JP2009251297A - Culture observation device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a culture observation device in which environmental changes in a culture space are suppressed as much as possible by automatically opening and closing an opening and closing cap of a desired sample container by a simple configuration and control, as well as, an operation of a chemical liquid to a desired sample container is carried out when the device is used in a time-lapse observation system. <P>SOLUTION: Upon operating a chemical liquid to a desired sample container 420, a sample tray 400 holding a plurality of sample containers 420 having hinge-type opening/closing caps 422 is moved over the range of observable trajectory by a moving means so as to position the designated desired sample container 420 at a chemical liquid operation position P2; the opening and closing cap 422 is automatically opened and closed by a cap opening and closing mechanism 440 that is linked to the movement of the sample tray; and the chemical liquid operation is carried out through an operation aperture at the chemical liquid operation position P2. <P>COPYRIGHT: (C)2010,JPO&INPIT

Description

  The present invention relates to a culture observation apparatus for observing cultured cells while culturing them.

  In research on molecular organisms and biophysics, research using cultured cells has become popular. Since the culture environment of the cultured cells is different from the normal indoor environment in which the optical microscope is used, the cultured cells are adversely affected in the normal indoor environment. For this reason, there has been proposed a culture microscope apparatus that enables observation of cells while culturing cells by combining an optical microscope with a culture apparatus that maintains an environment suitable for cell culture (for example, patents). Reference 1). When such a culture experiment is performed for a long period of time, a time lapse observation method is often used in which the cultured cells in the specimen container are photographed intermittently and time-sequentially with a microscope at intervals of a predetermined waiting time.

  In such cultured cell experiments, a chemical solution is administered or stirred to give an external stimulus to the cultured cells under experiment, or in a long-term experiment, the deteriorated medium is replaced. There is a case where the user wants to perform operations (collectively, these operations are called chemical operation). Since the inside (culture space) of the culture device is maintained in an environment suitable for cell culture, a pipette was used after opening the door provided on the culture device and manually opening the lid of the specimen container. In the chemical solution operation, the environment in the culture space is greatly changed, which causes great damage to cells sensitive to the environmental change.

  Therefore, in order to enable a chemical solution operation on the cultured cells without changing the environment of the culture space, a culture in which an attachment / detachment mechanism for attaching / detaching the lid member of the specimen container by an operation from the outside of the culture device is provided at a predetermined position in the culture device. A microscope apparatus has been proposed (see, for example, Patent Document 2).

JP 2006-11415 A JP 2007-111034 A

  However, in the device described in Patent Document 2, after moving the specimen container to the attachment / detachment position and manually operating the attachment / detachment mechanism to remove the lid member, the specimen container is moved to the liquid supply position where the liquid supply means is set. Then, the liquid must be supplied, the specimen container must be returned to the attaching / detaching position after the liquid supply, and the lid member must be attached by the attaching / detaching mechanism. For this reason, since the sample container is moved with the lid member removed even in the culture space, there is a possibility that foreign matter is mixed in or water is evaporated, which is not preferable. In particular, environmental changes during experiments in time-lapse observation affect the reliability of the experiments, which is not preferable. Further, the chemical solution operation is complicated and the movement control of the sample container is also complicated. Further, the device described in Patent Document 2 is for manually attaching / detaching the lid member, and after confirming that the target specimen container has moved to the attachment / detachment position of the lid member, the attachment / detachment operation of the lid member is performed. Must be performed carefully, and the operability is poor. Therefore, it is difficult to use together when observing by the time lapse observation method according to the set time schedule.

  The present invention has been made in view of the above, and time-lapse observation while suppressing the environmental change in the culture space as much as possible by automatically opening and closing the opening and closing lid of the desired specimen container with a simple configuration and control. It is an object of the present invention to provide a culture observation apparatus that can be used for a chemical solution operation on a desired specimen container in combination with a method.

  In order to solve the above-mentioned problems and achieve the object, the culture observation apparatus according to the present invention is a culture observation apparatus for observing cultured cells while culturing them, and cultures the cultured cells while managing the environment. A culture apparatus that forms a culture space and has an operation opening in a part thereof, and a plurality of specimen containers that are accommodated in the culture space and that contain culture cells and culture media and are closed by a hinged lid A specimen tray that is held in place, a microscope that intermittently observes cultured cells in the specimen container that are sequentially positioned at an observation position on the optical axis across a waiting period, and the specimen tray that is orthogonal to the optical axis of the microscope A moving means that moves two-dimensionally along a plane to be moved, and any specimen in which a chemical liquid operation is designated at a chemical liquid operation position that is set outside the range of a trajectory that can be observed by the microscope and that can be operated from the operation opening Yong A lid opening / closing mechanism that opens and closes the opening / closing lid of the specimen container in conjunction with movement of the specimen tray beyond the observable locus of the specimen tray by the moving means for positioning, and a chemical operation is instructed with the designation of the specimen container And a lid opening / closing control means for controlling the moving means in order to operate the lid opening / closing mechanism.

