JP2020005553A - Incubation system and incubator - Google Patents

Abstract

To provide incubation systems that enables efficient cell observation in a large-scale culture.SOLUTION: An incubation system 10 is equipped with a translucent member 21 that partitions at least the bottom surface of a space in which a translucent culture container 27 in which cells 28 are cultured is accommodated, a plurality of incubators 20 having a thermo-hygrostat 39 that maintains constant-temperature and -humidity of the space, an imaging device 70 capable of optically imaging the culture container 27 through the translucent member 21, in a space below the incubator 20, and a moving device 80 that moves the imaging device 70 below the plurality of incubators 20.SELECTED DRAWING: Figure 2

Description

  The present invention relates to an incubation system and an incubator capable of observing cells in a culture vessel while maintaining a culture vessel for culturing cells at a constant temperature and humidity.

  2. Description of the Related Art Conventionally, cells have been cultured for treating diseases or producing or evaluating pharmaceuticals. The cells are cultured in an incubator while keeping the temperature and humidity constant. In culturing cells, an operation of transferring some or all of the cultured cells to a new culture container such as a petri dish or a bag is performed. Such an operation is generally called passaging. Passaging is performed, for example, in a sterile environment called an isolator. The isolator is provided with a half suit or glove that can be worn by an operator from the outside (see Patent Document 1).

Japanese Patent No. 4403455

  For example, when culturing cells collected from a patient for treatment of a disease, it is required that the cells being cultured are not confused with cells of another patient. Therefore, it is preferable that only one culture vessel for culturing cells of one patient is accommodated in one incubator.

  In addition, in the passage, it is required to work the required amount of cells at once. The passage timing is determined by imaging the culture vessel in the incubator with an optical microscope or the like, analyzing the captured image, and the like.

  However, in order to take out the culture vessel from the incubator and take an image, it is necessary to provide a clean room for that purpose and prepare an environment in the clean room so that cells are not contaminated. In addition, every time an operator for imaging enters or leaves the clean room, it is necessary to check whether the environment in the clean room is well maintained. On the other hand, providing an imaging device individually for each incubator is not suitable for mass production of culturing cells of many patients, because the cost is great.

  The present invention has been made in view of the above-mentioned circumstances, and an object of the present invention is to provide an incubation system and an incubator that can efficiently observe cells in a large-scale culture.

  (1) An incubation system according to the present invention, a translucent member that partitions at least the bottom surface of a space in which a translucent culture container in which cells are cultured is accommodated, and a constant temperature that maintains the space at a constant temperature and humidity. A plurality of incubators having a constant humidity machine, and an imaging device arranged in a space below the incubator and capable of optically imaging the culture vessel through the translucent member, and moving the incubator or the imaging device And a moving device for causing the moving device to move.

  Since the incubator or the imaging device is moved by the moving device in the space below the plurality of incubators, it is not necessary to arrange the imaging device for each incubator. Further, the cells in the culture container in the incubator are imaged by the imaging device without removing the culture container from the incubator.

  (2) Preferably, the incubator has a cover movable to an open position exposing the translucent member to the outside and a closed position covering the translucent member to the outside.

  According to the above configuration, when it is not necessary to image the culture vessel with the imaging device, by covering the translucent member with a cover, heat conduction through the translucent member can be suppressed, or the light can pass through the translucent member. Invasion of external light can be suppressed.

  (3) Preferably, the incubation system further includes a support that horizontally supports the plurality of incubators, and the moving device includes a lower part of the plurality of incubators supported by the support. In the space, the imaging device is moved relatively to the incubator.

  Since the moving device moves not the relatively heavy incubator but the relatively light imaging device, the torque and energy required for the moving device are small. Thereby, the moving device can be downsized. Further, since the incubator is not moved, vibrations and the like are not easily transmitted to cells in the incubator.

  (4) Preferably, the imaging device is an illumination light source, an illumination optical system that emits illumination light from the illumination light source to the cells of the culture vessel that is the observation target, and the illumination light passes through the observation target. An observation optical system that forms an optical image of the observation target object from the reflected light reflected by the observation optical system, wherein the observation optical system changes a phase of the directly reflected light reflected by a reflection surface among the reflected lights. Provided with a plate, both the illumination optical system and the observation optical system are an epi-illumination phase contrast microscope arranged below the observation object, and photoelectrically convert the optical image obtained by the observation optical system. And an imaging element for creating image data of the cells, wherein the illumination light emitted from the illumination optical system is parallel light.

  According to the above configuration, parallel illumination light is emitted from the illumination optical system, and parallel reflection light enters the observation optical system. Therefore, the reflection light is parallel to the optical axis, and the distance from the phase plate to the reflection surface is large. Does not change the convergence point of the directly reflected light on the phase plate. Thereby, even if the distance from the phase plate to the reflection surface varies, the contrast of the image data can be kept constant, and a good optical image can be stably obtained.

  (5) Preferably, the present incubation system further includes a heater for heating the translucent member.

