WO2019064984A1 - Phase difference observation device and cell treatment device - Google Patents
Phase difference observation device and cell treatment device Download PDFInfo
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- WO2019064984A1 WO2019064984A1 PCT/JP2018/030464 JP2018030464W WO2019064984A1 WO 2019064984 A1 WO2019064984 A1 WO 2019064984A1 JP 2018030464 W JP2018030464 W JP 2018030464W WO 2019064984 A1 WO2019064984 A1 WO 2019064984A1
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Definitions
- the present invention relates to a phase contrast observation device and a cell processing device.
- 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.
- surface tension meniscus
- 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).
- 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.
- 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.
- 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.
- 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.
- FIG. 12 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.
- FIG. 15 is a perspective view showing an example of the configuration of the second area of the cell processing apparatus of Embodiment 2
- (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.
- the “Z-axis direction” refers to the direction perpendicular to the surface direction of the phase plate
- 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.
- 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.
- FIG. 1 is a schematic cross-sectional view showing the configuration of the phase difference observation apparatus 100 of the first embodiment.
- 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.
- 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.
- DMD digital micro mirror device
- 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.
- 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.
- 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.
- the illumination optical system 2 includes the light source lens 21, the DMD 22, and the condenser lens 23.
- 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.
- 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.
- 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, .
- 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.
- 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.
- 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.
- a known lens or lens system can be used as the condenser lens 23.
- 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.
- the imaging optical system 3 includes the objective lens 31 including the phase plate 32, the imaging lens 33, and the imaging device 34.
- 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.
- 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.
- 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.
- 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.
- a known lens or lens system can be used as the imaging lens 33.
- the imaging element 34 is, for example, an element that captures an optical image (phase difference image) of the object 42 to be observed.
- 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.
- CCD charge-coupled device
- CMOS complementary metal oxide semiconductor
- 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.
- 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.
- the position of the stage 4 is fixed.
- 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. .
- 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.
- 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.
- 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.
- the position in the phase contrast observation apparatus may be fixed or movable.
- 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 may further include, for example, temperature control means for adjusting the temperature of the cell culture vessel 41.
- temperature control means for adjusting the temperature of the cell culture vessel 41.
- 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.
- 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.
- pH adjustment means for adjusting the pH of the culture solution in the cell culture vessel 41.
- 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.
- plastics examples 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.
- PMMA polymethyl methacrylate
- polyvinylpyridine polymers poly (4-vinylpyridine), 4-vinylpyridine-styrene copolymer, etc.
- silicone resins Polymers (polydimethylsiloxane etc.), polyolefin polymers (polyethylene, polypropylene, polymethylpentene etc.), polyester polymers (polyethylene
- the cell culture vessel 41 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.
- a laser absorbing layer formed of a polymer containing a group or a photoacid generator which absorbs a laser and generates an acidic substance.
- 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.
- 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.
- control unit 5 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.
- control unit 5 controls each member according to a program. Control of each member by the control unit 5 will be described later.
- 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.
- the present invention is not limited to this.
- 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.
- 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.
- 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.
- 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
- 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.
- 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.
- 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.
- the optical path of the illumination light is omitted.
- 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.
- 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.
- 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.
- the image of the phase plate 32 and the image of the illumination light are imaged on the imaging device 34 as shown in FIG.
- the control unit 5 is electrically connected to the imaging device 34.
- the phase plate imaging lens 35 for example, a known lens or lens system can be used.
- 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.
- 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.
- 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.
- 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.
- the liquid surface of the culture solution forms a meniscus, and a lens effect 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 is not included in the phase plate image 32a, or is deformed or blurred.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- imaging target area 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.
- 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.
- 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.
- the control unit 5 synthesizes the tile image based on the obtained phase difference image (S4).
- 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. .
- the control unit 5 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.
- 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.
- 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.
- the stage 4 is, for example, a dark portion in the phase difference image.
- 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.
- 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.
- 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).
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the imaging optical system 3 is moved to a section for acquiring intensity distribution correction information (S21).
- 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.
- 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.
- the pixel block can be produced, for example, by dividing the image into squares.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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.
- the phase difference observation apparatus 100 can acquire the image of the said division simply, for example by unifying the area
- 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
- 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.
- 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.
- 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.
- the step S3 includes, for example, steps S31 to S37, S321, S351, and S371.
- the imaging optical system 3 is moved to a section for imaging (S31).
- 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.
- step S33 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.
- step S33 the number n of intensity distributions of illumination light applied at the time of imaging is set to 1 (S33).
- 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).
- 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.
- 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.
- 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.
- 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.
- the phase difference observation apparatus 100 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.
- 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
- 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
- FIG. 11 is a cross-sectional view of the first region viewed from II in FIG.
- FIG. 12 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
- 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
- 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
- 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.
- 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.
- 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.
- the third chamber 83 may be 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.
- the medium inside the cell culture vessel 41 contains the emission of the laser emitting unit 85c. Need to put the mouth.
- 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.
- each region 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.
- 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.
- the wavelength of the light may be, for example, a wavelength corresponding to the fluorescence to be detected.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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).
- 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
- 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.
- the gas outside the cell processing apparatus 200 and the dust contained therein can be prevented from flowing into the object processing chamber.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the storage container 815b is, for example, a rack in which the tip is stored.
- the cell processing apparatus 200 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.
- 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.
- each placement portion is along the long axis direction. It may not be arranged, and may not be arranged in this order.
- 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).
- 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.
- 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
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the sterilizing means is disposed on the wall above the opening 811a.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the container 41 can be suitably arranged.
- the circulation means 84 includes an intake part 84a, a circulation channel 84b, a gas supply part 84c, and an exhaust part 84d.
- 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.
- the intake portion 84 a is disposed in the vicinity (for example, immediately below) of the opening 811 a of the object processing chamber.
- 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.
- 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.
- 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.
- 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.
- 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.
- the circulation flow path 84b 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.
- the circulation means 84 includes the circulation flow passage 84b, but the circulation flow passage 84b may or may not be present.
- the intake part 84a is directly connected to, for example, the gas supply part 84c and the exhaust part 84d.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the gas cleaning means for example, dust and the like can be prevented from flowing into the observation object processing chamber.
- 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.
- 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.
- 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.
- the exhaust unit 84d may include, for example, the gas purification unit.
- the gas exhausted from the exhaust unit 84 d to the outside of the cell processing apparatus 200 passes through the gas purification unit.
- 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.
- 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
- 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.
- 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.
- the laser irradiation unit 85 includes a laser light source 85a, an optical fiber 85b, and a laser emission unit 85c.
- 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.
- 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.
- 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.
- the laser moving unit at the bottom of the second chamber 82 is disposed, for example, as an XY stage 71a
- the second moving unit 7 is disposed as an XY stage 71b.
- 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. .
- 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.
- the laser moving means doubles as, for example, a spot diameter adjusting means described later.
- 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.
- 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.
- 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.
- 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.
- 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.
- the magnifications of the plurality of types of objective lenses 31 are preferably different magnifications, such as 2 ⁇ , 4 ⁇ , and 8 ⁇ , for example.
- the imaging optical system 3 having the imaging means in the first chamber 81 and the objective lens 31 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.
- 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.
- 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.
- other means such as the laser moving means, the imaging optical system 3, and the second moving unit 7
- 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.
- 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.
- the laser moving unit preferably moves the laser emitting unit 85c.
- 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.
- 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.
- 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.
- 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).
- the positional relationship between the imaging optical system 3 and the laser irradiation unit 85 is not limited to this.
- the imaging optical system 3 of the observation unit has a large volume compared to the laser irradiation unit 85.
- 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. .
- 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.
- 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.
- 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.
- 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).
- 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 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.
- the diameter of the spot can be adjusted to an appropriate size, for example, the influence on the non-treated cells can be reduced.
- control unit 5 described later preferably controls the adjustment of the diameter of the spot by the spot diameter adjusting means.
- 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.
- a sealing member such as the packing and the sealing material described above.
- the third chamber 83 includes the control unit 5 and the power supply unit 57.
- 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.
- 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.
- 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.
- 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.
- control unit 5 controls the observation unit and the laser irradiation unit 85, but the control unit 5 may control either one.
- 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.
- 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.
- control unit 5 controls imaging in the object processing chamber by the camera 817 which is an imaging unit of the first chamber 81.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the control unit 5 moves the XY stage 71 and the carriage 711a around the processing target area.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- the cell processing apparatus 200 can process the object to be observed 42 such as cells and collect the processed object to be observed 42.
- 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
- processing such as sorting and recovery can be easily performed on the cells of the cell culture vessel 41.
- 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.
- 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.
- Example 1 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.
- the intensity distribution of the next illumination light was set by moving the illumination image.
- 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.
- 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
- the observable region of the dish is greatly expanded, and the effect is remarkable particularly when the diameter of the dish is small.
- 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.
- 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;
- 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 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.
- 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.
- the control unit controls movement by the first mobile unit.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
- 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.
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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
本発明は、位相差観察装置および細胞処理装置に関する。
The present invention relates to a phase contrast observation device and a cell processing device.
細胞培養容器の壁面の近傍を観察する場合、表面張力により培養液の液面が湾曲することにより(メニスカス)、光が屈折し、位相差画像に光が映り込むことがあった。また、前記位相差画像への光の映り込みが生じると、位相差画像の背景が暗く、細胞培養容器内の被観察物が明るく見えるという位相差効果が失われるという問題が生じていた(位相差画像の劣化)。
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).
前記位相差画像の劣化を防止する方法として、位相差画像の劣化に対して位相差観察装置のリングスリット(開口リング)およびコンデンサレンズを移動させることにより対応する装置が提案されている(特許文献1)。
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).
しかしながら、特許文献1の装置では、リングスリットおよびコンデンサレンズの位置を逐次的に調整するために、開口リングの像と、位相板の像とを撮像可能な撮像部を、観察用の撮像部とは別個に設け、得られた開口リングの像および位相板の像に基づき、リングスリットおよびコンデンサレンズの移動量を算出する。そして、得られた移動量に基づき、リングスリットおよびコンデンサレンズの位置を調整する。このため、リングスリットおよびコンデンサレンズの位置調整のために、追加の構成が必要となり、位相差観察装置が大型化するという問題があった。
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.
そこで、本発明は、例えば、追加の撮像部の構成が不要であることにより、装置を小型化でき、かつ前記メニスカスによる位相差画像の劣化を抑制可能な位相差観察装置を提供することを目的とする。
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.
前記目的を達成するために、本発明の位相差観察装置は、光源と、
前記光源からの照明光を細胞培養容器内の被観察体に導光する照明光学系と、
前記被観察体の光学像を撮像素子に結像させる結像光学系と、
制御ユニットとを含み、
前記照明光学系は、前記照明光の強度分布を変化させる空間変調素子を含み、
前記制御ユニットは、
前記細胞培養容器に対する前記結像光学系の位置と、前記結像光学系の位置における照明光の強度分布とを関連付けた強度分布補正情報を含み、
前記結像光学系の位置である結像系位置情報を取得し、
前記結像系位置情報および前記強度分布補正情報に基づき、前記空間変調素子における照明光の強度分布を変化させることを特徴とする。 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.
前記光源からの照明光を細胞培養容器内の被観察体に導光する照明光学系と、
前記被観察体の光学像を撮像素子に結像させる結像光学系と、
制御ユニットとを含み、
前記照明光学系は、前記照明光の強度分布を変化させる空間変調素子を含み、
前記制御ユニットは、
前記細胞培養容器に対する前記結像光学系の位置と、前記結像光学系の位置における照明光の強度分布とを関連付けた強度分布補正情報を含み、
前記結像光学系の位置である結像系位置情報を取得し、
前記結像系位置情報および前記強度分布補正情報に基づき、前記空間変調素子における照明光の強度分布を変化させることを特徴とする。 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.
本発明の細胞処理装置は、細胞培養容器内の被観察物を観察可能な観察ユニットと、
前記被観察物に対して、レーザを照射可能なレーザ照射ユニットと、
前記観察ユニットおよび前記レーザ照射ユニットの少なくとも一方を制御する制御ユニットとを含み、
前記観察ユニットは、前記本発明の位相差観察装置であることを特徴とする。 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.
前記被観察物に対して、レーザを照射可能なレーザ照射ユニットと、
前記観察ユニットおよび前記レーザ照射ユニットの少なくとも一方を制御する制御ユニットとを含み、
前記観察ユニットは、前記本発明の位相差観察装置であることを特徴とする。 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.
本発明の位相差観察装置によれば、例えば、追加の撮像部の構成が不要であることにより、装置を小型化でき、かつ前記メニスカスによる位相差画像の劣化を抑制できる。
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.
以下、本発明において、「Z軸方向」とは、前記位相板の面方向に対する垂直方向をいい、「X軸方向」とは、前記位相板の面(XY平面)方向における1方向をいい、「Y軸方向」とは、前記位相板の面方向において、前記X軸方向と直交する方向をいう。
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.
本発明において、「位相差画像の劣化」は、例えば、位相差画像における位相差効果が低減すること、すなわち、コントラストが低減することを意味する。
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.
以下、本発明の位相差観察装置および細胞処理装置について、図面を参照して詳細に説明する。ただし、本発明は、以下の説明に限定されない。なお、以下の図1~図19において、同一部分には、同一符号を付し、その説明を省略する場合がある。また、図面においては、説明の便宜上、各部の構造は適宜簡略化して示す場合があり、各部の寸法比等は、実際とは異なり、模式的に示す場合がある。また、各実施形態は、特に言及しない限り、互いにその説明を援用できる。
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.
(実施形態1)
本実施形態は、位相差観察装置の一例である。図1は、実施形態1の位相差観察装置100の構成を示す模式断面図である。図1に示すように、位相差観察装置100は、光源1、照明光学系2、結像光学系3、ステージ4、制御ユニット5、第1の移動ユニット6、および第2の移動ユニット7を主要な構成として含む。照明光学系2は、光源レンズ21、デジタルマイクロミラーデバイス(DMD)22、およびコンデンサレンズ23を含み、光源1から照射された照明光の光路に沿って、この順序で配置されている。前記空間変調素子であるDMD22は、一般的な位相差観察装置におけるリングスリットと同等の機能を発揮する構成として配置されている。光源1および照明光学系2の光源レンズ21およびDMD22は、開口を有する筐体86に収容されている。また、照明光学系2のコンデンサレンズ23は、DMD22から導光された照明光(反射光)を細胞培養容器41内の被観察体42に導光可能なように、筐体86の前記開口に配置されている。結像光学系3は、位相板32を備える対物レンズ31、結像レンズ33、および撮像素子34を含み、前記照明光の光路に沿って、この順序で配置されている。結像光学系3の結像レンズ33および撮像素子34は、筐体87に収容されている。対物レンズ31は、被観察体42の光学像を撮像素子34に結像可能なように、筐体87の開口に配置されている。ステージ4には、被観察体42を含む細胞培養容器41が配置されている。ステージ4は、前記照明光の光路において、照明光学系2と結像光学系3との間に配置されている。制御ユニット5は、DMD22、第1の移動ユニット6、および第2の移動ユニット7と電気的に接続されている。第1の移動ユニット6は、光源1および照明光学系2を移動可能なように、筐体86に接するように配置されている。第2の移動ユニット7は、結像光学系3を移動可能なように、筐体87に接するように配置されている。本実施形態の位相差観察装置100は、ステージ4、細胞培養容器41、第1の移動ユニット6、第2の移動ユニット7、筐体86、および筐体87を含むが、いずれも任意の構成であり、あってもよいし、なくてもよい。 (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 phasedifference 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.
本実施形態は、位相差観察装置の一例である。図1は、実施形態1の位相差観察装置100の構成を示す模式断面図である。図1に示すように、位相差観察装置100は、光源1、照明光学系2、結像光学系3、ステージ4、制御ユニット5、第1の移動ユニット6、および第2の移動ユニット7を主要な構成として含む。照明光学系2は、光源レンズ21、デジタルマイクロミラーデバイス(DMD)22、およびコンデンサレンズ23を含み、光源1から照射された照明光の光路に沿って、この順序で配置されている。前記空間変調素子であるDMD22は、一般的な位相差観察装置におけるリングスリットと同等の機能を発揮する構成として配置されている。光源1および照明光学系2の光源レンズ21およびDMD22は、開口を有する筐体86に収容されている。また、照明光学系2のコンデンサレンズ23は、DMD22から導光された照明光(反射光)を細胞培養容器41内の被観察体42に導光可能なように、筐体86の前記開口に配置されている。結像光学系3は、位相板32を備える対物レンズ31、結像レンズ33、および撮像素子34を含み、前記照明光の光路に沿って、この順序で配置されている。結像光学系3の結像レンズ33および撮像素子34は、筐体87に収容されている。対物レンズ31は、被観察体42の光学像を撮像素子34に結像可能なように、筐体87の開口に配置されている。ステージ4には、被観察体42を含む細胞培養容器41が配置されている。ステージ4は、前記照明光の光路において、照明光学系2と結像光学系3との間に配置されている。制御ユニット5は、DMD22、第1の移動ユニット6、および第2の移動ユニット7と電気的に接続されている。第1の移動ユニット6は、光源1および照明光学系2を移動可能なように、筐体86に接するように配置されている。第2の移動ユニット7は、結像光学系3を移動可能なように、筐体87に接するように配置されている。本実施形態の位相差観察装置100は、ステージ4、細胞培養容器41、第1の移動ユニット6、第2の移動ユニット7、筐体86、および筐体87を含むが、いずれも任意の構成であり、あってもよいし、なくてもよい。 (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
光源1は、例えば、細胞培養容器41で培養されている被観察体42に照射される照明光を生成する光源である。光源1は、特に制限されず、例えば、公知の光源が使用でき、具体例として、ハロゲンランプ、タングステンランプ、白色LED(Light Emitting Diode)、単色LED、半導体レーザ等があげられる。
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.