  Further, in the culture observation apparatus according to the present invention, in the above invention, the lid opening / closing control means indicates an observation state by the microscope prior to opening the opening / closing lid by the lid opening / closing mechanism of any specified specimen container. The start timing of the opening operation is determined after confirmation.

  Further, in the culture observation apparatus according to the present invention, in the above invention, the lid opening / closing mechanism is capable of changing an opening amount of the opening / closing lid in accordance with an amount of movement of the sample tray exceeding a range of an observable locus, The lid opening / closing control means controls the amount of movement of the sample tray by the moving means exceeding the range of the observable trajectory so as to be the opening amount of the opening / closing lid set at the time of designating the chemical liquid operation.

  Further, in the culture observation apparatus according to the present invention, in the above invention, the lid opening / closing mechanism is capable of changing an opening / closing speed of the opening / closing lid in accordance with a moving speed of the sample tray exceeding a range of an observable locus, The lid opening / closing control means controls the moving speed of the sample tray by the moving means to exceed the range of the observable trajectory so as to be the opening / closing speed of the opening / closing lid set at the time of designating the chemical liquid operation. To do.

  The culture observation apparatus according to the present invention positions a specimen tray holding a plurality of specimen containers having hinge-type opening / closing lids at a chemical liquid manipulation position when a chemical liquid manipulation is performed on a desired specimen container. The moving means is moved beyond the range of the observable trajectory, and the lid is automatically opened and closed by the lid opening / closing mechanism linked to the movement of the sample tray. Therefore, the environment in the culture space can be set by automatically opening and closing the opening and closing lid of the desired specimen container with a simple configuration and control that simply switches the movement range of the specimen tray by the moving means used for so-called time-lapse observation. While suppressing the change as much as possible, there is an effect that a chemical solution operation can be performed on a desired specimen container in combination with the time lapse observation method.

  Hereinafter, the best mode for carrying out the culture observation apparatus according to the present invention will be described with reference to the drawings.

  FIG. 1 is a longitudinal front view showing an outline of an overall configuration example of a culture observation apparatus according to an embodiment of the present invention, and FIG. 2 is a schematic longitudinal side view showing an enlarged part of the culture apparatus. . The culture observation apparatus 100 according to the present embodiment is an apparatus for observing cultured cells while culturing them. In general, a culture apparatus (incubator) 200 for culturing cultured cells, and observing the cultured cells. The microscope 300 is configured in combination.

The culture apparatus 200 is configured by forming a culture space 202 in which a cultured cell is cultured by being sealed by a main housing 104 and a sub-housing 106 fixedly mounted on a base housing 102. Here, the front front side of the main casing 104 constituting the culture apparatus 200 is opened, and as shown in FIG. 2, there are two of the glass inner door 108 and the outer casing door 110 made of, for example, iron positioned outside thereof. It is closed by a heavy door structure so that it can be opened and closed. Further, the main housing 104 includes a heater 204 embedded so as to cover the culture space 202 and a humidifying pad 206 that is disposed in the culture space 202 and contains humidified water. Furthermore, the culture space 202 is connected to the CO ₂ cylinder 210 via a valve 208.

  In the culture apparatus 200 having such a configuration, when the culture observation apparatus 100 is used, the environment of the culture space 202 is controlled, for example, at a temperature of 37 ° C., a humidity of 95%, and a carbon dioxide concentration of 5 by controlling a heater 204 and a valve 208 by a controller described later. %, And the internal temperature of the sub housing 106 is maintained at 37 ° C.

  The culture observation apparatus 100 also has a specimen tray 400 accommodated in the culture space 202 and a horizontal movement that is attached to the sub-housing 106 and serves as a moving means for moving the specimen tray 400 two-dimensionally in a horizontal plane. Mechanism 500. As will be described in detail later, the specimen tray 400 holds a plurality of specimen containers 420 containing the cultured cells and culture medium, in this embodiment, eight specimen containers 420 at equal intervals on the same circumference. The horizontal moving mechanism 500 is a motor 502 for rotating the sample tray 400 and a linear motor that integrally supports the sample tray 400 and the motor 502 and linearly moves the sample tray 400 in the X direction, which is the horizontal direction in FIG. 504. Thereby, the horizontal movement mechanism 500 is configured as an Xθ stage that rotates or linearly moves the sample tray 400. Note that the sub-housing 106 is formed with a through hole 106a for enabling movement of the shaft of the motor 502 in the X direction.