  According to the above configuration, by heating the translucent member that can be exposed to the outside by the heater, dew condensation is less likely to occur on the translucent member, and the inside of the incubator can be less affected by the external environment. .

  (6) The present invention provides a light-transmitting member that partitions at least a part of the bottom surface of a space in which a light-transmitting container is housed, holding means for holding the container on the light-transmitting member, and the space And a door for opening and closing the space, an open position for exposing the translucent member to the outside, and a closed position for covering the translucent member to the outside. And may be regarded as an incubator having a cover movable below.

  ADVANTAGE OF THE INVENTION According to the incubation system which concerns on this invention, mass culture of a cell can be performed efficiently.

FIG. 1 is an external view of an incubation system 10 according to the present embodiment. FIG. 2 is a block diagram illustrating a configuration of the incubation system 10 according to the present embodiment. FIG. 3 is an external view of the incubator 20 according to the present embodiment. FIG. 4 is a diagram showing the internal structure of the incubator 20 according to the present embodiment, and FIG. 4A is a perspective view showing the internal structure of the incubator 20 when the cover is in the open position, and FIG. FIG. 4 is a perspective view showing the internal structure of the incubator 20 when the cover is in the closed position. FIG. 5 is an external view of the culture container carrier 50 according to the present embodiment. FIG. 6 is an external view of the culture container stocker 51 according to the present embodiment. FIG. 7 is an external view of the imaging device 70 and the moving device 80 according to the present embodiment. FIG. 8 is a schematic diagram illustrating a configuration of an optical system of the imaging device 70 according to the present embodiment.

  Hereinafter, embodiments of the present invention will be described with reference to the drawings as appropriate. The embodiments described below are merely examples in which the present invention is embodied, and it is needless to say that the embodiments can be appropriately changed without changing the gist of the present invention.

  Hereinafter, in the description of the drawings, the front direction 14 and the rear direction 15 along the horizontal direction are defined as front and rear directions. The upward direction 16 and the downward direction 17 along the vertical direction are defined as the vertical direction. A right direction 18 and a left direction 19 orthogonal to the front-back direction along the horizontal direction are defined as left-right directions.

  A direction orthogonal to the front direction 14 and the right direction 18 is defined as a downward direction 17, and a direction opposite to the downward direction 17 is defined as an upward direction 16. A direction orthogonal to the front direction 14 and the downward direction 17 is defined as a right direction 18, and a direction opposite to the right direction 18 is defined as a left direction 19. A direction orthogonal to the right direction 18 and the downward direction 17 is defined as a front direction 14, and a direction opposite to the front direction 14 is defined as a rear direction 15.

  Hereinafter, in the description of the drawings, “facing in the forward direction” includes facing in the direction including the component of the front direction 14, and “facing in the rear direction” refers to the direction including the component of the rear direction 15. Including turning. Further, "facing downward" includes facing in a direction including a component of downward direction 17, and "facing upward" includes facing in a direction including a component of upward direction 16. Further, “turn right” includes turning in a direction including a component of right direction 18, and “turn left” includes turning in a direction including a component of left direction 19. For example, “the front faces in the front direction” may mean that the front faces in a direction along the front direction 14 or the front face may face in a direction inclined with respect to the front direction 14.

[Incubation system 10]
As shown in FIG. 1 and FIG. 2, the incubation system 10 according to the present embodiment includes a plurality of incubators 20, a support table 91 that supports the incubator 20, a transfer table 97 on which the incubator is mounted and moves, and an incubator The imaging device 70 includes an imaging device 70 that captures an image of the culture container 27 accommodated in the imaging device 20, and a moving device 80 that moves the imaging device 70. The incubation system 10 is a system in which the imaging device 70 moves with respect to each incubator 20 and images the culture vessels 27 accommodated in the culture chambers 25 of each incubator 20.

[Incubator 20]
As shown in FIG. 3, FIG. 4 (a) and FIG. 4 (b), the incubator 20 is a substantially rectangular parallelepiped having an inner space as a culture room 25, and includes a lid 30, a translucent member 21, a cover 23, a partition wall 26, a thermo-hygrostat 39, a culture vessel carrier 50, and a culture vessel stocker 51.

  The outer surface of the incubator 20 includes a partition wall 26, a lid 30, and a cover 23. Each side surface of the incubator 20 facing upward, right, left, front, and rear is constituted by a partition wall 26. A side surface of the incubator 20 facing the left direction 19 is formed by a lid 30. The lid 30 opens and closes an opening formed in the partition wall 26 and is rotatably connected to the partition wall 26.

  When the lid 30 opens the opening of the partition wall 26, the culture chamber 25 can be accessed from outside. When the lid 30 closes the opening of the partition wall 26, the culture chamber 25 is kept airtight. When the operator houses the culture container 27 in the culture room 25, the lid 30 is moved to the open state. When the accommodation operation is completed, the worker moves the lid 30 to the closed state, whereby the culture chamber 25 is kept airtight to the outside. The cover 30 may have a confirmation window 31. A translucent member such as glass is fitted in the confirmation window 31 so that the inside of the culture chamber 25 can be visually recognized through the confirmation window 31 when the lid 30 is in a closed state.