照明光学系2は、例えば、光源1からの照明光を細胞培養容器41内の被観察体42に導光する光学系である。本実施形態の位相差観察装置100において、照明光学系2は、光源レンズ21、DMD22、およびコンデンサレンズ23を含むが、光源レンズ21、およびコンデンサレンズ23は、任意の構成であり、あってもよいし、なくてもよい。照明光学系2は、例えば、視野絞り、開口絞り、リレーレンズ、コリメートレンズ、フライアイレンズ、拡散板等のレンズ、内部全反射プリズム(TIRプリズム)等のプリズム、ミラー等の他の構成を含んでもよい。
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.
光源レンズ21は、例えば、光源1から照射された照明光を集光するレンズである。光源レンズ21は、例えば、公知のレンズまたはレンズ系が使用でき、被観察体42が存在する面を均一に照明できるケーラー照明となるレンズまたはレンズ系が好ましい。
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.
DMD22は、例えば、光源1から照射された照明光の強度分布を変化させる。具体的には、DMD22は、例えば、光源1から照射された照明光を、被観察体42方向に反射することにより導光する。前述のように、DMD22は、一般的な位相差観察装置におけるリングスリットと同等の機能を発揮する。このため、DMD22は、例えば、位相板32との組合せにより、撮像素子34により撮像される位相差画像(撮像データ)において、位相差効果が得られるように、前記照明光の位置、形状、および大きさを整形することにより、前記照明光の強度分布を任意に変更する。前記照明光の位置、形状、および大きさは、例えば、後述のように、制御ユニット5により制御される。具体例として、細胞培養容器41における培養液の液面が水平の場合、前記照明光の形状は、例えば、リング状である。前記照明光の形状は、例えば、DMD22と対物レンズ31との間における光軸方向と垂直方向の平面における照明光の形を意味する。DMD22は、特に制限されず、公知のDMD素子が使用できる。DMD22は、例えば、互いに平行な回動軸線を中心として回動可能な複数の反射面を含む。この場合、例えば、前記複数の反射面の角度を、それぞれ変更することにより、前記照明光の位置、形状、および大きさが、整形される。
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.
位相差観察装置100におけるDMD22の位置は、特に制限されないが、例えば、被観察体42の位相差画像において、より大きなコントラスト(位相差効果)を得られることから、結像光学系3の瞳(瞳位置)と光学的に共役の位置、より具体的には、位相板32と光学的に共役の位置に配置されることが好ましい。
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.
本実施形態の位相差観察装置100は、前記空間変調素子として、DMD22を含むが、前記空間変調素子は、前記照明光の強度分布を変化させることが可能な任意の構成を採用できる。具体例として、前記空間変調素子は、例えば、液晶パネル、エレクトロクロミック素子がアレイ状に配置されたアレイ、有機エレクトロルミネッセンス(EL)素子等の発光素子がアレイ状に配置されたアレイ等があげられる。前記空間変調素子が前記液晶パネルまたは前記エレクトロクロミック素子の場合、光源1、照明光学系2、ステージ4、および結像光学系3は、例えば、この順序で直線上に配置される。前記空間変調素子が前記発光素子である場合、前記空間変調素子は、例えば、光源1を兼ねてもよい。前記空間変調素子は、例えば、DMD22と同様に、位相板32との組合せにより、撮像素子34により取得される画像において、位相差効果が得られるように、前記照明光の位置、形状、および大きさを整形することにより、前記照明光の強度分布を任意に変更する。前記空間変調素子の位置は、特に制限されず、例えば、DMD22の位置と同様である。
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.
コンデンサレンズ23は、例えば、前記照明光を被観察体42に集光するレンズである。コンデンサレンズ23は、例えば、公知のレンズまたはレンズ系が使用できる。
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.
結像光学系3は、例えば、被観察体42の光学像を撮像素子34に結像させる光学系である。本実施形態の位相差観察装置100において、結像光学系3は、位相板32を備える対物レンズ31、結像レンズ33、および撮像素子34を含むが、位相板32を備える対物レンズ31および結像レンズ33は、任意の構成であり、あってもよいし、なくてもよい。結像光学系3は、例えば、ミラー等の他の構成を含んでもよい。
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.
対物レンズ31は、例えば、被観察体42の像を目的の倍率に拡大するレンズである。対物レンズ31は、例えば、公知のレンズまたはレンズ系が使用でき、目的の倍率に応じて適宜選択できる。本実施形態の位相差観察装置100において、対物レンズ31は、1つであるが、対物レンズ31は、2つ以上でもよい。対物レンズ31が2つ以上の場合、各対物レンズ31は、互いに異なる倍率に拡大可能であることが好ましい。対物レンズ31は、位相板32を備えるが、前述のように、位相板32は、任意の構成であり、あってもよいし、なくてもよい。
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.
位相板32は、例えば、入射光の一部の位相を操作する。本実施形態の位相差観察装置100において、対物レンズ31が、位相板32を備えている、すなわち、対物レンズ31と位相板32とが一体として構成されているが、位相板32は、対物レンズ31と、独立して構成されてもよい。位相板32は、例えば、1/4波長板等の波長板と、ニュートラル・デンシティー(Neutral Density)フィルタ等の光吸収性フィルタ等とをリング状に形成することで構成できる。
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.
結像レンズ33は、例えば、被観察体42の光学像を撮像素子34に結像させるレンズである。結像レンズ33は、例えば、公知のレンズまたはレンズ系が使用できる。
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.
撮像素子34は、例えば、被観察体42の光学像(位相差像)を撮像する素子である。撮像素子34は、例えば、公知の撮像素子が使用でき、具体例として、Charge-Coupled Device(CCD、電荷結合素子)、Complementary Metal Oxide Semiconductor(CMOS)等を備える素子があげられる。撮像素子34の撮像面は、例えば、より明確な被観察体42の位相差画像を得られることから、被観察体42と光学的に共役な位置に配置されることが好ましい。撮像素子34は、例えば、制御ユニット5を介して、撮像された被観察体42の位相差画像を表示装置に出力してもよい。この場合、撮像素子34は、制御ユニット5と電気的に接続されている。
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.
ステージ4は、例えば、細胞培養容器41を支持する。ステージ4は、例えば、細胞培養容器41を配置可能な凹部を有し、細胞培養容器41と対応する位置に開口を有する。
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.
ステージ4は、位相差観察装置100における位置が固定されている、すなわち、移動しないが、移動可能であってもよい。ステージ4が移動可能な場合、ステージ4は、例えば、公知の移動手段(駆動手段)により移動する。ステージ4の移動方向は、特に制限されず、例えば、X軸方向、Y軸方向、およびZ軸方向のうちのいずれか1方向、2方向または全方向である。本実施形態の位相差観察装置100において、ステージ4は、その位置が固定されている。このため、本実施形態の位相差観察装置100は、例えば、ステージ4が移動可能な位相差観察装置において、ステージ4の移動時に生じる細胞培養容器41内の培養液の液面のゆれを防止できる。また、本実施形態の位相差観察装置100は、例えば、培養液の液面のゆれによる被観察体42の位相差画像の乱れを抑制できるため、細胞培養容器41の自動的な撮像に好適である。また、本実施形態の位相差観察装置100によれば、前記培養液の液面のゆれが収まるまでの待機時間を減らすことができるため、例えば、より短時間で細胞培養容器41の全面を撮像することができる。
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.
本実施形態の位相差観察装置100は、前記細胞培養容器配置ユニットとして、ステージ4を含むが、前記細胞培養容器配置ユニットは、細胞培養容器41を配置可能であり、細胞培養容器41内の被観察体42を観察可能な任意の構成を採用できる。本実施形態において、ステージ4は、開口を有するが、例えば、前記開口の結像光学系3側に光透過性材料が配置されてもよい。前記光透過性材料は、例えば、透明なガラス板、アクリル板等があげられる。前記細胞培養容器配置ユニットは、例えば、前記位相差観察装置における位置が固定されていてもよいし、移動可能であってもよい。前記細胞培養容器配置ユニットの移動は、例えば、前述のステージ4の移動の説明を援用できる。
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.
前記細胞培養容器配置ユニット(ステージ4)は、例えば、さらに細胞培養容器41の温度を調整する温度調整手段を含んでもよい。前記温度調整手段を含むことにより、細胞培養容器41内の細胞を処理する間の培養条件を一定にでき、例えば、被観察体42の撮像時および後述の細胞処理時の被観察体42へのダメージを低減できる。前記温度調整手段は、例えば、ヒータ等の加温手段があげられる。
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.
前記細胞培養容器配置ユニット(ステージ4)は、例えば、さらに細胞培養容器41内の培養液のpHを調整するpH調整手段を含んでもよい。前記pH調整手段を含むことにより、前記細胞培養容器内の細胞を処理する間の培養条件を一定にでき、例えば、細胞処理時の細胞へのダメージを低減できる。前記pH調整手段としては、例えば、二酸化炭素濃度調整手段等があげられ、具体例として、位相差観察装置100外の二酸化炭素供給手段と接続する接続部等があげられる。
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.
細胞培養容器41は、特に制限されず、例えば、細胞培養に用いられる公知のディッシュ、フラスコ等の培養容器があげられる。細胞培養容器41の形成材料は、特に制限されず、例えば、後述のレーザ照射ユニットにより照射されるレーザを透過する材料があげられ、具体例として、レーザを透過するプラスチック、ガラス等があげられる。プラスチックは、例えば、ポリスチレン系ポリマー、アクリル系ポリマー(ポリメタクリル酸メチル(PMMA)等)、ポリビニルピリジン系ポリマー(ポリ(4-ビニルピリジン)、4-ビニルピリジン-スチレン共重合体等)、シリコーン系ポリマー(ポリジメチルシロキサン等)、ポリオレフィン系ポリマー(ポリエチレン、ポリプロピレン、ポリメチルペンテン等)、ポリエステルポリマー(ポリエチレンテレフタレート(PET)、ポリエチレンナフタレート(PEN)等)、ポリカーボネート系ポリマー、エポキシ系ポリマー等があげられる。
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
細胞培養容器41内の被観察体42をレーザ処理する場合、細胞培養容器41は、例えば、細胞培養容器41の内側(被観察体42側)の底面に、前記レーザを吸収する色素構造(発色団)を含むポリマー、またはレーザを吸収し酸性物質を発生する光酸発生剤により形成されるレーザ吸収層を含むことが好ましい。前記色素構造および光酸発生剤は、例えば、特許6033980号公報の説明を援用できる。細胞培養容器41は、前記レーザ吸収層を含むことで、例えば、後述の細胞処理装置のレーザ照射ユニットでレーザを照射した際に、前記レーザのエネルギーを熱、酸等に変換し、前記レーザ吸収層の上部に存在する細胞を死滅、遊離等させることができる。
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.
被観察体42は、特に制限されず、細胞、細胞から構成される細胞塊、組織、臓器等でもよい。前記細胞は、例えば、培養細胞でもよいし、生体から単離した細胞でもよい。また、前記細胞塊、組織または臓器は、例えば、前記細胞から作製した細胞塊、組織または臓器でもよいし、生体から単離した細胞塊、組織または臓器でもよい。
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.
制御ユニット5は、パーソナルコンピュータ、サーバコンピュータ、ワークステーション等と類似する構成を含む。図2は、本実施形態の位相差観察装置100における制御ユニット5の一例を示すブロック図である。図2に示すように、制御ユニット5は、中央演算装置(CPU)51、メインメモリ52、補助記憶デバイス53、ビデオコーデック54、I/O(input-output)インターフェイス55等を含み、これらがコントローラ(システムコントローラ、I/Oコントローラ等)56により制御され、連携動作する。補助記憶デバイス53は、フラッシュメモリ、ハードディスクドライブ等の記憶手段があげられる。ビデオコーデック54は、CPU51より受けた描画指示をもとに表示する画面を生成し、その画面信号を、例えば、位相差観察装置100外の表示装置等に向けて送信するGPU(Graphics Processing Unit)、画面および画像のデータを一時的に記憶しておくビデオメモリ等を含む。I/Oインターフェイス55は、DMD22、第1の移動ユニット6、および第2の移動ユニット7等の各部材と通信可能に接続してこれらを制御するためのデバイスである。I/Oインターフェイス55は、サーボドライバ(サーボコントローラ)を含んでもよい。また、I/Oインターフェイス55は、例えば、位相差観察装置100外の入力手段と接続してもよい。表示装置は、映像により出力するモニター(例えば、液晶ディスプレイ(LCD)、ブラウン管(CRT)ディスプレイ等の各種画像表示装置等)等があげられる。入力装置は、使用者が手指で操作可能なタッチパネル、トラックパッド、マウス等のポインティングデバイス、キーボード、押下ボタン等があげられる。
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.
制御ユニット5が実行するプログラムおよび各情報は、補助記憶デバイス53に記憶されている。前記プログラムは、実行時にメインメモリ52に読み込まれ、CPU51によって解読される。そして、制御ユニット5は、プログラムに従い、各部材を制御する。制御ユニット5による各部材の制御については、後述する。
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.
本実施形態の位相差観察装置100は、制御ユニット5に、DMD22、第1の移動ユニット6、および第2の移動ユニット7の制御機能を持たせることで、各部材に制御ユニットを個別に設けなくてもよいため、装置の小型化を実現できる。ただし、本発明はこれに限定されない。本発明の位相差観察装置は、例えば、制御ユニット5として、DMD22、第1の移動ユニット6、および第2の移動ユニット7のそれぞれに制御ユニットを設け、各部材の制御ユニットにより、各部材を制御してもよい。また、本発明の位相差観察装置は、例えば、制御ユニット5と各部材の制御ユニットとを設け、共同して各部材を制御してもよい。また、制御ユニット5は、一つの半導体素子で構成しても良いし、複数の半導体素子をワンパッケージしたチップとしても良いし、複数の半導体素子を基板上に設けた構成でもよい。
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.
第1の移動ユニット6は、例えば、光源1および照明光学系2を移動可能な移動ユニットである。第1の移動ユニット6は、例えば、公知の移動手段(駆動手段)があげられる。第1の移動ユニット6の移動方向は、特に制限されず、例えば、X軸方向、Y軸方向、およびZ軸方向のうちのいずれか1方向、2方向または全方向である。第1の移動ユニット6は、例えば、DMD22と結像光学系3の瞳(瞳位置)とを光学的に共役な位置となるように、光源1および照明光学系2を移動でき、被観察体42の位相差画像において、より大きなコントラスト(位相差効果)を得られることから、Z軸方向に移動可能であることが好ましい。本実施形態の位相差観察装置100において、第1の移動ユニット6は、光源1および照明光学系2を移動可能であるが、第1の移動ユニット6は、例えば、光源1および照明光学系2のいずれか一方のみを移動可能に構成してもよい。第1の移動ユニット6は、例えば、光源1および照明光学系2を移動可能に配置されていればよく、使用する移動手段に応じて適宜配置できる。第1の移動ユニット6による移動は、例えば、後述するように、制御ユニット5により制御される。
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.
第2の移動ユニット7は、例えば、結像光学系3を移動可能な移動ユニットである。第2の移動ユニット7は、公知の移動手段(駆動手段)があげられる。第2の移動ユニット7の移動方向は、特に制限されず、例えば、X軸方向、Y軸方向、およびZ軸方向のうちのいずれか1方向、2方向または全方向である。第2の移動ユニット7は、例えば、撮像素子34の撮像面と被観察体42とを光学的に共役な位置となるように、結像光学系3を移動でき、より明確な被観察体42の位相差画像を得られることから、Z軸方向に移動可能であることが好ましい。第2の移動ユニット7は、例えば、結像光学系3を移動可能に配置されていればよく、使用する移動手段に応じて適宜配置できる。第2の移動ユニット7による移動は、例えば、後述するように、制御ユニット5により制御される。
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.
つぎに、本実施形態の位相差観察装置100における制御ユニット5の動作および位相差観察装置100を用いた位相差画像の撮像方法について説明する。
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.
本実施形態の制御ユニット5は、細胞培養容器41に対する結像光学系3の位置と、結像光学系3の位置における照明光の強度分布とを関連付けた強度分布補正情報を含む。このため、まず、前記強度分布補正情報を取得し、制御ユニット5の補助記憶デバイス53に記憶させる。前記強度分布補正情報は、例えば、予め他の位相差観察装置等で取得した情報でもよいし、本実施形態の位相差観察装置100の使用者が入力した情報でもよいし、本実施形態の位相差観察装置100で取得した情報でもよいが、メニスカスによる位相差画像の劣化をより効果的に抑制できることから、本実施形態の位相差観察装置100で取得した情報が好ましい。本実施形態の位相差観察装置100を用いて前記強度分布補正情報を取得する場合、前記強度分布補正情報は、具体的には、以下のように取得する。
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.
図3は、前記強度分布補正情報を取得する場合の位相差観察装置100の構成を示す模式図である。なお、図3では、前記照明光の光路は、省略している。図3に示すように、位相差観察装置100のステージ4には、被観察体42を含まない細胞培養容器41(補正用の細胞培養容器41)が配置されている。この際、補正用の細胞培養容器41内の培養液の液量および種類は、被観察体42を含む細胞培養容器41内の液量および種類と同じとする。また、位相差観察装置100の結像光学系3の結像レンズ33と、撮像素子34との間には、位相板撮像レンズ35が挿入されている。これにより、位相板32の像および照明光の像(DMD22の反射光の像)が、図3に示すように、撮像素子34に結像される。図示していないが、制御ユニット5は、撮像素子34と電気的に接続されている。位相板撮像レンズ35は、例えば、公知のレンズまたはレンズ系が使用できる。
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.
