, 2018, The entire disclosure is incorporated herein.

<Supplementary Note>
Some or all of the above embodiments and examples may be described as in the following appendices, but are not limited to the following.
(Supplementary Note 1)
Light source,
An illumination optical system for guiding illumination light from the light source to an object in a cell culture vessel;
An imaging optical system for forming an optical image of the object to be observed on an imaging device;
Including a control unit,
The illumination optical system includes a spatial modulation element that changes the intensity distribution of the illumination light,
The control unit
It includes intensity distribution correction information that associates the position of the imaging optical system with respect to the cell culture vessel and the intensity distribution of illumination light at the position of the imaging optical system,
Acquiring imaging system position information which is the position of the imaging optical system;
A phase difference observation apparatus characterized in that an intensity distribution of illumination light in the spatial modulation element is changed based on the imaging system position information and the intensity distribution correction information.
(Supplementary Note 2)
The imaging optical system includes a phase plate,
The intensity distribution of the illumination light in the intensity distribution correction information is the intensity of the illumination light included in the phase plate image, which is the image of the phase plate, of the illumination image, which is the image of the illumination light, at the position of the imaging optical system. The phase difference observation device according to appendix 1, which is a distribution.
(Supplementary Note 3)
The imaging optical system includes a phase plate,
The control unit is configured such that an illumination image, which is an image of the illumination light, is included in a phase plate image, which is an image of the phase plate, at the position of the imaging optical system with respect to the cell culture vessel that does not include the subject. The phase difference observation apparatus according to

Appendix

1 or 2, wherein the intensity distribution of illumination light is determined, and the obtained intensity distribution of illumination light and the position of the imaging optical system are associated and stored as the intensity distribution correction information.
(Supplementary Note 4)
The phase difference observation device according to any one of appendices 1 to 3, wherein the spatial modulation element includes a digital micromirror device that guides the illumination light by reflecting the illumination light toward the subject.
(Supplementary Note 5)
A first moving unit capable of moving the light source and the illumination optical system;
The phase difference observation device according to any one of appendices 1 to 4, wherein the control unit controls movement by the first mobile unit.
(Supplementary Note 6)
The imaging optical system includes a phase plate,
The phase difference observation apparatus according to claim 5, wherein the first moving unit is movable in a direction perpendicular to a surface of the phase plate.
(Appendix 7)
The control unit
Storing illumination system position correction information in which the position of the imaging optical system is associated with the positions of the light source and the illumination optical system;
The phase difference observation apparatus according to any one of appendices 1 to 6, wherein the positions of the light source and the illumination optical system are corrected based on the imaging system position information and the illumination system position correction information.
(Supplementary Note 8)
The control unit
Fractionating the imaging target area of the cell culture vessel into a plurality of sections;
Based on the imaging system position information and the intensity distribution correction information, the intensity distribution of the illumination light in the spatial modulation element is changed, and each section is imaged by the imaging element,
15. The phase contrast observation device according to any one of appendices 1 to 7, which produces an image of an imaging target region of the cell culture vessel based on the obtained image.
(Appendix 9)
The control unit
In the image of the imaging target area, it is determined whether there is a defective imaging site,
If there is the imaging failure site, the correction value of the intensity distribution correction information of the section including the imaging failure site is calculated,
Based on the imaging system position information, the intensity distribution correction information, and the correction value of the intensity distribution correction information, the imaging device re-images a section including the imaging failure portion,
The phase difference observation apparatus according to appendix 8, wherein the image of the section including the imaging failure region is changed to an image obtained by the reimaging, and an image of the imaging target area is produced.
(Supplementary Note 10)
The control unit
In the cell culture vessel not including the object, the intensity distribution of the illumination light included in the phase plate image, which is an image of the phase plate, is an illumination image, which is an image of the illumination light, at the position of the imaging optical system. Asking for
The intensity distribution of the illumination light in the spatial modulation element is changed to the intensity distribution of the illumination light obtained, and the image pickup device picks up an image, and determines whether the luminance value at the pixel of the obtained image is greater than the reference value of the luminance value And
When the luminance value in the image is equal to or more than the reference value of the luminance value, the illumination image, which is an image of the illumination light, is changed at the position of the imaging optical system in a region equal to or more than the reference value of the luminance value of the image. , Find the intensity distribution of the following illumination light,
11. The phase difference observation apparatus according to any one of appendices 1 to 9, wherein the obtained intensity distribution of each illumination light and the position of the imaging optical system are associated with each other and stored as the intensity distribution correction information.
(Supplementary Note 11)
The control unit
It is determined whether the intensity distribution correction information associated with the imaging system position information includes the intensity distribution of a plurality of illumination lights,
When the intensity distribution correction information includes the intensity distribution of a plurality of illumination light, the intensity distribution of the illumination light in the spatial modulation element is imaged by changing the intensity distribution of each illumination light,
10. The phase difference observation apparatus according to appendix 10, wherein in each of the obtained images, an area in which a luminance value of a pixel of the obtained image is less than a reference value of the luminance value is extracted and the extracted images are integrated.
(Supplementary Note 12)
A cell culture vessel placement unit capable of placing the cell culture vessel,
11. The phase contrast observation device according to any one of appendices 1 to 11, wherein the cell culture vessel placement unit is disposed between the illumination optical system and the imaging optical system in the light path of the illumination light.
(Supplementary Note 13)
15. The phase contrast observation device according to appendix 12, wherein the cell culture vessel placement unit has a fixed position in the phase contrast observation device.
(Supplementary Note 14)
A second moving unit capable of moving the imaging optical system;
The phase difference observation device according to any one of appendices 1 to 13, wherein the control unit controls movement of the second mobile unit.
(Supplementary Note 15)
The phase difference observation apparatus according to any one of appendices 1 to 14, wherein the spatial modulation element is disposed at a position optically conjugate with the pupil of the imaging optical system.
(Supplementary Note 16)
An observation unit capable of observing an object in the cell culture vessel;
A laser irradiation unit capable of irradiating a laser to the object to be observed;
A control unit that controls at least one of the observation unit and the laser irradiation unit;
The cell processing apparatus, wherein the observation unit is the phase contrast observation apparatus according to any one of appendices 1 to 15.
(Supplementary Note 17)
Comprising a first area, a second area and a third area,
The first area and the second area are continuously arranged,
The first region is an object processing chamber for processing an object in the cell culture vessel,
The subject processing chamber includes a cell culture vessel placement unit capable of placing the cell culture vessel,
The first area includes a light source and an illumination optical system in the observation unit,
The second area includes an imaging optical system in the observation unit and the laser irradiation unit.
The third area includes the control unit
17. The cell processing apparatus according to appendix 16, wherein the cell culture vessel placement unit is disposed adjacent to the second region in the subject treatment chamber.