  A part of the lower surface of the incubator 20 is constituted by a translucent member 21. The translucent member 21 partitions the culture room 25. The translucent member 21 is made of a material that transmits light, and is made of, for example, a polymer compound such as an acrylic resin or a polystyrene resin, or a silicate compound such as a glass. Light passes between the lower part of the incubator 20 and the culture chamber 25 through the translucent member 21. A cover 23 is provided below the translucent member 21. The covers 23 form a pair, and each is rotatably connected to the partition wall 26.

  As shown in FIG. 4A, the cover 23 is rotated to move to a closed position for covering the translucent member 21 from below and an open position for exposing the translucent member 21 to the outside. The cover 23 in the open position does not overlap the translucent member 21 in the vertical direction. As shown in FIG. 4B, when the cover 23 is in the closed position, light does not enter the culture chamber 25 from the outside through the translucent member 21. When the cover 23 is in the open position, light can enter the culture chamber 25 from outside through the translucent member 21.

  As shown in FIG. 2, the cover 23 is rotated by a cover drive unit 107 having a drive transmission mechanism such as a motor, a gear train, or a belt. The cover drive unit 107 is controlled by the cover control unit 106. The cover control unit 106 receives a control signal from the culture computer 36 and transmits a drive signal to the cover drive unit 107. That is, the opening and closing of the cover 23 is controlled through the culture computer 36.

[Constant temperature and humidity machine 39]
As shown in FIG. 2, FIG. 4A and FIG. 4B, the incubator 20 includes a thermo-hygrostat 39. The thermo-hygrostat 39 is a mechanism for controlling the atmosphere (temperature and humidity) of the culture room 25. Specifically, the constant temperature and humidity machine 39 includes a heating device 32, a temperature sensor 33, a humidifying device 34, a humidity sensor 35, a culture computer 36, an operation device 37, a display device 38, a heater temperature, A sensor 101, a heater control unit 102, a cover control unit 106, and a cover driving unit 107 are provided.

  The heating device 32 is for raising the room temperature of the culture room 25. The heating device 32 has, for example, a heating element that generates heat when energized. The temperature sensor 33 outputs an electric signal corresponding to the room temperature of the culture room 25. The humidifier 34 is for raising the humidity of the culture room 25. The humidifier 34 generates fine water droplets by, for example, heating or vibration. The humidity sensor 35 outputs an electric signal according to the humidity of the culture room 25.

  The culture computer 36 is a so-called arithmetic device, and has a program for controlling operations of the heating device 32, the humidifying device 34, the cover control unit 106, and the heater control unit 102. The operating device 37 is an input device for inputting target values of the temperature and the humidity of the culture room 25 and timing for opening and closing the cover 23 to the culture computer 36. The display device 38 is a display that displays the temperature detected by the temperature sensor 33, the humidity detected by the humidity sensor 35, the set value set by the operation device 37, and the like. The culture computer 36 operates the heating device 32 and controls the humidification device 34 so that the detected values of the temperature and humidity obtained by the temperature sensor 33 and the humidity sensor 35 match the set values of the set temperature and humidity. Control the operation. Thus, the atmosphere of the culture room 25 is controlled to a desired temperature and humidity.

[Heater 22]
As shown in FIGS. 2, 4A and 4B, the incubator 20 includes a heater 22. The heater 22 heats the translucent member 21 and is, for example, a glass heater having a transparent electrode film on which an indium oxide film (ITO film) is deposited. The operation of the heater 22 is controlled by the heater control unit 102. The heater control unit 102 controls the temperature of the light transmitting member 21 to a predetermined temperature based on an electric signal corresponding to the temperature of the light transmitting member 21 output from the heater temperature sensor 101 provided on the light transmitting member 21. Is controlled by the heater control unit 102.

[Culture container carrier 50]
As shown in FIG. 5, the culture container carrier 50 (an example of a holding unit) includes a culture container stocker 51 on which the culture container 27 is loaded, a fixing angle 52, and an evaporation pad 53.

  As shown in FIG. 6, the culture container stocker 51 includes a pair of fixing plates 55 and a plurality of pillars 56. Each of the pair of fixing plates 55 has a flat plate shape having a plurality of openings 54. In the present embodiment, four openings 54 are formed in a row in each fixing plate 55. Each opening 54 is circular, and the diameter thereof is slightly smaller than the diameter of the bottom surface of the thin columnar culture vessel 27.

  The pair of fixing plates 55 are vertically separated from each other, and are connected by a plurality of columns 56 extending in the vertical direction. A guide 57 extending in a circular shape protrudes from the upper surface of the fixing plate 55 located below along the periphery of each opening 54. The guide 57 extends along a circle slightly larger than the outer diameter of the culture vessel 27. The culture container 27 is loaded inside the guide 57. The bottom surface of the culture container 27 loaded inside the guide 57 is exposed downward through the opening 54.