つぎに、図3に示す位相差観察装置100を用いて、補正用の細胞培養容器41を複数の区分に分画して、各区分を撮影(タイリング撮影)することにより、補正用の位相板32の像および照明光の像を、撮像素子34により取得する。また、各撮像の際に、制御ユニット5は、結像光学系3の位置を、結像系位置情報として取得する。前記結像系位置情報(結像光学系3の位置)は、例えば、XYZ軸における座標(三次元座標)またはXY軸における座標(二次元座標)である。
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.
図4は、撮像素子34により撮像される補正用の位相板32の像および照明光の像の模式図である。補正用の細胞培養容器41内の培養液の水平面のZ軸方向に、照明光学系2および結像光学系3を配置する。この際に、DMD22と位相板32とを光学的に共役させ、撮像素子34により撮像すると、撮像された位相差画像(補正用画像)では、図4(A)に示すように、照明光の像22aが、位相板32の像(位相板像)32aに含まれる。しかしながら、前述のように、細胞培養容器41の壁面の近傍を観察する場合、培養液の液面がメニスカスを形成し、レンズ効果が生じる。このため、メニスカスを通過する照明光が、屈折し、DMD22と位相板32との光学的な共役関係が崩れる。そして、この際に、図4(B)に示すように、前記補正用画像において、照明光の像22aが、位相板像32aに含まれない、変形する、および/またはぼけが生じる等の問題が生じ、位相差効果が低減する。そこで、制御ユニット5は、図4(B)に示す問題が生じている際に、照明光の像22aと位相板像32aとの関係が、図4(A)の状態となるように補正するための情報を、強度分布補正情報として取得する。具体的には、制御ユニット5は、補正用の細胞培養容器41について、前記タイリング撮影時に、各撮像位置、すなわち各結像光学系3の位置において、照明光の像22aが、位相板像32aに含まれる照明光の強度分布を求める。すなわち、制御ユニット5は、照明光の像22aが位相板像32aに含まれる状態に照明光の強度分布が補正された際の被観察体42方向に照明光を反射するDMD22のミラーの位置情報と、被観察体42方向に照明光を反射しないDMD22のミラーの位置情報とを求める。
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.
制御ユニット5は、前記タイリング撮影時に、前記補正用画像において照明光の像22aが、位相板像32aに含まれるか否かを検出する。そして、照明光の像22aが位相板像32aに含まれない場合、すなわち、照明光の像22aと位相板32の像32aとにずれがある場合、制御ユニット5は、照明光の像22aが位相板像32aに含まれる、DMD22の反射光の位置および大きさを求める。具体的には、制御ユニット5は、前記補正用画像における明るいリング(照明光の像22a)および暗いリング(位相板像32a)を検出する。つぎに、制御ユニット5は、前記明るいリングおよび前記暗いリングが同心となり、かつ照明光の像22aが、位相板像32aに含まれるDMD22の反射光の位置および大きさを求める。具体的には、DMD22の反射光の位置をX軸方向およびY軸方向の少なくとも一方に所定範囲内で移動させ、また、あわせてDMD22の反射光の大きさを所定範囲内で変更することで、前記明るいリングおよび前記暗いリングが同心となるDMD22の反射光の位置および大きさを求める。
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.
つぎに、制御ユニット5は、前記補正用画像において、照明光の像22aが変形しているか否かを検出する。そして、照明光の像22aが変形している場合、制御ユニット5は、照明光の像22aがリングの外周および内周が略真円となる、DMD22の反射光の形状を求める。具体的には、制御ユニット5は、前記補正用画像における明るいリングの形状を検出する。つぎに、DMD22の反射光の形状を所定範囲内で変形させ、前記明るいリングの外周および内周が略真円となるDMD22の反射光の形状を求める。
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.
そして、制御ユニット5は、前記補正用画像の撮像位置の情報、すなわち、前記結像系位置情報を、前記照明光の強度分布であるDMD22の反射光の位置、形状および大きさの情報と関連づけて、前記強度分布補正情報として、補助記憶デバイス53に記憶する。補助記憶デバイス53に記憶する各情報は、例えば、後述の位相差観察装置100を用いた撮像時に、細胞培養容器41の種類および大きさ、培養液の種類および液量等の撮像対象の条件により、前記強度分布補正情報を指定できることから、細胞培養容器41の種類および大きさの情報、ならびに前記培養液の種類および液量等の撮像対象の条件と関連づけて記憶することが好ましい。
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.
さらに、制御ユニット5は、照明光の像22aのぼけが存在するか否かを検出してもよい。そして、照明光の像22aのぼけが存在する場合、制御ユニット5は、照明光の像22aのぼけが解消する光源1および照明光学系2の位置を求める。具体的には、制御ユニット5は、第1の移動ユニット6をZ軸方向に所定範囲内で移動させることで、光源1および照明光学系2を移動させ、照明光の像22aのぼけが解消する光源1および照明光学系2の位置を求める。また、制御ユニット5は、ぼけが解消する光源1および照明光学系2の位置である照明系位置情報を取得する。前記照明系位置情報(光源1および照明光学系2の位置)は、例えば、XYZ軸における座標(三次元座標)またはZ軸における座標である。そして、制御ユニット5は、前記結像系位置情報と、ぼけが解消する光源1および照明光学系2の位置である照明系位置情報とを関連づけて、照明系位置補正情報として、補助記憶デバイス53に記憶させる。なお、前記照明系位置補正情報は、例えば、前記強度分布補正情報と同様に、前記撮像対象の条件と関連付けて記憶することが好ましい。
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.
本実施形態の位相差観察装置100において、制御ユニット5は、前記照明光の強度分布の全体を補正することにより、前記強度分布補正情報を取得しているが、他の方法により、前記強度分布補正情報を取得してもよい。具体的には、制御ユニット5は、例えば、1つの結像光学系3の位置において、DMD22のミラーを1枚動作させ、動作されたミラーにより照明光を被観察体42方向に反射させる。この際に、制御ユニット5は、例えば、撮像素子34により前記補正用画像を撮像する。つぎに、制御ユニット5は、例えば、前記補正用画像において、前記ミラーからの反射光が結像した位置を特定し、動作させたミラーの位置情報と、その反射光の補正用画像における結像位置とを関連付ける。さらに、制御ユニット5は、例えば、これをDMD22の全てのミラーについて実施し、各ミラーの位置情報と、その反射光の補正用画像における結像位置とを関連付ける。そして、制御ユニット5は、各ミラーの反射光の補正用画像における結像位置のうち、位相板像32aに含まれる結像位置を抽出する。制御ユニット5が、例えば、抽出された結像位置に対応するミラーの位置情報に基づき、DMD22の対応する位置のミラーを動作させ、動作されたミラーにより被観察体42方向に照明光を反射させると、照明光の像22aが位相板像32aに含まれる。このため、制御ユニット5は、例えば、前記結像系位置情報と、前記抽出された結像位置に対応するミラーの位置情報とを関連付けて、強度分布補正情報として、補助記憶デバイス53に記憶する。制御ユニット5は、例えば、他の結像光学系3の位置において、同様の処理を実施し、各結像光学系3の位置における強度分布補正情報を取得する。
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.
なお、制御ユニット5が、前記強度分布補正情報を算出する場合を例にあげて説明したが、前記強度分布補正情報は、例えば、前記補正用画像に基づき、位相差観察装置100の使用者が、前述の入力手段等を用いて入力してもよい。
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.
このようにして、本実施形態の位相差観察装置100を用いて、前記強度分布補正情報を取得することができる。
Thus, the intensity distribution correction information can be acquired using the phase difference observation apparatus 100 of the present embodiment.
図5は、図1に示す位相差観察装置100を用いた位相差画像のタイル画像の撮像方法を示すフローチャートである。図5に示すように、本実施形態の位相差観察装置100を用いた位相差画像の撮像方法は、例えば、S1~S4工程を含む。前記撮像方法は、さらに、S5~S10工程を含んでもよい。なお、以下の説明では、細胞培養容器41の全面を撮像する場合をあげるが、位相差観察装置100により撮像される領域(撮像対象領域)はこれに限定されない。位相差観察装置100は、例えば、使用者等により設定された撮像対象領域を撮像する。前記撮像対象領域は、例えば、細胞培養容器41の一部または全部である。前記画像は、例えば、細胞培養容器41内の被観察体42を含む画像である。
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.
まず、位相差観察装置100の使用者が、撮像対象の細胞培養容器41のサイズ、細胞培養容器41内の培養液の種類、液量等の撮像対象の条件を選択する(S1)。そして、選択された撮像対象の条件を満たす、前記強度分布補正情報を補助記憶デバイス53から読み出す(S2)。また、前記照明系位置補正情報が補助記憶デバイス53に記憶されている場合、前記照明系位置補正情報を補助記憶デバイス53から読み出してもよい。
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.
つぎに、位相差観察装置100に配置された被観察体42を含む細胞培養容器41について、タイリング撮影を実施する(S3)。制御ユニット5は、各撮像時に、結像光学系3の位置である結像系位置情報を取得し、前記結像系位置情報および前記強度分布補正情報に基づき、結像光学系3の位置に対応するDMD22の反射光の位置、形状、および大きさとなるように、照明光の強度分布を補正する。そして、位相差観察装置100における撮像素子34により、位相差画像を撮像する。また、前記照明系位置補正情報を読み出している場合、制御ユニット5は、さらに、前記結像系位置情報および前記照明系位置補正情報に基づき、結像光学系3の位置に対応する第1の移動ユニット6の位置に、第1の移動ユニット6を移動させることにより、光源1および照明光学系2の位置を補正してもよい。さらに、位相差観察装置100における撮像素子34により、位相差画像を撮像してもよい。
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.
そして、制御ユニット5は、得られた位相差画像に基づき、タイル画像を合成する(S4)。このようにして、図1の位相差観察装置100を用いて、タイル画像を取得することができる。なお、タイル画像の取得を例にあげて説明したが、細胞培養容器41のうち、位相差観察装置100を用いて、被観察体42が存在する領域の位相差画像のみを取得してもよい。
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. .
図1の位相差観察装置100を用いてタイル画像を取得する場合、設定した強度分布補正情報における培養液の種類および液量、細胞培養容器41の種類および大きさ等と、撮像対象の細胞培養容器41における培養液の種類および液量、細胞培養容器41の種類および大きさ等の差異により生じる位相差効果の低減に対応できるため、制御ユニット5は、さらに、タイル画像の合成に用いた撮像画像における撮像不良部位を検出してもよい。この場合、制御ユニット5は、前記撮像不良部位と対応する細胞培養容器41の箇所(区分)について、撮像条件の補正値(強度分布補正情報の補正値)を算出し、撮像素子34による再撮像を実施する。
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.
具体的には、まず、制御ユニット5は、撮像不良部位があるか否かを検出する(S5)。Noの場合、制御ユニット5は、処理を終了する。他方、Yesの場合、S6に進む。前記撮像不良部位は、例えば、輝度が高くなることにより、コントラストが低下した領域を意味する。具体的には、制御ユニット5は、得られた各位相差画像について、1つの位相差画像内の輝度値の合計値を算出する。そして、制御ユニット5は、得られた輝度値の合計値が、閾値以上の場合、撮像不良部位であると判断する。前記閾値は、任意の値とすることができ、具体的には、細胞培養容器41の培養液が水平である領域で撮像した位相差画像の輝度値の合計値から所定倍以上(例えば、1.15倍以上)とすることができる。前記輝度値の合計値による撮像不良部位の検出は、例えば、得られた位相差画像の全部または一部に対して実施する。具体例として、前記位相差画像において、ステージ4は、例えば、前記位相差画像において暗部となる。このため、輝度値の合計値により撮像不良部位を検出する場合、例えば、ステージ4を含む位相差画像に対応するタイル画像の領域は、輝度値の合計値による撮像不良部位の検出対象から除外する等の処理を行なってもよい。前記除外処理された位相差画像に対応するタイル画像の領域は、例えば、他の撮像不良部位の検出方法により、撮像不良部位にあたるか否かを検出してもよい。
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.
つぎに、制御ユニット5は、前記撮像不良部位に対応する位相差画像の再撮像時の補正値を算出する。前記補正値は、例えば、前記照明光の強度分布、すなわち、DMD22の反射光の位置、大きさ、および形状を補正する補正値でもよいし、光源1および照明光学系2の位置を補正する補正値でもよい。前記補正値は、例えば、再撮像した位相差画像における輝度値の合計値が、前記閾値未満となるように設定される。
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.
制御ユニット5が、DMD22の反射光の位置、大きさ、および形状に関する補正値を算出する場合、前記補正値は、例えば、以下のように実施する。まず、前記撮影不良部位のうち、補正値を算出する1箇所(1つの位相差画像)を抽出する(S6)。前記強度分布補正情報におけるDMD22の反射光の位置、大きさ、および形状の求め方と同様にして、前記補正値を算出する(S7)。
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).
そして、制御ユニット5は、算出した補正値に基づき、再撮像を実施する(S8)。具体的には、制御ユニット5は、前記補正値に基づき、照明光の強度分布、DMD22の反射光の位置、大きさ、および形状を補正し、再撮像する。
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.
つぎに、制御ユニット5は、全ての撮像不良部位について再撮像が完了したかを確認する(S9)。Noの場合、S6に戻る。他方、Yesの場合、S10に進む。そして、制御ユニット5は、S10の再撮像された位相差画像(再撮像画像)を、元の位相差画像、すなわち、最初に撮像した位相差画像と置換後、得られた位相差画像に基づき、タイル画像を再合成する(S10)。そして、制御ユニット5は、処理を終了する。
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.
なお、DMD22の反射光の位置、大きさ、および形状に関する補正値を算出する場合を例にあげて説明したが、前述のように、光源1および照明光学系2の位置を補正する補正値であってもよい。この場合、前記補正値の算出は、例えば、以下のように実施できる。まず、前記撮影不良部位のうち、補正値を算出する1箇所(1つの位相差画像)を抽出する。つぎに、光源1および照明光学系2(照明系)のX軸方向の補正値、Y軸方向の補正値およびZ軸方向の補正値を算出する。具体的には、制御ユニット5は、第1の移動ユニット6により光源1および照明光学系2の位置を所定範囲内でX軸方向、Y軸方向および/またはZ軸方向に移動させる。この際に、制御ユニット5は、前記移動と並行して、撮像素子34により撮像される位相差画像における輝度値の合計値を算出する。そして、制御ユニット5は、各位相差画像における輝度値の合計値が、前記閾値以下となる位置、好ましくは、最小値となる位置を、補正値として算出する。前記位置は、例えば、XYZ軸における座標(三次元座標)である。
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.
位相差観察装置100を用いた撮影方法では、S3工程において、1つの区分に対して、複数の照明光の強度分布を用いて、撮像し、得られた画像のうち、位相差像として適した部分の画像を用いて、1つの区分の画像を作製してもよい。すなわち、1つの区分に対する強度分布補正情報は、複数の照明光の強度分布を含んでもよい。図6は、図3の位相差観察装置100を用いて、1または複数の照明光の強度分布を含む強度分布補正情報の取得方法の他の例を示すフローチャートである。図6に示すように、前記取得方法は、S21~S27工程およびS271工程を含む。前記強度分布補正情報の取得は、例えば、前記S1工程に先立ち実施される。
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.
まず、結像光学系3を、強度分布補正情報を取得する区分へ移動する(S21)。つぎに、前記区分において、制御ユニット5は、照明光の像22aが、位相板像32aに含まれる照明光の強度分布(1回目の照明光の強度分布)を求める(S22)。すなわち、制御ユニット5は、照明光の像22aが位相板像32aに含まれる状態に照明光の強度分布が補正された際の被観察体42方向に照明光を反射するDMD22のミラーの位置情報と、被観察体42方向に照明光を反射しないDMD22のミラーの位置情報とを求める。より具体的には、前述のように、制御ユニット5は、照明光の像22aが位相板像32aに含まれる、DMD22の反射光の位置および大きさを求める。
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.
つぎに、制御ユニット5は、DMD22における照明光の強度分布を、S22工程で得られた照明光の強度分布に変化させ、撮像素子34により、前記区分を撮像する(S23)。得られた画像の画素における輝度値が、輝度値の基準値以上かを判定する(S24)。前記画素は、前記画像における1画素でもよいし、複数画素でもよいが、輝度値の変化を検出しやすく、メニスカスによる影響を抑制できることから、後者が好ましい。後者の場合、前記画素は、画素ブロックということもできる。前記画素ブロックは、例えば、前記画像における任意の数の隣接する画素を1つのユニットとし、前記画像を複数のユニットに分割することにより作製できる。具体例として、前記画素ブロックは、例えば、前記画像をマス目状に分割することにより作製できる。前記画素が画素ブロックの場合、前記画素の輝度値は、例えば、前記画素ブロックの各画素の輝度値の合計値として算出できる。また、前記輝度値の基準値は、任意の値とでき、例えば、得られた画像において、輝度値が上限値に達している画素が含まれるように設定する。具体例として、前記輝度値の基準値(Bs)は、例えば、前記画像の画素において最低の輝度値(BL)を基準として、1.1×BL≦Bs≦1.2×BL、好ましくは、Bs=1.15×BLである。そして、Noの場合、すなわち、前記画像における輝度値が輝度値の基準値未満の場合、S26工程に進む。他方、Yesの場合、すなわち、前記画像における輝度値が輝度値の基準値以上の場合、前記区分において、制御ユニット5は、前記画像の輝度値の基準値以上の領域について、照明光の像22aを変化させ、つぎの照明光の強度分布(2回目の照明光の強度分布)を求める(S25)。照明光の像22aの変化は、例えば、照明光の像22aが、位相板像32aに含まれるように変化させてもよいし、その一部または全部が、位相板像32aに含まれないように変化させてもよい。照明光の像22aの変化は、例えば、照明光の像22aの位置を移動させることにより実施することが好ましい。この場合、照明光の像22aの大きさおよび形状は、変化させないことが好ましい。位相差観察装置100は、例えば、照明光の像22aの位置を移動させることにより、照明光の直接的な入射による輝度値の増加を抑制できる。このため、位相差観察装置100は、例えば、前記画像における輝度値の基準値以上の領域において、適切なコントラストの被観察体42の位相差像を取得可能な照明光の強度分布を求めることができる。
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.