According to the phase difference observation apparatus of the present invention, for example, since the configuration of the additional imaging unit is unnecessary, the apparatus can be miniaturized, and deterioration of the phase difference image due to the meniscus can be suppressed. For this reason, the present invention is extremely useful, for example, in the life science field for observing an object to be observed such as a cell, a tissue, etc., in the medical field, etc.

DESCRIPTION OF SYMBOLS 1 light source 2 illumination optical system 21 light source lens 22 digital micro mirror device 23 condenser lens 3 imaging optical system 31 objective lens 32 phase plate 33 imaging lens 34 imaging element 35 phase plate imaging lens 4 cell culture container arrangement unit (stage)
DESCRIPTION OF SYMBOLS 41 cell culture container 42 to-be-observed body 43 upper cover 44 bottom part 45 light transmission area | region 46 bottom wall 47 bottom plate 48 recessed part 49 protrusion part 5 control unit 51 CPU
52 main memory 53 auxiliary storage device 54 video codec 55 I / O interface 56 controller 57 power supply unit 6 first moving

unit

61, 71, 71a, 71b XY stage 62 arm 7 second moving

unit

711a, 711b truck 81

first Chamber

811a,

811b Opening

812a, 812b Door 813 Suction discharge unit 814a Drain container placement portion 814b Drain container 814c Tip member detachment means 815a Storage container placement portion 815b Storage container 816a Collection container placement portion 816b Collection container 817

Camera

818a, 818b Lighting 819 Germicidal light 82 Second chamber 83 Third chamber 84 Circulating means 84a Intake portion 84b Circulation flow path 84c Gas supply portion 84d Exhaust portion 85 Laser Morphism unit 85a the laser light source 85b fiber 85c