  The culture container stocker 51 includes a plurality of culture containers 27 stacked in the vertical direction as one group, and four groups are located between the fixing plates 55. Four openings 54 are formed in the fixing plate 55, and four groups of culture vessels 27 can be mounted directly above the openings 54. By mounting the plurality of culture containers 27 on the culture container stocker 51, the plurality of culture containers 27 can be integrally handled in the operation of taking the culture container 27 into and out of the culture room 25 of the incubator 20.

  As shown in FIG. 5, the fixing angle 52 fixes the plurality of culture vessel stockers 51 integrally. In the present embodiment, three culture container stockers 51 can be fixed to the fixing angle 52. The fixing angle 52 has a mounting plate on which the plurality of culture container stockers 51 can be mounted, and a guide that holds the positions of the plurality of culture container stockers 51 in the front-rear direction and the left-right direction on the mounting plate. Although not shown in the figure, the mounting plate of the fixing angle 52 is formed with an opening that exposes each opening 54 of the mounted culture vessel stocker 51 downward. The configuration of the fixing angle 52 is not limited to the configuration shown in the present embodiment.

  The fixing angle 52 is provided in the culture room 25. When placing the culture container stocker 51 in the culture room 25, the operator places the culture container stocker 51 on the mounting plate of the fixing angle 52. Thereby, in the culture room 25, the positioning of the culture container 27 in the front-back direction and the left-right direction is performed. An evaporation pad 53 is provided on the side of the fixing angle 52. The evaporation pad 53 is a container for storing water.

[Culture container 27]
As shown in FIGS. 2, 5 and 6, the culture vessel 27 is a so-called petri dish, and has a lower plate 27D and an upper plate 27U which are nested. The lower plate 27D and the upper plate 27U have a shallow cylindrical shape, and one end in the axial direction is open. The inner diameter of the opening of the upper plate 27U is slightly larger than the outer diameter of the opening of the lower plate 27D. One culture vessel 27 is formed by covering the lower plate 27D opening upward with the upper plate 27U opening downward. The lower plate 27D and the upper plate 27U are both made of a material that transmits light, for example, a polymer compound such as an acrylic resin or a polystyrene resin, or a silicate compound such as glass.

[Reflecting mirror 79]
As shown in FIGS. 2 and 8, the reflecting mirror 79 is provided on the upper surface of the lid of the upper plate 27U of the culture vessel 27. In the present embodiment, the reflecting mirror 79 has a substantially same circular shape as the bottom surface of the lower plate 27D. The lower surface 79a of the reflecting mirror 79 is a reflecting surface that reflects light. In the present embodiment, the reflecting mirror 79 is made of stainless steel or the like having excellent rust prevention and gloss, and a surface facing downward 17 is mirror-finished so as to reflect light. The reflecting mirror 79 may be configured as a lower surface of the lower plate 27D of the culture container 27. That is, in the culture containers 27 stacked vertically, the lower surface of the lower plate 27D of the culture container 27 positioned above may be configured to function as the reflecting mirror 79.

[Support base 91]
As shown in FIG. 1 and FIG. 2, the support base 91 is a frame formed in a rectangular parallelepiped shape, the moving device 80 is supported in an internal space 95 thereof, and a plurality of An incubator 20 is supported. By mounting the incubator 20 on the mounting surface 92, the two incubators 20 are aligned in the horizontal direction such that the direction in which the lid 30 of the incubator 20 faces the same direction. The imaging device 70 is mounted on the moving device 80. That is, by supporting the plurality of incubators 20, the moving device 80, and the imaging device 70 on the support base 91, the imaging device 70 can move in the space below the plurality of incubators 20. In the present embodiment, two incubators 20 can be supported on one support 91, but the number of incubators 20 that can be supported on one support 91 may be two or more.

  The transfer table 97 is used when moving the incubator 20 to place it on the support table 91. The transfer table 97 is a frame formed in a rectangular parallelepiped shape, and one incubator 20 can be supported on a mounting surface 98 as an upper surface of the frame. The height of the mounting surface 98 is equal to the height of the mounting surface 92 of the support base 91. A plurality of casters 99 are provided on the lower surface of the frame, and the transport table 97 can be smoothly moved on the floor surface by the rotation of the casters 99.

[Moving device 80]
As shown in FIGS. 1, 2, and 7, the moving device 80 includes a support frame 81, a lift 82, and a bogie mechanism 86.

  The support frame 81 is a rectangular frame. The support frame 81 is located in the internal space 95 of the support base 91 and can be moved in the internal space 95 by the transport mechanism 87. The elevating table 82 is vertically movable with respect to the support frame 81 by an elevating mechanism 84. In the lifting mechanism 84, a link mechanism that connects the support frame 81 and the lifting table 82 is moved by an actuator 85. The elevating table 82 has a rectangular dish shape equivalent to the support frame 81. An orthogonal robot 83 is supported on the upper surface of the lift 82. The orthogonal robot 83 supports the imaging device 70, and moves the imaging device 70 in the front-rear direction and the left-right direction. The operations of the actuator 85 and the orthogonal robot 83 are controlled by the movement control unit 46.