そして、1回目および2回目の照明光の強度分布を、結像光学系3の位置と関連付けて、前記強度分布補正情報として、記憶する(S26)。S26工程では、例えば、各回の照明光の強度分布と、前記画像の輝度値の基準値以上の領域とを関連付けて、前記強度分布補正情報として、記憶してもよい。これにより、位相差観察装置100は、例えば、撮像素子34による撮像後、各画像の輝度値の基準値未満の領域を統合することにより、前記区分の画像を簡易に取得できる。また、本実施形態の撮像方法における取得方法の説明では、S25工程後、S26工程に進むが、再度S23に戻り、1回目の照明光の強度分布に代えて、2回目の照明光の強度分布を用いて、S23工程からの工程を同様に実施してもよい。この場合、S24工程およびS25工程では、2回目の照明光の強度分布を適用して撮像することにより得られた画像のうち、1回目の照明光の強度分布を適用して撮像した際に、前記輝度値の基準値以上の領域(補正前領域)を対象にして実施する。そして、同様の工程を、各回のS23工程において得られた画像の補正前領域における画素における輝度値が、前記輝度値の基準値未満となるまで繰り返し実施してもよい。すなわち、本実施形態の撮像方法は、各回で撮像した画像において、前記輝度値の基準値未満の領域を統合した際に、撮像対象の区分全体の画像が得られるまで実施してもよい。
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.
S27工程では、全ての区分について、前記強度分布補正情報の取得が完了したかを判定する。Noの場合、すなわち、全ての区分について、前記強度分布補正情報の取得が完了していない場合、結像光学系3をつぎの区分へ移動し(S271)、再度S22工程を実施する。他方、Yesの場合、すなわち、全ての区分について、前記強度分布補正情報の取得が完了している場合、前記強度分布補正情報の取得方法を終了する。
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.
つぎに、前記取得方法により得られた前記強度分布補正情報および位相差観察装置100を用いた撮像方法について説明する。図7は、図1に示す位相差観察装置100を用いた位相差画像のタイル画像の撮像方法におけるS3工程の他の例を示すフローチャートである。図7に示すように、S3工程は、例えば、S31~S37工程、S321工程、S351工程、およびS371工程を含む。
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.
まず、結像光学系3を撮像する区分へ移動する(S31)。つぎに、前記区分と対応する前記強度分布補正情報、すなわち、結像光学系3の位置と関連付けられた強度分布補正情報を取得し、前記強度分布補正情報が、複数の照明光の強度分布を含むかを判定する(S32)。Noの場合、すなわち、前記強度分布補正情報が、1つの照明光の強度分布を含む場合、DMD22における照明光の強度分布を、S32工程で得られた照明光の強度分布に変化させ、撮像素子34により、前記区分を撮像し(S321)、S37工程に進む。他方、Yesの場合、すなわち、前記強度分布補正情報が、複数の照明光の強度分布を含む場合、S33工程に進む。以下、複数の照明光の強度分布が、N回分ある場合を例にあげて説明する。前記Nは、2以上の整数を意味する。S33工程では、撮像時に適用する照明光の強度分布の回数nを1にセットする(S33)。つぎに、DMD22における照明光の強度分布を、1回目の照明光の強度分布に変化させ、撮像素子34により、前記区分を撮像する(S34)。
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).
前記撮像後、N回目の照明光の強度分布のうち、適用していない照明光の強度分布があるかを判定する。すなわち、N=nかを判定する。そして、Noの場合、すなわち、N≠nの場合、nをn+1にセットし(この場合、n=2、S351)、S34工程に戻る。そして、2回目の照明光の強度分布を適用してS34工程からの工程を同様に実施する。そして、前記撮像方法では、N=nとなるまでS34およびS35工程を繰り返し実施する。他方、Yesの場合、すなわち、N=nの場合、得られたN枚の画像の画素における輝度値が、前記輝度値の基準値未満の領域(補正対象領域)を抽出し、抽出された画像を統合する。これにより、前記区分を撮像した、1枚の画像を取得する(S36)。
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).
そして、S37工程では、全ての区分について撮像が完了したかを判定する。Noの場合、次の区分へ移動し(S371)、S32工程からの工程を同様にして実施する。他方、Yesの場合、S3工程を終了する。
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.
S3工程において、1または複数の照明光の強度分布を含む強度分布補正情報を適用し、撮像することにより、1つの区分において、1つの照明光の強度分布を含む強度分布補正情報に基づく補正のみで難しい場合においても、位相差画像の劣化が抑制された画像を取得することができる。
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.
本実施形態の位相差観察装置100によれば、前記特許文献1のような、メニスカスによる位相差画像の劣化を抑制するための追加の撮像部の構成が不要である。このため、本実施形態の位相差観察装置100によれば、装置を小型化できる。また、本実施形態の位相差観察装置100は、例えば、被観察体42を含まない細胞培養容器41を用いて取得した強度分布補正情報に基づき、撮像時の照明光の強度分布を補正するため、例えば、撮像時に補正値を算出し、補正を実施する特許文献1の装置と比較して、より短い時間で撮像が可能である。また、本実施形態の位相差観察装置100は、強度分布補正情報に基づき、撮像時の照明光の強度分布を補正するため、メニスカスによる位相差画像の劣化を抑制することができる。このため、本実施形態の位相差観察装置100によれば、コントラストが大きな位相差画像を撮像できる。これらの効果は、後述の細胞処理装置においても同様である。
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.
(実施形態2)
本実施形態は、細胞処理装置の一例である。図8~図17に、本実施形態の細胞処理装置の構成の一例を示す。図8は、本実施形態の細胞処理装置の構成の一例を示す斜視図であり、図9は、本実施形態の細胞処理装置における第1領域、第2領域、および第3領域の構成を示す模式断面図であり、図10は、本実施形態の細胞処理装置の第1領域の構成の一例を示す斜視図であり、図11は、図8におけるI-I方向からみた第1領域の断面図であり、図12において、(a)は、本実施形態の細胞処理装置における培養容器配置部の一例を示す分解斜視図であり、(b)は、図12(a)におけるIII-III方向からみた断面図であり、図13は、前記第1領域の外壁を外した場合における第1領域および循環手段の斜視図であり、図14は、図8におけるII-II方向からみた前記第1領域の上部および前記循環手段の断面図であり、図15において、(a)は、本実施形態の細胞処理装置の第2領域の構成の一例を示す斜視図であり、(b)は、前記第2領域の構成の他の例を示す斜視図であり、図16は、本実施形態の細胞処理装置における制御ユニットの一例を示すブロック図であり、図17は、本実施形態の細胞処理装置の構成の他の例を示す斜視図である。 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.
本実施形態は、細胞処理装置の一例である。図8~図17に、本実施形態の細胞処理装置の構成の一例を示す。図8は、本実施形態の細胞処理装置の構成の一例を示す斜視図であり、図9は、本実施形態の細胞処理装置における第1領域、第2領域、および第3領域の構成を示す模式断面図であり、図10は、本実施形態の細胞処理装置の第1領域の構成の一例を示す斜視図であり、図11は、図8におけるI-I方向からみた第1領域の断面図であり、図12において、(a)は、本実施形態の細胞処理装置における培養容器配置部の一例を示す分解斜視図であり、(b)は、図12(a)におけるIII-III方向からみた断面図であり、図13は、前記第1領域の外壁を外した場合における第1領域および循環手段の斜視図であり、図14は、図8におけるII-II方向からみた前記第1領域の上部および前記循環手段の断面図であり、図15において、(a)は、本実施形態の細胞処理装置の第2領域の構成の一例を示す斜視図であり、(b)は、前記第2領域の構成の他の例を示す斜視図であり、図16は、本実施形態の細胞処理装置における制御ユニットの一例を示すブロック図であり、図17は、本実施形態の細胞処理装置の構成の他の例を示す斜視図である。 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.
図8に示すように、本実施形態の細胞処理装置200は、第1領域である第1室81と、第2領域である第2室82と、第3領域である第3室83と、循環手段84とを含み、第1室81と、第2室82と、第3室83とが、この順番で上から下方向に連続して配置されている。本実施形態の細胞処理装置200は、循環手段84を含むが、循環手段84は、任意の構成であり、あってもよいし、なくてもよい。また、第1室81、第2室82および第3室83の位置関係は、第1室81と第2室82とが連続(隣接)して配置されていればよく、第3室83は、任意の位置に配置できる。第3室83は、例えば、図17に示すように、第1室81と第2室82とは別個に配置してもよい。図17に示すように、第3室83が、第1室81および第2室82と別個に配置されている場合、細胞処理装置200は、例えば、細胞処理システムということもできる。前記細胞処理システムは、例えば、卓上型のシステムとしてもよい。第1室81は、第2室82の上部に配置されていることが好ましい。細胞培養容器41の上部から後述するレーザ照射ユニット85によりレーザを照射する場合、レーザ照射ユニット85の焦点位置を安定化させるために、細胞培養容器41内の培地内に、レーザ出射部85cの出射口を配置する必要がある。しかしながら、この状態でレーザ照射を行なうと、前記培地の成分が、レーザ出射部85cの出射口に固着する、焼き付く等の問題が生じ、レーザ出射部85cの出射口に汚れが生じる。このため、本実施形態の細胞処理装置200のように配置することで、例えば、後述するレーザ照射ユニット85で、細胞培養容器41内の細胞を照射する際に、レーザ照射ユニット85のレーザ出射口の汚れを抑制できる。したがって、本発明の細胞処理装置200によれば、例えば、レーザ照射ユニット85から出射されるレーザの出力を安定化することができ、効率よく細胞を処理できる。各領域(各室)の形成材料は、特に制限されず、例えば、ステンレス板、防錆処理された鉄板、真空成型、射出成型、圧空成型等による成型が可能な樹脂板等があげられる。各領域の形成材料は、後述する観察ユニットにより、細胞培養容器41内の細胞をより明確に撮像できることから、非透光性の材料であることが好ましい。前記「非透光性」は、例えば、前記観察ユニットによる撮像に影響を与える波長の光の透過を抑制することを意味する。前記観察ユニットが蛍光観察可能な場合、前記光の波長は、例えば、検出する蛍光に対応する波長があげられる。具体例として、前記非透光性の材料は、例えば、前述の各領域の形成材料等があげられる。各領域の大きさおよび形状は、特に制限されず、各領域内に配置する部材(手段)の大きさおよび形状に応じて適宜設定できる。本実施形態の細胞処理装置200において、第1室81と第2室82とは、別個の筐体で構成し、第1室81を構成する筐体および第2室82を構成する筐体を隣接して配置しているが、これに限定されず、第1室81と第2室82とを1つの筐体で構成し、1つの筐体内で、第1室81と第2室82とを区分けすることで構成してもよい。本実施形態の細胞処理装置200は、第1室81および第2室82を別個の筐体で構成することにより、例えば、細胞処理装置200内の各部材のメンテナンスを容易に実施でき、また、細胞処理装置200の組み立てが容易となる。
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.
つぎに、図9に示すように、本実施形態の細胞処理装置200は、光源1、照明光学系2、結像光学系3、細胞培養容器配置ユニット4、および制御ユニット5、ならびに後述の第1の移動ユニット6および第2の移動ユニット7を主要な構成として含む観察ユニットと、レーザ照射ユニット85とを主要な構成として含む。前記観察ユニットの細胞培養容器配置ユニット4以外の構成は、前記実施形態1の位相差観察装置100と同様であり、その説明を援用できる。なお、図9において、後述する第1室81の二重壁の外壁は、省略している。レーザ照射ユニット85は、レーザ光源85a、光ファイバ85b、およびレーザ出射部85cを含み、レーザ光源85aおよびレーザ出射部85cは、光ファイバ85bにより光学的に接続されている。第1室81には、筐体86に収容された光源1および照明光学系2と、第1の移動ユニット6とが配置されている。光源1および照明光学系2を収容する筐体86は、第1の移動ユニット6により移動可能である。第2室82には、筐体87に収容された結像光学系3と、第2の移動ユニット7と、レーザ照射ユニット85とが配置されている。結像光学系3と、レーザ照射ユニット85におけるレーザ出射部85cとは、第2の移動ユニット7により移動可能である。第3室83には、制御ユニット5および電源ユニット57が配置されている。細胞培養容器配置ユニット4は、第1室81と第2室82の間の隔壁の一部として形成されている。本実施形態の細胞処理装置200は、細胞培養容器配置ユニット4、細胞培養容器41、第1の移動ユニット6、第2の移動ユニット7、筐体86、筐体87、第1室81、第2室82、および第3室83を含むが、いずれも任意の構成であり、あってもよいし、なくてもよい。
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.
第1室81は、その前面(図8において手前側)に作業用の開口部811aを含み、またその側面にメンテナンスが可能な開口部811bを含む。開口部811aは、第1室81である被観察体処理室内で被観察体処理に関連する作業を行なうための開口部である。開口部811bは、前記被観察体処理室のメンテナンスが可能な開口部である。開口部811aの開口面積は、例えば、メンテナンス作業が容易になることから、開口部811bの開口面積より小さいことが好ましい。開口部811aならびに開口部811bの大きさおよび数は、特に制限されず、例えば、安全キャビネットにおける作業用の開口部およびメンテナンスが可能な開口部の大きさおよび数を参照できる。具体例として、開口部811aならびに開口部811bの大きさおよび数は、例えば、EN規格であるEN12469:2000で特定される安全キャビネットの規格を参照できる。開口部811bの数は、特に制限されず、任意の数とできるが、例えば、メンテナンスがより容易となることから、2以上が好ましい。第1室81における開口部811aおよび開口部811bの配置箇所は、特に制限されず、任意の場所とできるが、開口部811aと開口部811bとは、第1室81の異なる場所(例えば、異なる側面)に配置することが好ましい。本実施形態において、開口部811bは、細胞処理装置200内のメンテナンスを容易に行うことを主目的としたが、他の目的でも使用してもよい。本実施形態の細胞処理装置200は、例えば、開口部811bから内部の各部材の動き等を観察可能とすることで、細胞処理装置200にトラブルが発生した場合に不具合箇所を直接観察でき、対応策を検討することができる。
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.
第1室81の前面の壁は、外壁および内壁を有する二重壁となっており、扉812aは、前記外壁と前記内壁との間の空間に配置されたレールを昇降することにより、開口部811aの開口を開閉する。開口部811bは、その開口を、前記開口を覆う扉812bの着脱により開閉可能である。開口部811bは、例えば、前記被観察体処理室内で細胞の処理を行なう際に、その開口が扉812bに封止されていることが好ましい。これにより、例えば、細胞処理装置200外の気体およびそれに含まれる埃の前記被観察体処理室内への流入を防止できる。本実施形態の細胞処理装置200において、開口部811aおよびその扉812a、ならびに開口部811bおよびその扉812bは、任意の構成であり、あってもよいし、なくてもよいし、いずれかの開口部およびその扉のみを含んでもよい。また、第1室81の壁は、二重壁でもよいし、一重壁でもよいが、他の部材を内部に配置することで、細胞処理装置200の大きさを小さくできることから前者が好ましい。また、第1室81の壁が一重壁の場合、扉812aは、例えば、扉812bのように第1室81の外部に配置される。前記扉の開閉の形式は、特に制限されず、例えば、扉812aのように昇降式でもよいし、扉812bのように外付け式でもよいし、その他の形式でもよい。前記その他の形式は、例えば、観音開き式、アコーディオン式、引き扉式等があげられる。前記扉の形成材料は、特に制限されず、例えば、前述の各領域の形成材料を援用でき、非透光性の材料が好ましい。
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.
図10に示すように、本実施形態の細胞処理装置200の第1室81の内部は、被観察体を処理する被観察体処理室であり、扉812a、812bを閉めることにより閉鎖可能である、すなわち開閉可能である。前記被観察体処理室は、XYステージ61およびアーム62を含む第1移動ユニット6、吸引吐出ユニット813と、照明光学系2を収容する筐体86と、排液容器配置部814aと、収容容器配置部815aと、細胞培養容器配置ユニット4と、回収容器配置部816aとを含む。本実施形態において、前記被観察体処理室は、XYステージ61およびアーム62を含む第1移動ユニット6、吸引吐出ユニット813、排液容器配置部814a、収容容器配置部815a、ならびに回収容器配置部816aを含むが、いずれも任意の構成であり、あってもよいし、なくてもよく、また、いずれか1つを含んでもよいし、2つ以上を含んでもよい。XYステージ61は、前記被観察体処理室の底面に配置されており、X軸方向およびY軸方向に移動可能なように配置されている。XYステージ61の上部には、一対のアームを含むアーム62が配置されている。アーム62の一方のアーム先端部分には、吸引吐出ユニット813が、その吸引吐出口を下方向に向けて配置されている。また、アーム62の他方のアーム先端部分には、照明光学系2を含む筐体86が、照明光を被観察体42方向に導光(照射)可能なように配置されている。排液容器配置部814a、収容容器配置部815a、細胞培養容器配置ユニット4、および回収容器配置部816aは、前記被観察体処理室の底面において、XYステージ61のX軸方向の移動方向に沿って、この順番で配置されている。排液容器配置部814aには、先端部材脱離手段814cを有する排液容器814bが配置され、収容容器配置部815aには、収容容器815bが配置され、回収容器配置部816aには、回収容器816bが配置されている。
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.