laser emitting unit

86, 87 the housing 100 phase contrast observation apparatus 200 cells processor

Claims

Light source,
An illumination optical system for guiding illumination light from the light source to an object in a cell culture vessel;
An imaging optical system for forming an optical image of the object to be observed on an imaging device;
Including a control unit,
The illumination optical system includes a spatial modulation element that changes the intensity distribution of the illumination light,
The control unit
It includes intensity distribution correction information that associates the position of the imaging optical system with respect to the cell culture vessel and the intensity distribution of illumination light at the position of the imaging optical system,
Acquiring imaging system position information which is the position of the imaging optical system;
A phase difference observation apparatus characterized in that an intensity distribution of illumination light in the spatial modulation element is changed based on the imaging system position information and the intensity distribution correction information.
The imaging optical system includes a phase plate,
The intensity distribution of the illumination light in the intensity distribution correction information is the intensity of the illumination light included in the phase plate image, which is the image of the phase plate, of the illumination image, which is the image of the illumination light, at the position of the imaging optical system. The phase difference observation apparatus according to claim 1, which is a distribution.
The imaging optical system includes a phase plate,
The control unit is configured such that an illumination image, which is an image of the illumination light, is included in a phase plate image, which is an image of the phase plate, at the position of the imaging optical system with respect to the cell culture vessel that does not include the subject. The phase difference observation apparatus according to claim 1 or 2, wherein the intensity distribution of illumination light is obtained, and the obtained intensity distribution of illumination light and the position of the imaging optical system are associated with each other and stored as the intensity distribution correction information.
The phase contrast observation apparatus according to any one of claims 1 to 3, wherein the spatial modulation element includes a digital micromirror device that guides the illumination light by reflecting the illumination light toward the subject.
A first moving unit capable of moving the light source and the illumination optical system;
The phase difference observation apparatus according to any one of claims 1 to 4, wherein the control unit controls movement by the first mobile unit.
The imaging optical system includes a phase plate,
The phase contrast observation apparatus according to claim 5, wherein the first moving unit is movable in a direction perpendicular to the surface of the phase plate.
The control unit
Storing illumination system position correction information in which the position of the imaging optical system is associated with the positions of the light source and the illumination optical system;
The phase difference observation apparatus according to any one of claims 1 to 6, wherein the positions of the light source and the illumination optical system are corrected based on the imaging system position information and the illumination system position correction information.
The control unit
Fractionating the imaging target area of the cell culture vessel into a plurality of sections;
Based on the imaging system position information and the intensity distribution correction information, the intensity distribution of the illumination light in the spatial modulation element is changed, and each section is imaged by the imaging element,
The phase contrast observation apparatus according to any one of claims 1 to 7, wherein an image of an imaging target region of the cell culture vessel is produced based on the obtained image.
The control unit
In the image of the imaging target area, it is determined whether there is a defective imaging site,
If there is the imaging failure site, the correction value of the intensity distribution correction information of the section including the imaging failure site is calculated,
Based on the imaging system position information, the intensity distribution correction information, and the correction value of the intensity distribution correction information, the imaging device re-images a section including the imaging failure portion,
The phase contrast observation apparatus according to claim 8, wherein the image of the section including the imaging failure portion is changed to an image obtained by the reimaging, and an image of the imaging target area is created.
The control unit
In the cell culture vessel not including the object, the intensity distribution of the illumination light included in the phase plate image, which is an image of the phase plate, is an illumination image, which is an image of the illumination light, at the position of the imaging optical system. Asking for
The intensity distribution of the illumination light in the spatial modulation element is changed to the intensity distribution of the illumination light obtained, and the image pickup device picks up an image, and determines whether the luminance value at the pixel of the obtained image is greater than the reference value of the luminance value And
When the luminance value in the image is equal to or more than the reference value of the luminance value, the illumination image, which is an image of the illumination light, is changed at the position of the imaging optical system in a region equal to or more than the reference value of the luminance value of the image. , Find the intensity distribution of the following illumination light,
The phase difference observation apparatus according to any one of claims 1 to 9, wherein the obtained intensity distribution of each illumination light and the position of the imaging optical system are associated with each other and stored as the intensity distribution correction information.
The control unit
It is determined whether the intensity distribution correction information associated with the imaging system position information includes the intensity distribution of a plurality of illumination lights,
When the intensity distribution correction information includes the intensity distribution of a plurality of illumination light, the intensity distribution of the illumination light in the spatial modulation element is imaged by changing the intensity distribution of each illumination light,
The phase difference observation apparatus according to claim 10, wherein in each of the obtained images, a region in which the brightness value at a pixel of the obtained image is less than the reference value of the brightness value is extracted, and the extracted images are integrated.
A cell culture vessel placement unit capable of placing the cell culture vessel,
The phase contrast observation device according to any one of claims 1 to 11, wherein the cell culture vessel placement unit is disposed between the illumination optical system and the imaging optical system in the light path of the illumination light. .
The phase contrast observation apparatus according to claim 12, wherein the cell culture vessel placement unit has a fixed position in the phase contrast observation apparatus.
A second moving unit capable of moving the imaging optical system;
The phase difference observation apparatus according to any one of claims 1 to 13, wherein the control unit controls movement of the second mobile unit.
The phase contrast observation device according to any one of claims 1 to 14, wherein the spatial modulation element is disposed at a position optically conjugate with a pupil of the imaging optical system.
An observation unit capable of observing an object in the cell culture vessel;
A laser irradiation unit capable of irradiating a laser to the object to be observed;
A control unit that controls at least one of the observation unit and the laser irradiation unit;
The cell processing device, wherein the observation unit is the phase contrast observation device according to any one of claims 1 to 15.
Comprising a first area, a second area and a third area,
The first area and the second area are continuously arranged,
The first region is an object processing chamber for processing an object in the cell culture vessel,
The subject processing chamber includes a cell culture vessel placement unit capable of placing the cell culture vessel,
The first area includes a light source and an illumination optical system in the observation unit,
The second area includes an imaging optical system in the observation unit and the laser irradiation unit.
The third area includes the control unit
The cell processing apparatus according to claim 16, wherein the cell culture vessel placement unit is disposed adjacent to the second region in the subject treatment chamber.