  A carriage mechanism 86 is provided in the internal space 95 of the support base 91. The bogie mechanism 86 moves the support frame 81 in the horizontal direction, and includes, for example, a rail extending in the horizontal direction, a bogie movable on the rail, and a drive mechanism for driving the bogie. The drive mechanism of the bogie mechanism unit 86 includes, for example, a motor and a drive transmission mechanism such as a gear and a belt. The operation of the bogie mechanism unit 86 is controlled by controlling the rotation of the motor by the movement control unit 46. Is done.

[Microscope 60]
As shown in FIGS. 2 and 8, the microscope 60 includes an illumination light source 63, an illumination optical system 71, an observation optical system 72, and an image sensor 73. The illumination optical system 71 emits the illumination light 61 from the illumination light source 63 to a cell as an observation target. The observation optical system 72 forms an optical image of the cell from the reflected light 62 that is obtained by the illumination light 61 passing through the cell and reflected by the reflection surface 79a. The imaging element 73 photoelectrically converts the optical image of the cell to create image data of the cell.

  The illumination light source 63 is a light source that emits the illumination light 61. The illumination light source 63 is a variable wavelength light source, and can change the wavelength of the illumination light 61. The switching of the wavelength is performed by the user operating the operation device 47. When the target value of the wavelength is designated by operating the operation device 47, the light source control unit 44 controls the illumination light 61 such that the illumination light 61 of the wavelength of the target value is emitted.

  The microscope 60 is an epi-illumination type microscope, and the illumination optical system 71 and the observation optical system 72 are arranged on the same side in the up-down direction with respect to the cell, and in the present embodiment, in the downward direction 17 with respect to the cell. Accordingly, the illumination light source 63 is also arranged on the same side as the illumination optical system 71 and the observation optical system 72, that is, in the downward direction 17 with respect to the cells. Since the illumination optical system 71 and the observation optical system 72 are not disposed on both sides of the cell, the microscope 60 can be made compact.

  The illumination optical system 71 includes a half mirror 74, a first objective lens 75, and a second objective lens. The half mirror 74 reflects the illumination light 61 from the illumination light source 63 and emits the light toward the position where the cells are arranged. In the present embodiment, the light is emitted in the upward direction 16. A first objective lens 75 and a second objective lens 76 are arranged between the half mirror 74 and the cell. The first objective lens 75 is arranged on the side closer to the half mirror 74, and the second objective lens 76 is arranged on the side closer to the cell. After being refracted by the first objective lens 75 and the second objective lens 76, the illumination light 61 from the half mirror 74 passes through the translucent member 21 and the lower plate 27D, passes through the cells, and reaches the reflection surface 79a. To reach. The optical axis 69 is a vertical axis, a central axis of the light beam of the illumination light 61 emitted from the half mirror 74, and also a central axis of the first objective lens 75 and the second objective lens.

  The illumination light 61 emitted from the second objective lens 76 forms parallel light parallel to the optical axis 69 and along the vertical direction. The optical characteristics of the illumination light source 63 of the microscope 60, the half mirror 74, the first objective lens 75 and the second objective lens 76, and the first objective lens 75 and the second objective lens 76 so that such parallel light is emitted to the cells. The optical refractive index of the objective lens 76 is set in advance, and each component is arranged in advance.

  The illumination light 61 passes through the cells and is reflected on the reflection surface 79a to become reflected light 62. The reflected light 62 is directly diffracted inside the cell when passing through the cell, and is directly diffracted inside the cell when diffusing inside the cell when passing through the cell. Diffracted reflected light 62B reflected by the reflecting surface 79a with a delay of ４ wavelength. 8, the illumination light 61 is indicated by a solid line, the directly reflected light 62A is indicated by a broken line, and the diffracted reflected light 62B is indicated by a dashed line. The reflected light 62 is a general term for the direct reflected light 62A and the diffracted reflected light 62B.

  In the present embodiment, since the reflecting mirror 79 is disposed above the cells, more light is reflected on the reflecting surface 79a of the reflecting mirror 79, so that the amount of the reflected light 62 increases. The directly reflected light 62A is light that has passed through the cell twice before and after the reflection and has not been diffracted in the cell, of the light reflected by the reflection surface 79a of the reflection mirror 79. The diffracted reflected light 62B is, of the light reflected by the reflecting surface 79a of the reflecting mirror 79, light that passes through the cell twice before and after the reflection and is diffracted inside the cell.

  The observation optical system 72 includes a first objective lens 75, a second objective lens 76, a half mirror 74, a phase plate 77, and an image plane 78. The imaging surface 78 is a light receiving surface formed on the surface of the image sensor 73 in the present embodiment. Here, the first objective lens 75, the second objective lens 76, and the half mirror 74 are shared by the illumination optical system 71 and the observation optical system 72. The reflected light 62 from the surface of the reflection surface 79a is refracted by the first objective lens 75 and the second objective lens 76 via the translucent member 21 and the lower plate 27D, and then is refracted by the half mirror 74 and the phase plate 77. And reaches the imaging plane 78.