本実施形態の細胞処理装置200は、第1の移動ユニット6であるXYステージ61およびアーム62により、照明光学系2および吸引吐出ユニット813を移動可能であるが、吸引吐出ユニット813は、第1の移動ユニット6以外の駆動手段により移動可能であってもよい。この場合、吸引吐出ユニット813を移動可能な駆動手段の移動方向は、特に制限されず、例えば、X軸方向、Y軸方向、およびZ軸方向のうちのいずれか1方向、2方向または全方向である。本実施形態において、XYステージ61は、例えば、リニアモータ台車等を介して、対象物をX軸方向およびY軸方向に沿って高速かつ精密に移動可能な公知のものである。アーム62は、上下方向(Z軸方向)に伸縮可能であるが、アーム62は、固定されていてもよい。後者の場合、第1の移動ユニット6は、XY平面上のみにおいて、すなわち、図10において、X軸方向およびY軸方向のみに、吸引吐出ユニット813を移動可能である。
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.
吸引吐出ユニット813は、例えば、細胞培養容器41内の培地、細胞等を吸引および吐出する。吸引吐出ユニット813は、例えば、その吸引吐出口側に、後述する先端部材を装着して使用する。吸引吐出ユニット813は、特に制限されず、例えば、公知の吸引吐出手段が利用でき、具体例として、電動ピペッタ、電動シリンジポンプ等があげられる。
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.
排液容器配置部814aは、吸引吐出ユニット813により吸引した吸引液を排液する排液容器814bを配置可能な領域である。本実施形態において、排液容器配置部814aには、排液容器814bが配置されているが、排液容器814bは、任意の構成であり、あってもよいし、なくてもよい。本実施形態において、排液容器814bは、上部開口の箱であり、収容容器配置部815a側の壁が上方向に伸びており、その上端に、半円状の凹部(切り欠き)として形成されている先端部材脱離手段814cを含む、前記被観察体処理室の底面に対して略平行方向な壁(上面)を有する。排液容器814bは、吸引吐出ユニット813から脱離した先端部材を回収可能であることから、例えば、先端部材回収容器ということもでき、また、排液容器配置部814aは、先端部材回収容器配置部ということもできる。先端部材脱離手段814cは、排液容器814bに形成されているが、別個に配置されてもよい。また、先端部材脱離手段814cは、吸引吐出ユニット813の近傍、具体的には、吸引吐出ユニット813が配置されている第1移動ユニット6のアーム62に配置されてもよい。
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.
収容容器配置部815aは、吸引吐出ユニット813に着脱可能な先端部材が収容された収容容器815bを配置可能な領域である。本実施形態において、収容容器配置部815aには、収容容器815bが配置されているが、収容容器815bは、任意の構成であり、あってもよいし、なくてもよい。前記先端部材は、特に制限されず、吸引吐出ユニット813により吸引された液体を内部に貯留可能な部材であればよく、例えば、吸引吐出ユニット813がピペッタの場合、チップがあげられる。収容容器815bは、例えば、前記チップが収容されたラックがあげられる。本実施形態の細胞処理装置200は、先端部材脱離手段814cおよび収容容器配置部815aを含むことで、細胞培養容器41内の培地、細胞等を吸引および吐出する際の移動を簡素化(短く)できる。
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.
回収容器配置部816aは、吸引吐出ユニット813により回収した細胞を含む吸引液を回収する回収容器816bを配置可能な領域である。本実施形態において、回収容器配置部816aには、回収容器816bが配置されているが、回収容器816bは、任意の構成であり、あってもよいし、なくてもよい。回収容器816bは、例えば、公知のディッシュ、フラスコ等の培養容器等があげられる。
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.
本実施形態において、前記被観察体処理室の底面には、細胞培養容器配置ユニット4の配置面、すなわち、XY平面において、XYステージ61の長軸方向(X軸方向)の移動方向に沿って、排液容器配置部814a、収容容器配置部815a、細胞培養容器配置ユニット4、および回収容器配置部816aが、この順番で配置されているが、各配置部は、前記長軸方向に沿って配置されていなくてもよく、また、この順序で配置されていなくてもよい。本実施形態において、排液容器配置部814a、収容容器配置部815a、細胞培養容器配置ユニット4、および回収容器配置部816aが、前述の順序で配置されていることにより、例えば、吸引吐出ユニット813の移動を直線的にでき、細胞培養容器41内の培地、細胞等を吸引および吐出する際の移動を簡素化(短く)できる。
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).
また、図11に示すように、本実施形態の細胞処理装置200の前記被観察体処理室の前面側の壁には、開口部811aの上部に、カメラ817、照明灯818a、818bおよび殺菌灯819を含む。カメラ817のX軸方向の両側には、照明灯818a、818bが配置されており、また、上部には、殺菌灯819が配置されている。
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.
本実施形態において、第1室81の撮像手段として、カメラ817を設けているが、第1室81の撮像手段は、任意の構成であり、あってもよいし、なくてもよい。また、第1室81の撮像手段は、カメラに限定されず、第1室81内、すなわち、前記被観察体処理室内を撮像可能であればよい。第1室81の撮像手段は、特に制限されず、顕微鏡、カメラ等の公知の撮像手段が使用でき、また公知の撮像手段と、CCDやCMOS(Complementary MOS)等の固体撮像素子(イメージセンサ)とを組合せたものでもよい。本実施形態において、カメラ817は、前記被観察体処理室内の前面の壁に配置されているが、カメラ817の位置は、特に制限されず、任意の位置とでき、前記被観察体処理室内の広い範囲を撮像可能なように配置することが好ましい。具体的には、本実施形態の細胞処理装置200のように、前記被観察体処理室において、細胞培養容器配置ユニット4の奥側(図10において左上側)に、第1の移動ユニット6であるXYステージ61およびアーム62と吸引吐出ユニット813とが配置されている場合、前記被観察体処理室内の広い範囲を撮像可能であることから、前記被観察体処理室の手前側(図10において右下側)に配置することが好ましい。前記第1の撮像手段は、複数の倍率(例えば、異なる倍率)で撮像可能なことが好ましいが、1つの倍率で撮像可能であってもよい。前記倍率は、例えば、撮像倍率を意味する。具体例として、カメラ817は、例えば、複数の倍率(例えば、異なる倍率)のレンズを含む。第1室81の撮像手段は、例えば、光学ズーム、デジタルズーム等が可能であってもよい。本実施形態の細胞処理装置200はカメラ817を含むことで、例えば、前記被観察体処理室内の作業を確認可能であり、作業の確実性が向上する。前記被観察体処理室内に配置される第1室81の撮像手段の数は、特に制限されず、1つでもよいし、複数でもよい。
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.
本実施形態において、照明手段として、照明灯818a、818bを設けているが、前記照明手段は、任意の構成であり、あってもよいし、なくてもよい。また、前記照明手段は、照明灯に限定されず、前記被観察体処理室内に投光(照明)可能であればよい。前記照明手段は、特に制限されず、例えば、蛍光灯、LED灯等の公知の照明が使用できる。本実施形態において、照明灯818a、818bは、前記被観察体処理室内の前面の壁に配置されているが、照明灯818a、818bの位置は、特に制限されず、任意の位置とでき、前記被観察体処理室内の広い範囲に投光可能、すなわち、前記被観察体処理室内に影ができにくいように配置することが好ましい。具体的には、本実施形態の細胞処理装置200のように、前記被観察体処理室において、細胞培養容器配置ユニット4の奥側(図10において左上側)に、第1の移動ユニット6であるXYステージ61およびアーム62と吸引吐出ユニット813とが配置されている場合、前記被観察体処理室内の広い範囲に投光可能であることから、前記被観察体処理室の手前側(図10において右下側)に配置することが好ましい。本実施形態の細胞処理装置200は照明灯818a、818bを含むことで、例えば、前記被観察体処理室内の作業を確認可能であり、作業の確実性が向上する。前記被観察体処理室内に配置される照明手段の数は、特に制限されず、1つでもよいし、複数でもよい。
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.
本実施形態において、殺菌手段として、殺菌灯819を設けているが、前記殺菌手段は、任意の構成であり、あってもよいし、なくてもよい。また、前記殺菌手段は、殺菌灯に限定されず、前記被観察体処理室内、特に、細胞培養容器配置ユニット4周囲を殺菌可能であればよい。前記殺菌手段は、特に制限されず、例えば、殺菌灯、紫外LED灯等の公知の殺菌手段が使用できる。本実施形態において、殺菌灯819は、前記被観察体処理室内の前面の壁に配置されているが、殺菌灯819の位置は、特に制限されず、任意の位置とできる。殺菌灯819の位置は、例えば、細胞処理装置200外の埃等は、開口部811a、811bから流入することから、開口部811a、811b近傍を殺菌可能に配置されていることが好ましい。具体的には、本実施形態の細胞処理装置200のように、前記被観察体処理室の前面側の壁に、開口部811aが設けられている場合、前記被観察体処理室の前面側の壁において、開口部811aの上部に前記殺菌手段を配置することが好ましい。本実施形態の細胞処理装置200のように、前記被観察体処理室の側面側の壁に、開口部811bが設けられている場合、前記被観察体処理室の側面側の壁において、開口部811bの上部に前記殺菌手段を配置することが好ましい。また、細胞処理装置200が前記照明手段および前記殺菌手段を含む場合、両者を前記被観察体処理室の同じ壁、例えば、開口部811aが設けられている壁に配置することが好ましい。この場合、前記殺菌手段を、前記照明手段の上方に設けることが好ましい。本実施形態の細胞処理装置200は殺菌灯819を含むことで、例えば、前記被観察体処理室内の清浄性が向上する。前記被観察体処理室内に配置される殺菌手段の数は、特に制限されず、1つでもよいし、複数でもよい。
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.
本実施形態において、第1室81である前記被観察体処理室の大きさ、形状、構造等は、例えば、前記安全キャビネットの大きさ、形状、構造等を参照でき、具体例として、前述のEN12469:2000で特定される安全キャビネットの規格を参照できる。
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.
図12に示すように、本実施形態の細胞処理装置200の細胞培養容器配置ユニット4は、上蓋43および底部44を含み、上蓋43は、底部44に着脱可能に装着される。本実施形態において、細胞培養容器配置ユニット4は、上蓋43および底部44を含む箱であり、その内部に細胞培養容器41が配置されているが、細胞培養容器配置ユニット4は、これに限定されず、細胞培養容器41を配置可能であり、前記被観察体処理室において第2室82と隣接するように配置され、かつ細胞培養容器配置ユニット4における第2室82との隣接部(図12において、底板47)が透光可能であればよい。前記「透光」は、例えば、第2室82のレーザ照射ユニット85から照射されるレーザが透過することを意味する。また、前記観察ユニットの撮像素子34が、底板47を介して撮像可能であることを意味する。上蓋43は、細胞培養容器41に対して、光源1から照明光を照射可能なように、透光領域45が設けられている。透光領域45は、例えば、透明なガラス板、アクリル板等から形成される。底部44は、底壁46および透光性の底板47を含む。透光性の底板47は、例えば、透明なガラス板、アクリル板等から形成される。底板47は、第2室82と隣接している。このため、細胞培養容器配置ユニット4の第2室82との隣接部、すなわち、底板47は、前記被観察体処理室の壁の一部として形成されているということもできる。底板47と前記被観察体処理室の壁との接触部は、例えば、パッキン、シール材等の封止部材で封止されていることが好ましい。これにより、例えば、第2室82内の気体およびそれに含まれる埃等が細胞培養容器配置ユニット4および前記被観察体処理室に流入することを防止できる。底壁46は、4つの細胞培養容器41をそれぞれ配置可能な、4つの凹部48を含み、各凹部48の側面は、前記被観察体処理室の内部から前記被観察体処理室の外部方向(図12(b)において、上から下方向)に向かって狭まる逆テーパ状である。また、各凹部48は、その底板47端側において、凹部48の内側方向に突出する突出部49を含む。細胞培養容器41は、その底部端が、突出部49と接触する。本実施形態の細胞処理装置200において、底壁46は、4つの凹部48を有するが、底壁46が有する凹部48の数は、これに限定されず、配置する細胞培養容器41の数に応じて適宜設定できる。凹部48の大きさは、配置する細胞培養容器41の大きさに応じて適宜設定できる。本実施形態の細胞培養容器配置ユニット4は、凹部48が上述の構造を有することで、例えば、細胞培養容器41の側面の形状によらず、細胞培養容器配置ユニット4に細胞培養容器41を配置可能となる。本実施形態の細胞処理装置200において、底壁46は、その底面の壁と、その側面の壁とが一体形成されているが、底壁46は、これに限定されず、それぞれを別部材としてもよい。底壁46を別部材で構成することにより、例えば、異なる数および大きさの凹部48を有する、複数の底壁46の底面の壁の部材を準備しておくことができる。これにより、例えば、細胞培養容器41の大きさおよび数に応じて、細胞培養容器41の配置に適した大きさおよび数を有する底壁46の底面の壁の部材に取替えることができ、細胞培養容器41を好適に配置することができる。
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.
図13および14に示すように、本実施形態の細胞処理装置200において、循環手段84は、吸気部84aと、循環流路84bと、気体供給部84cと、排気部84dとを含む。これにより、循環手段84は、前記被観察体処理室内の気体を循環させる。
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.
吸気部84aは、前記被観察体処理室内の気体を吸気する。吸気部84aは、前記被観察体処理室内の気体に代えて、または加えて細胞処理装置200外の気体を吸気してもよい。本実施形態において、吸気部84aは、前記被観察体処理室の開口部811aの近傍(例えば、直下)に配置されている。具体的には、吸気部84aは、その上面に複数の開口(例えば、スリット)が形成されており(図示せず)、前記開口が開口部811aと連通するように、開口部811aの下側に配置されている。このように、前記被観察体処理室の開口部811aの近傍に吸気部84aを配置することで、例えば、扉812aを開き、作業者が前記被観察体処理室内で作業をする際に、細胞処理装置200外の気体およびそれに含まれる埃等が前記被観察体処理室内に流入することを防止できる。吸気部84aは、開口部811aに代えて、または加えて開口部811bの近傍に配置されてもよい。吸気部84aは、例えば、ファン等の送風手段により前記被観察体処理室内の気体を吸気してもよい。
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.
循環流路84bは、吸気部84aと気体供給部84cおよび排気部84dとを接続する。本実施形態において、循環流路84bは、前記外壁と前記内壁との間の空間および第1室81の上部に配置されている。循環流路84bは、例えば、中空の筒である。また、循環流路84bは、その一端が吸気部84aと連通し、その他端が、気体供給部84cおよび排気部84dと連通している。本実施形態の細胞処理装置200のように、循環流路84bを、前記外壁と前記内壁との間の空間に配置することで、例えば、細胞処理装置200の大きさを小さくできる。また、本実施形態において、循環手段84は、循環流路84bを含むが、循環流路84bはあってもよいし、なくてもよい。後者の場合、吸気部84aは、例えば、気体供給部84cおよび排気部84dと直接的に接続している。循環流路84bは、例えば、ファン等の送風手段により、吸気部84aにより吸気された気体を、気体供給部84cおよび排気部84dに送風してもよい。
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.
循環流路84bが前記送風手段を含む場合、前記送風手段は、吸気部84a、気体供給部84c、または排気部84dの近傍に配置してもよいし、これらの中央部等のその他の位置に配置してもよいが、吸気部84aからの吸気がよくなり、例えば、後述する気体供給部84cにより生じるダウンフローと比較して、埃等が前記被観察体処理室内に流入することをより効果的に防止できることから、吸気部84aの近傍に配置することが好ましい。前記送風手段が吸気部84aの近傍に配置される場合、前記送風手段は、例えば、第2室82または第3室83に配置されることが好ましい。具体例として、本実施形態の細胞処理装置200において、循環流路84bが、さらに前記送風手段を含む場合、前記送風手段は、第2室82または第3室83内において、手前側(図8における左下側)、すなわち、吸気部84aの下側に配置されている。そして、この場合、循環流路84bは、吸気部84aと前記送風手段の吸気側とを接続し、かつ前記送風手段の送風側と気体供給部84cおよび排気部84dとを接続する。すなわち、循環流路84bは、第2室82、または第2室82および第3室83と、前記外壁と前記内壁との間の空間と、第1室81の上部とに配置されている。
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.
気体供給部84cは、吸気部84aが吸気した気体の一部を前記被観察体処理室内に供給する。本実施形態において、気体供給部84cは、第1室81の上端と、吸気部84aより吸気した気体を前記被観察体処理室内に供給可能なように連通されている。気体供給部84cは、例えば、ファン等の送風手段により気体を前記被観察体処理室内に供給してもよい。また、気体供給部84cは、例えば、気体清浄化手段を含んでもよい。この場合、気体供給部84cから前記被観察体処理室内に供給される気体は、前記気体清浄化手段を通過する。前記気体清浄化手段を含むことにより、例えば、埃等が前記被観察体処理室内に流入することを防止できる。前記気体清浄化手段は、例えば、HEPAフィルタ(High Efficiency Particulate Air Filter)、ULPAフィルタ(Ultra Low Penetration Air Filter)等の微粒子捕集用フィルタ等があげられる。本実施形態の細胞処理装置200は、前記被観察体処理室の上部において気体供給部84cと接続していることにより、例えば、気体供給部84cからの送風によりダウンフローが生じ、これにより開口部811aから埃等が前記被観察体処理室内に流入することをより効果的に防止できる。
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.