  The phase plate 77 has a phase film region 77A for shifting the phase and a transmission region 77B for shifting the phase. The phase film region 77A is a circular region around the optical axis 69 in the phase plate 77. The transmission region 77B is an annular region around the optical axis 69 in the phase plate 77, and is outside the outer periphery of the phase film region 77A in the horizontal direction. The phase film region 77A and the transmission region 77B are made of a material that transmits light. When the light passes through the phase film region 77A, the phase of the light is shifted by １ ／ wavelength. In the present embodiment, the phase is delayed by １ ／ wavelength, but the phase may be advanced by １ ／ wavelength. On the other hand, the transmission region 77B does not shift the phase of light passing through the transmission region 77B.

  The refractive indices and arrangements of the first objective lens 75 and the second objective lens 76 and the size (radius) of the phase film region 77A are determined in advance so that the directly reflected light 62A of the reflected light 62 can pass through the phase film region 77A. Is set to Therefore, when the directly reflected light 62A passes through the phase difference film region 77A, the phase of the directly reflected light 62A is shifted in the phase film region 77A. On the other hand, since the diffracted reflected light 62B passes through the transmission area 77B, the phase of the diffracted reflected light 62B is not changed. When the phase of the directly reflected light 62A is delayed by １ ／ wavelength, the phase difference between the directly reflected light 62A and the diffracted reflected light 62B becomes 0 after passing through the phase plate 77. When the phase of the directly reflected light 62A is advanced by １ ／ wavelength, the phase difference between the directly reflected light 62A and the diffracted reflected light 62B becomes 波長 wavelength after passing through the phase plate 77.

  The luminous flux of the reflected light 62 reflected by passing through each position of the cell converges on the image plane 78. When the phase of the directly reflected light 62A is delayed by １ ／ wavelength, the direct reflected light 62A and the diffracted reflected light 62B reinforce each other on the imaging surface 78, so that the cell is bright and the background has a dark contrast. An image is obtained. When the phase of the directly reflected light 62A is advanced by １ ／ wavelength, on the contrary, an optical image of dark contrast with dark cells and a bright background is obtained.

  The optical image formed on the image forming surface 78 is photoelectrically converted by the image sensor 73 to create image data. The image data is displayed on the display device 48 after being processed by the image processing unit 45.

[Image processing unit 45]
As shown in FIG. 2, the image processing unit 45 has a filter function for removing noise information from image data obtained by the image sensor 73. This filter function is a function of removing noise information due to dust or the like attached to the second objective lens 76 from the image data based on two image data obtained by the illumination light 61 having two different wavelengths.

  Changing the wavelength of the illumination light 61 is equivalent to changing the optical path length from the cell to the imaging plane 78. Due to this change, the position where the reflected light 62 converges changes. When the reflected light 62 converges on the image plane 78, the optical image is in focus, but when the reflected light 62 does not converge on the image plane 78, the optical image is out of focus. In other words, in-focus image data and out-of-focus image data are created by the two different wavelengths of illumination light 61. Here, the optical image due to dust or the like adhering to the second objective lens 76 is not affected by the change in the optical path length, and thus does not change in luminance. Therefore, in two pieces of image data obtained by changing the wavelength, noise information included in the image data can be specified by specifying pixels in a portion where the fluctuation in luminance is relatively small. Further, by deleting the information of the pixel including the noise information, the image data from which the noise information has been deleted can be created.

  Prior to causing the image processing unit 45 to perform the filter function, the light source control unit 44 controls the illumination light source 63 to change the wavelength of the illumination light 61. In this manner, image data obtained with the illumination light 61 of the first wavelength and image data obtained with the illumination light of the second wavelength are obtained. Here, the image data is an aggregate of pixels arranged in two-dimensional coordinates. Each pixel has luminance value information as color information.

  The image processing unit 45 includes a calculation unit 41, a recognition unit 42, and a creation unit 43. The calculation unit 41 calculates a difference in luminance value for each pixel of the same coordinates for two image data obtained by the two different wavelengths of illumination light 61. The recognition unit 42 recognizes a pixel whose difference is smaller than a predetermined value as a noise pixel containing erroneous information. The creating unit 43 creates two corrected image data by deleting noise pixels from the two image data. In this manner, the presence or absence of noise information included in the image data is recognized, and image data from which the noise information has been deleted is created.

[Operation of the incubation system]
Incubation system 10 operates as follows. The subculture operation for cell culture is performed in an isolator (not shown). The plurality of culture vessels 27 in which the cells are seeded in the culture medium 28 are loaded on the culture vessel stocker 51, and the plurality of culture vessel stockers 51 are mounted on the culture vessel carrier 50 and accommodated in the culture room 25 of the incubator 20. The culture computer 36 maintains the culture room 25 of the incubator 20 containing the culture container 27 at a constant temperature and humidity by a constant temperature and humidity machine 39. Further, the culture computer 36 sets the cover 23 of the incubator 20 to the closed position during the cell culture. The cover 23 may be set to the closed position or the open position manually by an operator.