排気部84dは、吸気部84aが吸気した気体の残部を前記被観察体処理室外、具体的には、細胞処理装置200外に排気する。本実施形態において、排気部84dは、吸気部84aより吸気した気体を細胞処理装置200の外部に排気可能なように、細胞処理装置200の上端(最上部)に配置されている。このように排気部84dを細胞処理装置200の最上部に設けることにより、例えば、細胞処理装置200の大きさを小さくでき、かつ排気によって舞い上がった埃が、前記被観察体処理室内に流入することを防止できる。排気部84dは、例えば、ファン等の送風手段により気体を細胞処理装置200の外部に排気してもよい。また、排気部84dは、例えば、前記気体清浄化手段を含んでもよい。この場合、排気部84dから細胞処理装置200外に排出される気体は、前記気体清浄化手段を通過する。前記気体清浄化手段を含むことにより、例えば、前記被観察体処理室内で生じた微粒子等の細胞処理装置200外への流出を防止できる。
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.
循環手段84において、各部の大きさ、形状、構造等は、例えば、前記安全キャビネットの大きさ、形状、構造等を参照でき、具体例として、前述のEN12469:2000で特定される安全キャビネットの規格を参照できる。
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
図15(a)に示すように、本実施形態の細胞処理装置200において、第2室82は、第2の移動ユニット7、結像光学系3が収容された筐体87およびレーザ照射ユニット85を含む。本実施形態の細胞処理装置200は、第2の移動ユニット7を含むが、前述のように、第2の移動ユニット7は、任意の構成であり、あってもよいし、なくてもよく、またいずれか一方を含んでもよい。第2の移動ユニット7は、XYステージ71および台車711a、711bを含む。XYステージ71は、細胞培養容器配置ユニット4の配置面、すなわち、XY平面と略平行な第2室82の底面に配置されている。XYステージ71において、Y軸方向の共通のレール(移動路)上には、2つのX軸方向のレールが、前記共通のレール上を移動可能に配置されている。前記2つのX軸方向のレール上には、それぞれ、台車711a、711bがレールを移動可能なように配置されている。レーザ照射ユニット85は、レーザ光源85a、光ファイバ85b、およびレーザ出射部85cを含む。XYステージ71の上部には、結像光学系3が収容された筐体87が、結像光学系3の対物レンズ31を上方向(Z軸方向)に向けて台車711bに、また、レーザ照射ユニット85のレーザ出射部85cのレーザ出射口を上方向(Z軸方向)に向けて、台車711aに配置されている。台車711aは、上下方向(Z軸方向)に昇降可能である。レーザ光源85aは、第2室82において、XYステージ71の可動範囲と重ならない領域において、第2室82の底面に配置されている。光ファイバ85bは、その一端がレーザ光源85aと、その他端がレーザ出射部85cと、光学的に接続している。
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.
本実施形態の細胞処理装置200は、第2の移動ユニット7であるXYステージ71により結像光学系3およびレーザ照射ユニット85を移動可能であるが、レーザ照射ユニット85は、第2の移動ユニット7以外の駆動手段により移動可能であってもよい。この場合、レーザ照射ユニット85を移動可能な駆動手段の移動方向は、特に制限されず、例えば、X軸方向、Y軸方向、およびZ軸方向のうちのいずれか1方向、2方向または全方向である。また、本実施形態において、レーザ照射ユニット85を移動可能な駆動手段(レーザ移動手段)および第2の移動ユニット7は、Y軸方向(第1方向)のレールを共有しているが、前記レーザ移動手段および第2の移動ユニット7は、独立していてもよい。具体例として、図15(b)に示すように、第2室82の底面の前記レーザ移動手段は、例えば、XYステージ71aとして配置され、第2の移動ユニット7は、XYステージ71bとして配置されてもよい。前記レーザ移動手段および第2の移動ユニット7の移動方向は、特に制限されず、例えば、X軸方向、Y軸方向、およびZ軸方向のうちのいずれか1方向、2方向または全方向である。前記レーザ移動手段が、例えば、細胞培養容器配置ユニット4の配置面、すなわち、細胞培養容器41の底面に対し、略直交方向に、レーザ照射ユニット85を移動可能な場合、前記レーザ移動手段は、後述するスポット径を調整可能である。この場合、前記レーザ移動手段は、例えば、後述するスポット径調整手段を兼ねる。本実施形態において、XYステージ71は、例えば、リニアモータ台車等を介して、対象物をX軸方向およびY軸方向に沿って高速かつ精密に移動可能な公知のものである。
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.
前記レーザ移動手段および第2の移動ユニット7は、本実施形態のXYステージ71のように、細胞培養容器配置ユニット4の配置面に対し略平行な平面において、それぞれ、第1方向(例えば、図15(a)における矢印Y方向)に、レーザ照射ユニット85および前記第2の撮像手段を移動可能であり、かつ前記レーザ移動手段によるレーザ照射ユニット85の第1方向の移動と、第2の移動ユニット7による結像光学系3の第1方向の移動とが、同一直線上であることが好ましい。このように、同一直線上で、レーザ照射ユニット85および結像光学系3が移動することで、例えば、細胞培養容器41内の細胞を結像光学系3で撮像後、レーザ照射ユニット85で処理する等の細胞処理を行なう際に各手段の移動回数を低減でき、処理時間を低減できる。また、本実施形態のXYステージ71のように、前記レーザ移動手段は、レーザ照射ユニット85を配置する台車711a、および台車711aが移動し、かつ前記第1方向に沿って配置された移動路(レール)を含み、第2の移動ユニット7は、結像光学系3を配置する台車711bおよび、台車711bが移動し、かつ前記第1方向に沿って配置された移動路(レール)を含み、前記レーザ移動手段の移動路と、結像光学系3の移動路が同じであることが好ましい。このように構成することにより、結像光学系3で撮像後、レーザ照射ユニット85で処理する等の細胞処理を行なう際に各手段の移動回数をさらに低減でき、処理時間をさらに低減できる。第2の移動ユニット7は、本実施形態の細胞処理装置200のように、第1の移動ユニット6と独立して移動可能に構成されていることが好ましい。
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.
本実施形態の細胞処理装置200は、結像光学系3として、1種類の倍率の対物レンズ31を有する結像光学系3を収容する筐体87が配置されているが、これに限定されず、複数種類の対物レンズ31を有する結像光学系3を収容する筐体87を配置してもよい。この場合、複数種類の対物レンズ31の倍率は、例えば、それぞれ、2倍、4倍および8倍等の異なる倍率であることが好ましい。また、本実施形態の細胞処理装置200のように、第1室81の撮像手段および対物レンズ31を有する結像光学系3を含む場合、細胞培養容器41内の細胞をより明確に撮像できることから、結像光学系3の対物レンズ31の倍率は、第1室81の撮像手段の倍率より高倍率であることが好ましい。
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.
本実施形態の細胞処理装置200において、レーザ照射ユニット85は、レーザ光源85a、レーザ出射部85cおよび光ファイバ85bを含むが、レーザ照射ユニット85は、これに限定されず、細胞培養容器配置ユニット4に配置された細胞培養容器41にレーザを照射可能であればよい。レーザ照射ユニット85は、例えば、レーザ光源85aを含み、レーザ光源85aから直接的に細胞培養容器41にレーザを照射してもよい。また、レーザ光源85aのレーザをレーザ出射部85cに導光する場合、光ファイバ85bに代えて、ミラー、MEMS(Micro Electro Mechanical Systems)等の導光手段を用いて、導光してもよいが、第2室82内におけるレーザ光源85aの配置を自由に設定でき、例えば、第2室82において、前記レーザ移動手段、結像光学系3、および第2の移動ユニット7等の他の手段が配置されておらず、また他の手段の可動範囲と重ならない領域にレーザ光源85aを配置することで、細胞処理装置200の大きさを小さくでき、かつ他の導光手段と比較して細胞処理装置200の重量を低減できることから、光ファイバ85bが好ましい。
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.
レーザ光源85aは、例えば、連続波レーザまたはパルスレーザを発振する装置である。レーザ光源85aは、例えば、連続波に近い、パルス幅の長い高周波レーザでもよい。レーザ光源85aから発振されるレーザの出力は、特に制限されず、例えば、処理および細胞に応じて、適宜決定できる。レーザ光源85aが発振するレーザの波長は、特に制限されず、例えば、405nm、450nm、520nm、532nm、808nm等の可視光レーザ、赤外線レーザ等があげられる。前述のように、細胞培養容器41にレーザ吸収層を設けている場合、レーザ光源85aは、例えば、前記レーザ吸収層が吸収可能な波長を発振する。レーザ光源85aは、細胞への影響を抑制できることから、波長が380nmより長いレーザを発振することが好ましい。具体例として、レーザ光源85aは、波長が405nm近傍にある最大出力5Wの連続波ダイオードレーザがあげられる。
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.
レーザ照射ユニット85がレーザ出射部85cを含む場合、前記レーザ移動手段は、レーザ出射部85cを移動させることが好ましい。また、前記レーザ移動手段がレーザ出射部85cを上下方向(図15において、矢印Z方向)に移動させる場合、レーザ出射部85cのレーザ出射口が、前記被観察体処理室の底面、好ましくは、細胞培養容器配置ユニット4の底面と接触しないように移動させることが好ましい。具体例として、前記レーザ移動手段は、レーザ出射部85cのレーザ出射口を、細胞培養容器配置ユニット4の底面を基準として、1mm以内に接近しないように移動させることが好ましい。このような範囲で、前記レーザ移動手段がレーザ出射部85cを移動させることにより、例えば、レーザ出射部85cと細胞培養容器配置ユニット4の底面との接触で生じる、細胞培養容器配置ユニット4に配置された細胞培養容器41内の培地の揺れを防止できる。
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.
本実施形態において、結像光学系3は、手前側(図15において、左下側)に配置され、レーザ照射ユニット85は、奥側(図15において、右上側)に配置されている。ただし、結像光学系3およびレーザ照射ユニット85との位置関係は、これに限定されず、例えば、結像光学系3を奥側に配置し、レーザ照射ユニット85を手前側に配置してもよい。一般的に観察ユニットの結像光学系3は、レーザ照射ユニット85と比較して、その体積が大きい。このため、第1室81において細胞培養容器配置ユニット4が手前側に配置されている場合、結像光学系3を奥側に配置し、レーザ照射ユニット85は、手前側に配置することで、細胞処理装置200の大きさを小さくすることができる。
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.
本実施形態の細胞処理装置200は、さらに、前記レーザが被照射物の被照射部に形成するスポットの径を調整するスポット径調整手段を含んでもよい。前記スポット径は、前記レーザと前記被照射物との接触部におけるレーザのビーム径を意味する。前記スポット径は、例えば、レーザ照射ユニット85のレーザ集光レンズおよびコリメータレンズ(コリメーションレンズ)の少なくとも一方の切替え、またはレーザ照射ユニット85と前記被照射物との距離を変更することにより、調整できる。前者の場合、レーザ照射ユニット85は、例えば、複数のレンズを含み、前記スポット径調整手段は、前記レンズを変更することにより、前記スポットの径を調整することが好ましい。前記複数のレンズは、例えば、複数の集光レンズでもよいし、複数のコリメータレンズでもよいし、1以上の集光レンズと1以上のコリメータレンズとの組合せでもよい。前記複数の集光レンズは、例えば、互いに異なる焦点距離を有する。前記複数のコリメータレンズは、例えば、互いに異なる焦点距離を有する。前記レンズの変更は、例えば、手動で行なってもよいし、後述する制御ユニット5により、変更されてもよい。後者の場合、例えば、レンズの変更手段を含み、前記変更手段により、レンズが変更される。また、前記スポット径調整手段が距離を変更する場合、前記スポット径調整手段は、レーザ照射ユニット85と、前記被照射物との距離を調整することにより、前記スポットの径を調整することが好ましい。レーザ照射ユニット85と、前記被照射物との距離は、例えば、細胞培養容器配置ユニット4の配置面、すなわち、細胞培養容器41の底面に対し、略直交方向の距離を意味する。また、レーザ照射ユニット85がレーザ出射部85cを含む場合、レーザ照射ユニット85と、前記被照射物との距離は、レーザ出射部85cと、前記被照射物との距離を意味する。レーザ照射ユニット85と、前記被照射物との距離は、例えば、前記レーザ移動手段により調整できる。具体例として、前記レーザ移動手段による矢印Z方向の移動により、前記被照射物である細胞培養容器41の底面との距離を調整できる。本実施形態の細胞処理装置200において、前記レーザ移動手段を兼ねるXYステージ71の台車711aは、上下方向(矢印Z方向)に昇降可能である。このため、本実施形態におけるレーザ移動手段は、例えば、スポット径調整手段ということもできる。前記スポット径調整手段は、例えば、小さいスポット径が好ましい細胞処理を実施する場合、例えば、細胞塊の分割、特定領域の細胞または細胞塊の切り出し等を実施する場合、スポット径を小さく調整する。また、前記スポット径調整手段は、例えば、大きいスポット径が好ましい細胞処理を実施する場合、例えば、特定領域の細胞の死滅等を実施する場合、スポット径を大きく調整する。前記スポット径の大きさは、特に制限されず、例えば、細胞処理の種類、細胞の大きさ等に応じて、適宜設定できる。本実施形態の細胞処理装置200はスポット径調整手段を含むことで、例えば、細胞に対して行なう処理によりスポット径を適切な大きさに調整することができ、迅速に細胞処理を実施できる。また、適切な大きさのスポット径に調整できるため、例えば、処理を行なわない細胞への影響を低減することができる。
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.
本実施形態の細胞処理装置200が前記スポット径調整手段を含む場合、後述する制御ユニット5が、前記スポット径調整手段による前記スポットの径の調整を制御することが好ましい。
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.
本実施形態の細胞処理装置200において、前記被観察体処理室と第2室82との間において、気体の移動が抑制されていることが好ましい。前記気体の移動の抑制は、例えば、前記被観察体処理室における第2室82との隣接部を、前述のパッキン、シール材等の封止部材で封止することにより実施できる。このように気体の移動を抑制することで、例えば、前記気体に含まれる埃の前記被観察体処理室内への流入を防止できる。
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.
本実施形態の細胞処理装置200において、第3室83は、制御ユニット5および電源ユニット57を含む。図16に示すように、本実施形態の制御ユニット5において、I/Oインターフェイス55は、DMD22、第1の移動ユニット6、第2の移動ユニット7、吸引吐出ユニット813、カメラ817、前記観察ユニット、およびレーザ照射ユニット85等の各部材と通信可能に接続してこれらを制御するためのデバイスである。この点を除き、本実施形態の制御ユニット5は、実施形態1の制御ユニット5と同様の構成を有し、その説明を援用できる。
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.
本実施形態の細胞処理装置200は、制御ユニット5に、DMD22、第1の移動ユニット6、第2の移動ユニット7、吸引吐出ユニット813、カメラ817、前記観察ユニット、およびレーザ照射ユニット85の制御機能を持たせることで、各部材に制御ユニットを個別に設けなくてもよいため、装置の小型化を実現できる。ただし、本発明はこれに限定されない。本発明の細胞処理装置は、例えば、制御ユニット5として、DMD22、第1の移動ユニット6、第2の移動ユニット7、吸引吐出ユニット813、カメラ817、前記観察ユニット、およびレーザ照射ユニット85のそれぞれに制御ユニットを設け、各部材の制御ユニットにより、各部材を制御してもよい。また、本発明の細胞処理装置は、例えば、制御ユニット5と各部材の制御ユニットとを設け、共同して各部材を制御してもよい。
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.
本実施形態において、制御ユニット5は、前記観察ユニットおよびレーザ照射ユニット85を制御するが、制御ユニット5は、いずれか一方を制御してもよい。
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.
本実施形態において、制御ユニット5は、レーザ照射ユニット85によるレーザ照射ならびに前記レーザ移動手段を兼ねるXYステージ71および台車711aによるレーザ照射ユニット85のレーザ出射部85cの移動を制御するが、制御ユニット5は、いずれか一方を制御してもよい。
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.
本実施形態において、制御ユニット5は、吸引吐出ユニット813による吸引吐出ならびに第1移動ユニット6であるXYステージ61およびアーム62による吸引吐出ユニット813の移動を制御するが、制御ユニット5は、いずれか一方を制御してもよい。
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.
本実施形態において、制御ユニット5は、第1室81の撮像手段であるカメラ817による前記被観察体処理室内の撮像を制御する。
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.
本実施形態において、制御ユニット5は、光源1のON/OFF、第1移動ユニット6であるXYステージ61およびアーム62による照明光学系2の移動、結像光学系3の撮像素子34による被観察体の撮像、および第2の移動ユニット7であるXYステージ71および台車711bによる結像光学系3の移動を制御するが、制御ユニット5、いずれか1つまたは2つ以上を制御してもよい。
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 .
電源ユニット57は、特に制限されず、公知の電源を使用できる。電源ユニット57は、例えば、レーザ照射ユニット85、前記観察ユニット、第1移動ユニット6、第2の移動ユニット7、吸引吐出ユニット813、循環手段84、前記照明手段、前記殺菌手段、制御ユニット5等の電力により稼働する部材(手段)に電力を供給する。このため、電源ユニット57は、例えば、前記電力により稼働する部材(手段)と、電気的に接続されている。電源ユニット57は、例えば、100Vの電圧で電力を供給する。これにより、例えば、一般的な電力環境においても、細胞処理装置200が使用可能となる。本実施形態の細胞処理装置200は、全体の電源供給を、電源ユニット57に担わせることによって、各部材にそれぞれ、個別に電源ユニットを設けなくてもよいので、例えば、細胞処理装置200の小型化や軽量化を実現することができる。ただし、本発明はこれに限定されず、例えば、各手段の少なくとも一つに専用の電源ユニットを設けてもよい。
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.
本実施形態の細胞処理装置200は、さらに、第3室83に、通信部(図示せず)を設けてもよい。前記通信部は、例えば、有線もしくは無線により、パーソナルコンピュータ、移動体通信機器等の外部の機器とデータの送受信機能またはインターネット等との接続機能を有する。前記通信部は、例えば、既存の通信モジュール等があげられる。このように通信部を設けることで、外部と細胞処理装置200を接続できるようになるため、例えば、外部から細胞処理装置200を操作すること、または外部からのデータを細胞処理装置200が受信することができる。また、細胞処理装置200内のデータを、例えば、外部から接続することで閲覧可能とすることができる。
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.
つぎに、本実施形態の細胞処理装置200を用いた細胞の処理および処理された細胞の回収について、例をあげて説明する。
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.
まず、殺菌灯819を消灯し、照明灯818a、818bを点灯させる。また、制御ユニット5により、カメラ817を起動させ、前記被観察体処理室内の撮像を開始する。カメラ817により撮像された前記被観察体処理室内の画像は、例えば、制御ユニット5を介して、前記表示装置に出力される。つぎに、循環手段84を稼働させ、前記被観察体処理室内の気体を循環させる。さらに、作業者が、開口部811aの扉812aを開け、細胞培養容器配置ユニット4に細胞培養容器41を配置し、また、回収容器配置部816aに回収容器816bを配置する。細胞培養容器41の底面には、前記レーザ吸収層が形成されている。前記配置後、前記作業者は、開口部811aの扉812aを閉じる。
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.
つぎに、制御ユニット5により、XYステージ71および台車711bが移動するよう制御され、結像光学系3を収容する筐体87が、細胞培養容器41の底面の下側に移動する。また、制御ユニット5により、XYステージ61が移動するように制御され、照明光学系2を収容する筐体86が、細胞培養容器41の上面の上部、すなわち、細胞培養容器配置ユニット4の上部に移動する。そして、前記実施形態1の位相差観察装置100の撮像方法と同様にして、細胞培養容器41のタイル画像を取得する。前記タイル画像の取得は、例えば、処理対象の被観察体42の大きさに応じて、異なる倍率の対物レンズ31を用いて行なってもよい。撮像素子34により撮像される画像は、例えば、位相差顕微鏡により撮像された位相差画像があげられる。結像光学系3が蛍光観察可能な場合、撮像素子34により撮像される画像は、蛍光画像であってもよい。前記撮像された画像は、例えば、制御ユニット5を介して、前記表示装置に出力される。
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.
作業者が、例えば、前記撮像されたタイル画像に基づき、処理対象領域(例えば、回収する細胞領域)を前記入力装置により指定すると、制御ユニット5により、XYステージ71および台車711aが移動するように制御され、レーザ出射部85cは、細胞培養容器41の底面の下側において、前記処理対象領域の周囲の細胞等の対象物にレーザを照射可能な位置に移動する。そして、制御ユニット5により、レーザ光源85aが、レーザを発振するように制御される。発振されたレーザは、光ファイバ85bにより導光され、レーザ出射部85cから照射される。また、前記レーザ照射とともに、制御ユニット5により、XYステージ71および台車711aが、前記処理対象領域の周囲を移動する。この際に、前記処理対象領域の周囲の対象物の大きさに応じて、台車711aを昇降させることで、前記スポット径の大きさを適切な大きさに調整し、前記処理対象領域内の被観察体42に影響が出ないようにする。照射されたレーザは、細胞培養容器41の底面に形成された前記レーザ吸収層に吸収され、前記レーザ吸収層から生じる熱等により、前記処理対象領域の周囲の対象物を死滅させる。これにより、前記処理対象領域を切り出すことができる。
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.
つぎに、制御ユニット5により、XYステージ61が移動するように制御され、吸引吐出ユニット813が、収容容器815bの上部に移動する。制御ユニット5により、アーム62が降下および上昇するように制御され、吸引吐出ユニット813の吸引吐出口側に、前記先端部材であるチップが装着される。さらに、制御ユニット5により、XYステージ61が移動するように制御され、吸引吐出ユニット813は、細胞培養容器41の上部において、前記処理対象領域の上部に移動する。制御ユニット5により、アーム62が降下するように制御され、前記チップの開口を前記処理対象領域の近傍に配置する。この状態で、制御ユニット5により、吸引吐出ユニット813が吸引するように制御され、前記処理対象領域の被観察体42を周囲の培地とともに前記チップ内に吸引する。
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.
さらに、制御ユニット5により、アーム62が上昇し、かつ、XYステージ61が移動するように制御され、吸引吐出ユニット813は、回収容器816bの上部に移動する。また、制御ユニット5により、アーム62が降下するように制御され、回収容器816bの内部に前記チップの開口が移動する。この状態で、制御ユニット5により、吸引吐出ユニット813が吐出するように制御され、前記チップ内の前記処理対象領域の被観察体42を含む培地を、回収容器816b内に吐出する。
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.
前記吐出後、制御ユニット5により、アーム62が上昇し、かつ、XYステージ61が移動するように制御され、吸引吐出ユニット813は、排液容器814bの上部に移動する。さらに、制御ユニット5により、アーム62が降下し、かつ、XYステージ61が移動するように制御され、前記チップの上側端を、排液容器814bに設けられた上面の凹部である先端部材脱離手段814cに引っかける。この状態で、制御ユニット5により、アーム62が上昇するように制御され、吸引吐出ユニット813から前記チップが脱離する。
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.
そして、作業者が、開口部811aの扉812aを開け、細胞培養容器配置ユニット4から細胞培養容器41を回収し、また、回収容器配置部816aから回収容器816bを回収する。このようにすることで、実施形態の細胞処理装置200により、細胞等の被観察体42の処理および処理された被観察体42の回収を実施できる。
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.
本実施形態の細胞処理装置200によれば、メニスカスによる位相差画像の劣化を抑制できるため、前記観察ユニットにより、コントラストが大きな位相差画像を撮像できる。前記コントラストが大きな位相差画像に基づけば、レーザ処理を実施する被観察体42、領域等を明確にすることができるため、精度よくレーザ処理を実施できる。このため、本実施形態の細胞処理装置200によれば、例えば、レーザ処理時のダメージを低減できる。
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.
本実施形態の細胞処理装置200によれば、細胞培養容器41の細胞に対し、例えば、選別、回収等の処理を簡易に実施できる。また、本実施形態の細胞処理装置200は、作業者自身ではなく、レーザ照射ユニット85により細胞を処理するため、例えば、作業者の技術レベルによる影響を受けない。このため、例えば、処理後に得られる細胞の品質が、安定する。
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.
次に、本発明の実施例について説明する。ただし、本発明は、下記実施例により制限されない。市販の試薬は、特に示さない限り、それらのプロトコルに基づいて使用した。
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.
[実施例1]
本発明の位相差観察装置を用いて、細胞培養容器内の細胞を撮像することにより、メニスカスによる位相差画像の劣化を抑制できることを確認した。 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.
本発明の位相差観察装置を用いて、細胞培養容器内の細胞を撮像することにより、メニスカスによる位相差画像の劣化を抑制できることを確認した。 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.
35mm(直径)ディッシュ(IWAKI社製)に培養液3mLを導入すると、図18の矢印で示すように、メニスカスにより壁面から約7mmの範囲(破線と実線の間の領域)で位相差画像の劣化が生じる。そこで、実施例1では、位相差観察装置100を用いて、前記培養液が導入されたディッシュの図18に示す領域a~dを撮像した。具体的には、領域a~dについて、図6に示す取得方法により、強度分布補正情報を取得後、図7に示す撮像方法により撮像した。なお、図6に示す取得方法における各工程は使用者により実施した。また、つぎの照明光の強度分布は、照明像を移動させることにより設定した。領域a~dにおいて、領域b~dが、メニスカスにより、位相差画像の劣化が生じる領域である。このため、領域bは、1つの照明光の強度分布を含む強度分布補正情報を用いて補正し、画像(補正画像1)を取得した。領域cは、2つの照明光の強度分布を含む強度分布補正情報を用いて補正し、2枚の画像(補正画像2~3)を取得後、前記補正対象領域を統合することにより、1枚の画像を取得した。領域dは、3つの照明光の強度分布を含む強度分布補正情報を用いて補正し、3枚の画像(補正画像4~6)を取得後、前記補正対象領域を統合することにより、1枚の画像を取得した。これらの結果を図19に示す。
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.
図19は、位相差観察装置100により撮像した位相差画像を示す写真である。図19に示すように、メニスカスが生じていない領域aでは、補正がない状態においても位相差画像の劣化が見られなかった。他方、メニスカスが生じている領域b~dでは、位相差画像の劣化が生じていたが、前記強度分布補正情報による補正により得られた画像(補正画像2~6)を採用または統合することにより、これらの領域における位相差画像の劣化をより一層抑制できた。
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.
これらの結果から、本発明の位相差観察装置を用いて、細胞培養容器内の細胞を撮像することにより、メニスカスによる位相差画像の劣化を抑制できることがわかった。
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.
[実施例2]
本発明の位相差観察装置により、メニスカスによる位相差画像の劣化が抑制された画像が撮像可能な領域が拡大することを確認した。 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.
本発明の位相差観察装置により、メニスカスによる位相差画像の劣化が抑制された画像が撮像可能な領域が拡大することを確認した。 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.
培養液3mLが導入された35mmディッシュの全面について、前記実施例1と同様にして、位相差画像を撮像した。また、コントロールは、前記強度分布補正情報による補正および前記強度分布補正情報の補正値を用いなかった以外は、同様にして位相差像を撮像した。そして、各位相差像において、メニスカスが抑制されている領域、すなわち、ディッシュ内の細胞が観察可能な領域の面積を算出した。また、前記強度分布補正情報の補正値による補正前後の画像について、35mmディッシュの壁面から前記観察可能な領域までの距離の平均値(RA)を算出した。
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.
つぎに、60mm(直径)ディッシュ(φ60、IWAKI社製)および100mm(直径)ディッシュ(φ100、IWAKI社製)について、35mmディッシュにおける前記壁面から前記観察可能な領域までの距離の平均値(RA)に基づき、前記強度分布補正情報の補正値による補正前後におけるディッシュ内の細胞が観察可能な領域の面積を算出した。この結果を下記表1に示す。
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.
前記表1に示すように、位相差観察装置100によれば、ディッシュにおける観察可能な領域が格段に拡大し、特にディッシュの直径が小さいときに、その効果が顕著であった。
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.
これらの結果から、本発明の位相差観察装置により、メニスカスによる位相差画像の劣化が抑制された画像が撮像可能な領域が拡大することがわかった。
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.
以上、実施形態を参照して本発明を説明したが、本発明は、上記実施形態に限定されるものではない。本発明の構成や詳細には、本発明のスコープ内で当業者が理解しうる様々な変更をすることができる。
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.
この出願は、2017年9月28日に出願された日本出願特願2017-189170、および2018年6月8日に出願された日本出願特願2018-110020を基礎とする優先権を主張し、その開示のすべてをここに取り込む。
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.
<付記>
上記の実施形態および実施例の一部または全部は、以下の付記のように記載されうるが、以下には限られない。
(付記1)
光源と、
前記光源からの照明光を細胞培養容器内の被観察体に導光する照明光学系と、
前記被観察体の光学像を撮像素子に結像させる結像光学系と、
制御ユニットとを含み、
前記照明光学系は、前記照明光の強度分布を変化させる空間変調素子を含み、
前記制御ユニットは、
前記細胞培養容器に対する前記結像光学系の位置と、前記結像光学系の位置における照明光の強度分布とを関連付けた強度分布補正情報を含み、
前記結像光学系の位置である結像系位置情報を取得し、
前記結像系位置情報および前記強度分布補正情報に基づき、前記空間変調素子における照明光の強度分布を変化させることを特徴とする、位相差観察装置。
(付記2)
前記結像光学系は、位相板を含み、
前記強度分布補正情報における照明光の強度分布は、前記結像光学系の位置において、前記照明光の像である照明像が、前記位相板の像である位相板像に含まれる照明光の強度分布である、付記1記載の位相差観察装置。
(付記3)
前記結像光学系は、位相板を含み、
前記制御ユニットは、前記被観察体を含まない細胞培養容器について、前記結像光学系の位置において、前記照明光の像である照明像が、前記位相板の像である位相板像に含まれる照明光の強度分布を求め、得られた照明光の強度分布と前記結像光学系の位置とを関連付けて、前記強度分布補正情報として記憶する、付記1または2記載の位相差観察装置。
(付記4)
前記空間変調素子は、前記照明光を前記被観察体方向に反射することにより導光するデジタルマイクロミラーデバイスを含む、付記1から3のいずれかに記載の位相差観察装置。
(付記5)
前記光源と前記照明光学系とを移動可能な第1の移動ユニットを含み、
前記制御ユニットは、前記第1の移動ユニットによる移動を制御する、付記1から4のいずれかに記載の位相差観察装置。
(付記6)
前記結像光学系は、位相板を含み、
前記第1の移動ユニットは、前記位相板の面に対する垂直方向に移動可能である、付記5記載の位相差観察装置。
(付記7)
前記制御ユニットは、
前記結像光学系の位置と、前記光源および前記照明光学系の位置とを関連づけた照明系位置補正情報を記憶し、
前記結像系位置情報および前記照明系位置補正情報に基づき、前記光源と前記照明光学系の位置を補正する、付記1から6のいずれかに記載の位相差観察装置。
(付記8)
前記制御ユニットは、
前記細胞培養容器の撮像対象領域を複数の区分に分画し、
前記結像系位置情報および前記強度分布補正情報に基づき、前記空間変調素子における照明光の強度分布を変化させ、前記撮像素子により各区分を撮像し、
得られた画像に基づき、前記細胞培養容器の撮像対象領域の画像を作製する、付記1から7のいずれかに記載の位相差観察装置。
(付記9)
前記制御ユニットは、
前記撮像対象領域の画像において、撮影不良部位があるかを判定し、
前記撮影不良部位がある場合、前記撮像不良部位を含む区分の強度分布補正情報の補正値を算出し、
前記結像系位置情報、前記強度分布補正情報、および前記強度分布補正情報の補正値に基づき、前記撮像素子により、前記撮像不良部位を含む区分を再撮像し、
前記撮像不良部位を含む区分の画像を、前記再撮像により得られた画像に変更し、前記撮像対象領域の画像を作製する、付記8記載の位相差観察装置。
(付記10)
前記制御ユニットは、
前記被観察体を含まない細胞培養容器について、前記結像光学系の位置において、前記照明光の像である照明像が、前記位相板の像である位相板像に含まれる照明光の強度分布を求め、
前記空間変調素子における照明光の強度分布を得られた照明光の強度分布に変化させ、前記撮像素子により撮像し、得られた画像の画素における輝度値が、輝度値の基準値以上かを判定し、
前記画像における輝度値が前記輝度値の基準値以上の場合、前記画像の輝度値の基準値以上の領域について、前記結像光学系の位置において、前記照明光の像である照明像を変化させ、つぎの照明光の強度分布を求め、
得られた各照明光の強度分布と前記結像光学系の位置とを関連付けて、前記強度分布補正情報として記憶する、付記1から9のいずれかに記載の位相差観察装置。
(付記11)
前記制御ユニットは、
前記結像系位置情報と関連する強度分布補正情報が、複数の照明光の強度分布を含むかを判定し、
前記強度分布補正情報が複数の照明光の強度分布を含む場合、前記空間変調素子における照明光の強度分布を、各照明光の強度分布を変化させ、前記撮像素子により撮像し、
得られた各画像において、得られた画像の画素における輝度値が、前記輝度値の基準値未満の領域を抽出し、抽出された画像を統合する、付記10記載の位相差観察装置。
(付記12)
前記細胞培養容器を配置可能な細胞培養容器配置ユニットを含み、
前記細胞培養容器配置ユニットは、前記照明光の光路において、前記照明光学系と前記結像光学系との間に配置される、付記1から11のいずれかに記載の位相差観察装置。
(付記13)
前記細胞培養容器配置ユニットは、前記位相差観察装置における位置が固定されている、付記12記載の位相差観察装置。
(付記14)
前記結像光学系を移動可能な第2の移動ユニットを含み、
前記制御ユニットは、前記第2の移動ユニットの移動を制御する、付記1から13のいずれかに記載の位相差観察装置。
(付記15)
前記空間変調素子は、前記結像光学系の瞳と光学的に共役の位置に配置される、付記1から14のいずれかに記載の位相差観察装置。
(付記16)
細胞培養容器内の被観察物を観察可能な観察ユニットと、
前記被観察物に対して、レーザを照射可能なレーザ照射ユニットと、
前記観察ユニットおよび前記レーザ照射ユニットの少なくとも一方を制御する制御ユニットとを含み、
前記観察ユニットは、付記1から15のいずれかに記載の位相差観察装置であることを特徴とする、細胞処理装置。
(付記17)
第1領域、第2領域および第3領域を含み、
前記第1領域および前記第2領域は、連続して配置され、
前記第1領域は、前記細胞培養容器内の被観察体を処理する被観察体処理室であり、
前記被観察体処理室は、前記細胞培養容器を配置可能な細胞培養容器配置ユニットを含み、
前記第1領域は、前記観察ユニットにおける光源および照明光学系を含み、
前記第2領域は、前記観察ユニットにおける結像光学系と、前記レーザ照射ユニットとを含み、
前記第3領域は、前記制御ユニットを含み、
前記細胞培養容器配置ユニットは、前記被観察体処理室において、前記第2領域と隣接するように配置されている、付記16記載の細胞処理装置。 <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 toappendix 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 ofappendices 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 ofappendices 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 toclaim 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 ofappendices 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 ofappendices 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 ofappendices 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 ofappendices 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 ofappendices 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 ofappendices 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 ofappendices 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.