  During the cell culture, the culture container 27 accommodated in the culture room 25 of the incubator 20 is observed using the microscope 60. Specifically, the operation device 47 receives a user's input, and moves the moving device 80 to a position directly below the incubator 20 that houses the culture container 27 to be observed. Subsequently, the culture computer 36 sets the cover 23 of the incubator 20 immediately above the moving device 80 to the open position. Thereby, the translucent member 21 of the incubator 20 is exposed to the outside. The culture computer 36 energizes the heater 22 to heat the translucent member 21.

  Subsequently, the operating device 47 operates the orthogonal robot 83 to move the microscope 60 in the culture room 25 of the incubator 20 directly below the culture container 27 to be observed. In addition, the elevating mechanism 84 is operated, and the microscope 60 moves up just below the culture container 27 to be observed so as to approach the translucent member 21. The relative positions of the microscope 60 and the culture vessel 27 to be observed in the up-down direction are set in advance so that good image data of the cells to be observed can be obtained.

  The microscope 60 operated by the operation device 47 operates as follows. The light source controller 44 illuminates the culture vessel 27 to be observed from below with the illumination light source 63, and the microscope 60 captures an optical image of the culture vessel 27. The image sensor 73 photoelectrically converts an optical image captured by the microscope 60 to create image data. The image processing unit 45 receives and stores image data output from the image sensor 73 and performs data processing necessary for display and the like. The image data processed by the image processing unit 45 is displayed on the display device 48. The user determines the passage timing by observing the image displayed on the display device 48.

  When the observation is completed, the operation device 47 operates the elevating mechanism 84 to separate the microscope 60 from the translucent member 21 of the incubator 20. Further, the culture computer 36 closes the cover 23 and stops energizing the heater 22.

  When the observation of the culture container 27 accommodated in the incubator 20 to be observed is completed, the user can observe the culture container 27 accommodated in another incubator 20 in the same manner.

[Operation and effect of the present embodiment]
According to the incubation system 10, by providing the moving device 80 for moving the imaging device 70 relative to the incubator 20 in the downward direction 17 of the plurality of incubators 20, the culture accommodated in the culture room 25 of the incubator 20 is provided. The cells cultured in the culture container 27 can be observed with the microscope 60 without removing the container 27 from the culture chamber 25. Further, the cultured cells in the culture container 27 are observed by the microscope 60 without removing the culture container 27 accommodated in the culture room 25 of another incubator 20 using the same imaging device 70 from the culture room 25. Therefore, the cells in the culture vessels 27 in the plurality of incubators 20 can be observed by one imaging device 70. Therefore, in the passage, it is possible to prevent cell contamination and reduce equipment costs. In addition, since the efficiency of the operation is improved, cells of many patients can be cultured in a large amount.

  Further, according to the incubation system 10, by providing the cover 23, the translucent member 21 can be covered and closed with the cover 23 except when the cells in the culture container 27 are observed. This can prevent external light from entering the culture chamber 25 from the translucent member 21.

  Further, according to the incubation system 10, the illumination light emitted from the illumination optical system of the microscope 60 of the imaging device 70 is made into parallel light, so that parallel reflected light enters the observation optical system. It is parallel to the axis, and even if the distance from the phase plate to the reflection surface changes, the convergence point of the directly reflected light on the phase plate does not change. For this reason, even if the distance from the phase plate to the reflection surface varies, the contrast of the image data can be kept constant, and the number of cells can be counted efficiently.

  Further, according to the incubation system 10, the light-transmitting member 21 can be heated by the heater 22, and dew condensation on the surface of the light-transmitting member 21 inside the incubator 20 can be suppressed. Further, the influence of the external environment on the inside of the incubator 20 can be reduced.

[Modification]
In the present embodiment, the incubator system 10 is a device for moving the microscope 60, but is not limited to this configuration. The moving device 80 may be a device that relatively moves the incubator 20 and the microscope 60. The moving device 80 may be a device that moves the incubator 20 instead of moving the microscope 60. In this embodiment, a moving device 80 controlled by the movement control unit 46 to move the microscope 60 is provided. The movement control unit 46 is not an essential component, but may be a movement device 80 for manually moving the microscope 60 or the incubator 20.

  In the present embodiment, in the incubator system 10, the moving device 80 moves in the same direction as the direction in which the incubator 20 is aligned with the mounting surface 92 of one support base 91. The direction in which the moving device 80 moves is not limited to the direction of the mounting surface 92 of the support table 91. For example, as shown in FIG. 1, when the supports 91 placed on the incubator 20 in the left and right directions 18 and 19 are arranged in parallel in the front and rear directions 14 and 15, the moving device 80 moves in the front and rear directions 14 and 15. May be. Further, the support base 91 is not limited to the one that supports the plurality of incubators 20. For example, a support base or a support leg may be provided for each incubator 20 so as to secure a space in which the imaging device 70 can be arranged below.

  In the present embodiment, the cover 23 of the incubator 20 may be any mechanism that moves between the open position and the closed position. In the present embodiment, the cover 23 is moved to the open position and the closed position by being rotated by the cover driving unit 107. The cover driving unit 107 is not an essential component, but may be a mechanism for manually moving the cover 23 between the open position and the closed position.

  In the present embodiment, the cover 23 of the incubator 20 is moved to an open position and a closed position by rotating. The rotation of the cover 23 is not an essential component, but may be a mechanism in which the cover 23 slides in a horizontal direction to move to the open position and the closed position.

  In the present embodiment, the cover 23 of the incubator 20 has a member that rotates in the front direction 14 and a member that rotates in the rear direction 15 as shown in FIG. The member of the cover 23 does not need to be divided into two members, and may be only a member that rotates in one of the front direction 14 and the rear direction 15.

  In the present embodiment, a reflecting mirror 79 having a reflecting surface 79a on the lower surface is provided. The reflecting mirror 79 is not an essential component, and the reflecting mirror 79 may not be provided. In this case, an optical image of the cell is formed on the microscope 60 based on the reflected light 62 that has passed through the cell and has been reflected on the surface of another reflection surface. For example, the lower surface of the upper plate 27U in FIG. 2 or the lower surface of the lower plate 27D when the lower plate 27D is further placed on the upper direction 16 of the upper plate 27U can be used as the reflecting surface similarly to the reflecting surface 79a. It is.

  In the present embodiment, the reflecting mirror 79 is a plate fixed to the upper plate 27U of the culture vessel 27, but is not limited to this configuration. The reflecting mirror 79 only needs to be arranged in the upward direction 16 of the cell and reflect the illumination light 61, and the position where the reflecting mirror 79 is arranged and the object to which the reflecting mirror 79 is fixed are not limited. The reflector 79 may be arranged in the culture room 25 and fixed to a part of the partition wall 26 of the incubator 20.

In the present embodiment, by switching the wavelength of the illumination light 61 between a plurality of different wavelengths,
The obtained image can be blurred, and image data of a foreign substance such as dust can be extracted. However, the present invention is not limited to this. It can be adjusted, the position (convergence point) where the directly reflected light 62A converges on the phase plate 77 can be changed, and the image plane can be focused.

10 Incubation system 20 Incubator 21 Translucent member 22 Heater 23 Cover 27 Culture container 28 Cells 39 Constant temperature and humidity machine 60 ..Microscope, epi-illumination phase contrast microscope 61 ... Illumination light 62 ... Reflection light 62A ... Direct reflection light 63 ... Illumination light source 70 ... Imaging device 71 ... Illumination optical system 72 ... Observation optical system 73 Image sensor 77 Phase plate 79a Reflecting surface 80 Moving device 91 Support

The illumination light 61 passes through the cells and is reflected on the reflection surface 79a to become reflected light 62. The reflected light 62 is a direct reflected light 62A that does not diffract inside the cell when passing through the cell, and a diffracted reflected light 62B that is diffracted inside the cell when passing through the cell and the phase is delayed by １ ／ wavelength. , Consisting of In FIG. 8, the directly reflected light 62A is indicated by a solid line , and the diffracted reflected light 62B is indicated by a broken line . The reflected light 62 is a general term for the direct reflected light 62A and the diffracted reflected light 62B.

Claims (7)

A translucent member that partitions at least the bottom surface of a space in which a culture vessel having translucency in which cells are cultured is accommodated, and a plurality of incubators having a thermo-hygrostat that maintains the space at a thermo-hygrostat,
An imaging device arranged in the space below the incubator and capable of optically imaging the culture vessel through the translucent member,
A moving device for moving the incubator or the imaging device.   2. The incubator according to claim 1, wherein the incubator has a cover that is movable between an open position where the translucent member is exposed to the outside and a closed position that covers the translucent member against the outside. 3. Incubation system. It further comprises a support base for supporting the plurality of incubators in a horizontal direction,
The incubation system according to claim 1, wherein the moving device moves the imaging device relative to the incubator in a space below the plurality of incubators supported by the support base. The imaging device includes an illumination light source, an illumination optical system that emits illumination light from the illumination light source to cells in the culture container that is an observation target, and reflected light that is reflected by the illumination light passing through the observation target. An observation optical system that forms an optical image of the observation object from
The observation optical system includes a phase plate that changes the phase of the directly reflected light reflected on the reflecting surface of the reflected light,
An epi-illumination phase contrast microscope in which both the illumination optical system and the observation optical system are arranged below the observation target,
The photoelectric conversion of the optical image obtained by the observation optical system, an imaging device to create image data of the cells, comprising,
4. The incubation system according to claim 1, wherein the illumination light emitted from the illumination optical system is parallel light.   The incubation system according to claim 1, further comprising a heater that heats the translucent member. A light-transmissive member that partitions the bottom surface of at least a part of a space in which the light-transmissive container is housed,
Holding means for holding the container on the translucent member,
A thermo-hygrostat that maintains the space at a thermo-hygrostat;
A door that opens and closes the space,
An incubator having a cover movable below an open position for exposing the translucent member to the outside and a closed position for covering the translucent member to the outside. The incubator according to claim 6, further comprising a heater that heats the translucent member.