上記の実施形態および実施例の一部または全部は、以下の付記のように記載されうるが、以下には限られない。
(付記1)
光源と、
前記光源からの照明光を細胞培養容器内の被観察体に導光する照明光学系と、
前記被観察体の光学像を撮像素子に結像させる結像光学系と、
制御ユニットとを含み、
前記照明光学系は、前記照明光の強度分布を変化させる空間変調素子を含み、
前記制御ユニットは、
前記細胞培養容器に対する前記結像光学系の位置と、前記結像光学系の位置における照明光の強度分布とを関連付けた強度分布補正情報を含み、
前記結像光学系の位置である結像系位置情報を取得し、
前記結像系位置情報および前記強度分布補正情報に基づき、前記空間変調素子における照明光の強度分布を変化させることを特徴とする、位相差観察装置。
(付記2)
前記結像光学系は、位相板を含み、
前記強度分布補正情報における照明光の強度分布は、前記結像光学系の位置において、前記照明光の像である照明像が、前記位相板の像である位相板像に含まれる照明光の強度分布である、付記1記載の位相差観察装置。
(付記3)
前記結像光学系は、位相板を含み、
前記制御ユニットは、前記被観察体を含まない細胞培養容器について、前記結像光学系の位置において、前記照明光の像である照明像が、前記位相板の像である位相板像に含まれる照明光の強度分布を求め、得られた照明光の強度分布と前記結像光学系の位置とを関連付けて、前記強度分布補正情報として記憶する、付記1または2記載の位相差観察装置。
(付記4)
前記空間変調素子は、前記照明光を前記被観察体方向に反射することにより導光するデジタルマイクロミラーデバイスを含む、付記1から3のいずれかに記載の位相差観察装置。
(付記5)
前記光源と前記照明光学系とを移動可能な第1の移動ユニットを含み、
前記制御ユニットは、前記第1の移動ユニットによる移動を制御する、付記1から4のいずれかに記載の位相差観察装置。
(付記6)
前記結像光学系は、位相板を含み、
前記第1の移動ユニットは、前記位相板の面に対する垂直方向に移動可能である、付記5記載の位相差観察装置。
(付記7)
前記制御ユニットは、
前記結像光学系の位置と、前記光源および前記照明光学系の位置とを関連づけた照明系位置補正情報を記憶し、
前記結像系位置情報および前記照明系位置補正情報に基づき、前記光源と前記照明光学系の位置を補正する、付記1から6のいずれかに記載の位相差観察装置。
(付記8)
前記制御ユニットは、
前記細胞培養容器の撮像対象領域を複数の区分に分画し、
前記結像系位置情報および前記強度分布補正情報に基づき、前記空間変調素子における照明光の強度分布を変化させ、前記撮像素子により各区分を撮像し、
得られた画像に基づき、前記細胞培養容器の撮像対象領域の画像を作製する、付記1から7のいずれかに記載の位相差観察装置。
(付記9)
前記制御ユニットは、
前記撮像対象領域の画像において、撮影不良部位があるかを判定し、
前記撮影不良部位がある場合、前記撮像不良部位を含む区分の強度分布補正情報の補正値を算出し、
前記結像系位置情報、前記強度分布補正情報、および前記強度分布補正情報の補正値に基づき、前記撮像素子により、前記撮像不良部位を含む区分を再撮像し、
前記撮像不良部位を含む区分の画像を、前記再撮像により得られた画像に変更し、前記撮像対象領域の画像を作製する、付記8記載の位相差観察装置。
(付記10)
前記制御ユニットは、
前記被観察体を含まない細胞培養容器について、前記結像光学系の位置において、前記照明光の像である照明像が、前記位相板の像である位相板像に含まれる照明光の強度分布を求め、
前記空間変調素子における照明光の強度分布を得られた照明光の強度分布に変化させ、前記撮像素子により撮像し、得られた画像の画素における輝度値が、輝度値の基準値以上かを判定し、
前記画像における輝度値が前記輝度値の基準値以上の場合、前記画像の輝度値の基準値以上の領域について、前記結像光学系の位置において、前記照明光の像である照明像を変化させ、つぎの照明光の強度分布を求め、
得られた各照明光の強度分布と前記結像光学系の位置とを関連付けて、前記強度分布補正情報として記憶する、付記1から9のいずれかに記載の位相差観察装置。
(付記11)
前記制御ユニットは、
前記結像系位置情報と関連する強度分布補正情報が、複数の照明光の強度分布を含むかを判定し、
前記強度分布補正情報が複数の照明光の強度分布を含む場合、前記空間変調素子における照明光の強度分布を、各照明光の強度分布を変化させ、前記撮像素子により撮像し、
得られた各画像において、得られた画像の画素における輝度値が、前記輝度値の基準値未満の領域を抽出し、抽出された画像を統合する、付記10記載の位相差観察装置。
(付記12)
前記細胞培養容器を配置可能な細胞培養容器配置ユニットを含み、
前記細胞培養容器配置ユニットは、前記照明光の光路において、前記照明光学系と前記結像光学系との間に配置される、付記1から11のいずれかに記載の位相差観察装置。
(付記13)
前記細胞培養容器配置ユニットは、前記位相差観察装置における位置が固定されている、付記12記載の位相差観察装置。
(付記14)
前記結像光学系を移動可能な第2の移動ユニットを含み、
前記制御ユニットは、前記第2の移動ユニットの移動を制御する、付記1から13のいずれかに記載の位相差観察装置。
(付記15)
前記空間変調素子は、前記結像光学系の瞳と光学的に共役の位置に配置される、付記1から14のいずれかに記載の位相差観察装置。
(付記16)
細胞培養容器内の被観察物を観察可能な観察ユニットと、
前記被観察物に対して、レーザを照射可能なレーザ照射ユニットと、
前記観察ユニットおよび前記レーザ照射ユニットの少なくとも一方を制御する制御ユニットとを含み、
前記観察ユニットは、付記1から15のいずれかに記載の位相差観察装置であることを特徴とする、細胞処理装置。
(付記17)
第1領域、第2領域および第3領域を含み、
前記第1領域および前記第2領域は、連続して配置され、
前記第1領域は、前記細胞培養容器内の被観察体を処理する被観察体処理室であり、
前記被観察体処理室は、前記細胞培養容器を配置可能な細胞培養容器配置ユニットを含み、
前記第1領域は、前記観察ユニットにおける光源および照明光学系を含み、
前記第2領域は、前記観察ユニットにおける結像光学系と、前記レーザ照射ユニットとを含み、
前記第3領域は、前記制御ユニットを含み、
前記細胞培養容器配置ユニットは、前記被観察体処理室において、前記第2領域と隣接するように配置されている、付記16記載の細胞処理装置。 <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
(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
(Supplementary Note 4)
The phase difference observation device according to any one of
(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
(Supplementary Note 6)
The imaging optical system includes a phase plate,
The phase difference observation apparatus according to
(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
(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
(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
(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
(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
(Supplementary Note 15)
The phase difference observation apparatus according to any one of
(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
(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.
本発明の位相差観察装置によれば、例えば、追加の撮像部の構成が不要であることにより、装置を小型化でき、かつ前記メニスカスによる位相差画像の劣化を抑制できる。このため、本発明は、例えば、細胞、組織等の被観察体を観察する生命科学分野、医療分野等において、極めて有用である。
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.
1 光源
2 照明光学系
21 光源レンズ
22 デジタルマイクロミラーデバイス
23 コンデンサレンズ
3 結像光学系
31 対物レンズ
32 位相板
33 結像レンズ
34 撮像素子
35 位相板撮像レンズ
4 細胞培養容器配置ユニット(ステージ)
41 細胞培養容器
42 被観察体
43 上蓋
44 底部
45 透光領域
46 底壁
47 底板
48 凹部
49 突出部
5 制御ユニット
51 CPU
52 メインメモリ
53 補助記憶デバイス
54 ビデオコーデック
55 I/Oインターフェイス
56 コントローラ
57 電源ユニット
6 第1の移動ユニット
61、71、71a、71b XYステージ
62 アーム
7 第2の移動ユニット
711a、711b 台車
81 第1室
811a、811b 開口部
812a、812b 扉
813 吸引吐出ユニット
814a 排液容器配置部
814b 排液容器
814c 先端部材脱離手段
815a 収容容器配置部
815b 収容容器
816a 回収容器配置部
816b 回収容器
817 カメラ
818a、818b 照明灯
819 殺菌灯
82 第2室
83 第3室
84 循環手段
84a 吸気部
84b 循環流路
84c 気体供給部
84d 排気部
85 レーザ照射ユニット
85a レーザ光源
85b 光ファイバ
85c レーザ出射部
86、87 筐体
100 位相差観察装置
200 細胞処理装置 DESCRIPTION OFSYMBOLS 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 OFSYMBOLS 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
52main 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
2 照明光学系
21 光源レンズ
22 デジタルマイクロミラーデバイス
23 コンデンサレンズ
3 結像光学系
31 対物レンズ
32 位相板
33 結像レンズ
34 撮像素子
35 位相板撮像レンズ
4 細胞培養容器配置ユニット(ステージ)
41 細胞培養容器
42 被観察体
43 上蓋
44 底部
45 透光領域
46 底壁
47 底板
48 凹部
49 突出部
5 制御ユニット
51 CPU
52 メインメモリ
53 補助記憶デバイス
54 ビデオコーデック
55 I/Oインターフェイス
56 コントローラ
57 電源ユニット
6 第1の移動ユニット
61、71、71a、71b XYステージ
62 アーム
7 第2の移動ユニット
711a、711b 台車
81 第1室
811a、811b 開口部
812a、812b 扉
813 吸引吐出ユニット
814a 排液容器配置部
814b 排液容器
814c 先端部材脱離手段
815a 収容容器配置部
815b 収容容器
816a 回収容器配置部
816b 回収容器
817 カメラ
818a、818b 照明灯
819 殺菌灯
82 第2室
83 第3室
84 循環手段
84a 吸気部
84b 循環流路
84c 気体供給部
84d 排気部
85 レーザ照射ユニット
85a レーザ光源
85b 光ファイバ
85c レーザ出射部
86、87 筐体
100 位相差観察装置
200 細胞処理装置 DESCRIPTION OF
DESCRIPTION OF
52
Claims (17)
- 光源と、
前記光源からの照明光を細胞培養容器内の被観察体に導光する照明光学系と、
前記被観察体の光学像を撮像素子に結像させる結像光学系と、
制御ユニットとを含み、
前記照明光学系は、前記照明光の強度分布を変化させる空間変調素子を含み、
前記制御ユニットは、
前記細胞培養容器に対する前記結像光学系の位置と、前記結像光学系の位置における照明光の強度分布とを関連付けた強度分布補正情報を含み、
前記結像光学系の位置である結像系位置情報を取得し、
前記結像系位置情報および前記強度分布補正情報に基づき、前記空間変調素子における照明光の強度分布を変化させることを特徴とする、位相差観察装置。 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. - 前記結像光学系は、位相板を含み、
前記強度分布補正情報における照明光の強度分布は、前記結像光学系の位置において、前記照明光の像である照明像が、前記位相板の像である位相板像に含まれる照明光の強度分布である、請求項1記載の位相差観察装置。 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. - 前記結像光学系は、位相板を含み、
前記制御ユニットは、前記被観察体を含まない細胞培養容器について、前記結像光学系の位置において、前記照明光の像である照明像が、前記位相板の像である位相板像に含まれる照明光の強度分布を求め、得られた照明光の強度分布と前記結像光学系の位置とを関連付けて、前記強度分布補正情報として記憶する、請求項1または2記載の位相差観察装置。 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. - 前記空間変調素子は、前記照明光を前記被観察体方向に反射することにより導光するデジタルマイクロミラーデバイスを含む、請求項1から3のいずれか一項に記載の位相差観察装置。 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.
- 前記光源と前記照明光学系とを移動可能な第1の移動ユニットを含み、
前記制御ユニットは、前記第1の移動ユニットによる移動を制御する、請求項1から4のいずれか一項に記載の位相差観察装置。 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. - 前記結像光学系は、位相板を含み、
前記第1の移動ユニットは、前記位相板の面に対する垂直方向に移動可能である、請求項5記載の位相差観察装置。 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. - 前記制御ユニットは、
前記結像光学系の位置と、前記光源および前記照明光学系の位置とを関連づけた照明系位置補正情報を記憶し、
前記結像系位置情報および前記照明系位置補正情報に基づき、前記光源と前記照明光学系の位置を補正する、請求項1から6のいずれか一項に記載の位相差観察装置。 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. - 前記制御ユニットは、
前記細胞培養容器の撮像対象領域を複数の区分に分画し、
前記結像系位置情報および前記強度分布補正情報に基づき、前記空間変調素子における照明光の強度分布を変化させ、前記撮像素子により各区分を撮像し、
得られた画像に基づき、前記細胞培養容器の撮像対象領域の画像を作製する、請求項1から7のいずれか一項に記載の位相差観察装置。 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. - 前記制御ユニットは、
前記撮像対象領域の画像において、撮影不良部位があるかを判定し、
前記撮影不良部位がある場合、前記撮像不良部位を含む区分の強度分布補正情報の補正値を算出し、
前記結像系位置情報、前記強度分布補正情報、および前記強度分布補正情報の補正値に基づき、前記撮像素子により、前記撮像不良部位を含む区分を再撮像し、
前記撮像不良部位を含む区分の画像を、前記再撮像により得られた画像に変更し、前記撮像対象領域の画像を作製する、請求項8記載の位相差観察装置。 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. - 前記制御ユニットは、
前記被観察体を含まない細胞培養容器について、前記結像光学系の位置において、前記照明光の像である照明像が、前記位相板の像である位相板像に含まれる照明光の強度分布を求め、
前記空間変調素子における照明光の強度分布を得られた照明光の強度分布に変化させ、前記撮像素子により撮像し、得られた画像の画素における輝度値が、輝度値の基準値以上かを判定し、
前記画像における輝度値が前記輝度値の基準値以上の場合、前記画像の輝度値の基準値以上の領域について、前記結像光学系の位置において、前記照明光の像である照明像を変化させ、つぎの照明光の強度分布を求め、
得られた各照明光の強度分布と前記結像光学系の位置とを関連付けて、前記強度分布補正情報として記憶する、請求項1から9のいずれか一項に記載の位相差観察装置。 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. - 前記制御ユニットは、
前記結像系位置情報と関連する強度分布補正情報が、複数の照明光の強度分布を含むかを判定し、
前記強度分布補正情報が複数の照明光の強度分布を含む場合、前記空間変調素子における照明光の強度分布を、各照明光の強度分布を変化させ、前記撮像素子により撮像し、
得られた各画像において、得られた画像の画素における輝度値が、前記輝度値の基準値未満の領域を抽出し、抽出された画像を統合する、請求項10記載の位相差観察装置。 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. - 前記細胞培養容器を配置可能な細胞培養容器配置ユニットを含み、
前記細胞培養容器配置ユニットは、前記照明光の光路において、前記照明光学系と前記結像光学系との間に配置される、請求項1から11のいずれか一項に記載の位相差観察装置。 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. . - 前記細胞培養容器配置ユニットは、前記位相差観察装置における位置が固定されている、請求項12記載の位相差観察装置。 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.
- 前記結像光学系を移動可能な第2の移動ユニットを含み、
前記制御ユニットは、前記第2の移動ユニットの移動を制御する、請求項1から13のいずれか一項に記載の位相差観察装置。 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. - 前記空間変調素子は、前記結像光学系の瞳と光学的に共役の位置に配置される、請求項1から14のいずれか一項に記載の位相差観察装置。 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.
- 細胞培養容器内の被観察物を観察可能な観察ユニットと、
前記被観察物に対して、レーザを照射可能なレーザ照射ユニットと、
前記観察ユニットおよび前記レーザ照射ユニットの少なくとも一方を制御する制御ユニットとを含み、
前記観察ユニットは、請求項1から15のいずれか一項に記載の位相差観察装置であることを特徴とする、細胞処理装置。 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. - 第1領域、第2領域および第3領域を含み、
前記第1領域および前記第2領域は、連続して配置され、
前記第1領域は、前記細胞培養容器内の被観察体を処理する被観察体処理室であり、
前記被観察体処理室は、前記細胞培養容器を配置可能な細胞培養容器配置ユニットを含み、
前記第1領域は、前記観察ユニットにおける光源および照明光学系を含み、
前記第2領域は、前記観察ユニットにおける結像光学系と、前記レーザ照射ユニットとを含み、
前記第3領域は、前記制御ユニットを含み、
前記細胞培養容器配置ユニットは、前記被観察体処理室において、前記第2領域と隣接するように配置されている、請求項16記載の細胞処理装置。 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.
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EP18863653.4A EP3677945B1 (en) | 2017-09-28 | 2018-08-17 | Phase difference observation device and cell treatment device |
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