  The microscope 300 is supported by the sub-case 106 and has an objective lens 302 whose optical axis is set in the vertical direction facing the sample tray 400, and a semi-focusing mechanism using a linear motor for moving the objective lens 302 up and down. 304, a variable magnification imaging lens 306 disposed on the optical axis, and a CCD camera 308 that captures an enlarged image of the cultured cells imaged by the variable magnification imaging lens 306. The variable magnification imaging lens 306 has an electric turret structure, and the magnification can be changed electrically. In addition, the microscope 300 includes an illumination optical system 312 including a red light emitting LED 310 for transmitting and illuminating cultured cells on the optical axis from above, and a blue light emitting device that selectively illuminates the cultured cells on the optical axis from below. And an illumination optical system 318 including an LED 314 and a green LED 316. The base casing 102 in which the CCD camera 308 and the like are built is maintained at a temperature of room temperature +5 to 10 ° C.

  Furthermore, the culture observation apparatus 100 of the present embodiment includes a controller 600 that controls the entire apparatus under a command from a personal computer 610 provided outside. The controller 600 controls, for example, the horizontal movement mechanism 500, the semi-focusing mechanism 304, the heater 204, the LEDs 310, 314, 316, and the like. Function.

  When observing the cultured cells, the cultured cells in the sample container 420 positioned at the observation position P1 on the optical axis of the objective lens 302 are observed by the microscope 300 as the sample tray 400 rotates. The specimen container 420 to be observed is switched by rotating the specimen tray 400 by a predetermined amount by the motor 502 of the horizontal movement mechanism 500. The observation location in the cultured cell is adjusted by moving the sample tray 400 in the X direction along a plane orthogonal to the optical axis of the objective lens 302 by the linear motor 504 of the horizontal movement mechanism 500. For example, if the cultured cells in the sample container 420 are 1 mm square, and the rotation trajectory of the sample container 420 is set to the cell center position, the sample container 420 positioned at the observation position P1 is used. If the linear motor 504 is linearly moved ± 0.5 mm in the X direction, the entire range of 1 mm square cultured cells can be observed.

  Next, the sample tray 400, the sample container 420, the lid opening / closing mechanism and the like will be described. 3 is an exploded perspective view showing a configuration example around the specimen tray 400, FIG. 4 is a perspective view showing a cover closed state, and FIG. 5 is a perspective view showing a cover open state. FIG. 3 shows only one specimen container 420 out of eight.

  First, a specimen container 420 for accommodating cultured cells and a medium is made of a petri dish-like plastic container having a diameter of 35 mm, for example, and an opening having a diameter of 10 mm is formed at the bottom thereof, and this opening is widely used in the microscope field. For example, it is closed with a cover glass having a thickness of 0.17 mm. The cultured cells and the medium are accommodated on this cover glass. The specimen container 420 includes a hinge-type opening / closing lid 422 that closes the upper opening. The opening / closing lid 422 includes an upper lid 426 and a lower lid 428 connected by a hinge pin 424. When the open / close lid 422 is closed, the upper surface of the lower lid 428 and the lower surface of the upper lid 286 are in close contact with each other, and the upper lid 426 is disposed so as to be rotatable about the hinge pin 424 with respect to the lower lid 428. . Here, the open / close lid 422 is installed so that the concave portion on the bottom surface side of the lower lid 428 to be fixed to the specimen tray 400 is placed on the specimen container 420. In addition, circular openings 426a and 428a are formed in the upper lid 426 and the lower lid 428, respectively. A transparent member 427 such as glass is fixed to the opening 426a of the upper lid 426 so as to close the opening 426a. With the opening / closing lid 422 closed, the cultured cells can be irradiated with transmitted illumination light during microscopic observation through the opening 426a (transparent member 427) and the opening 428a.

  The sample tray 400 includes a plurality of, for example, eight storage recesses 402 that hold the sample containers 420. These accommodating recesses 402 are formed in the same shape at equal intervals at positions on the outer circumference of the sample tray 400 so as to position and hold the eight sample containers 420 at equal intervals on the same circumference. An opening 403 for light transmission is formed at the center. In addition, the specimen tray 400 is disposed on both sides of the housing recess 402, and a spring-like member 404 such as a leaf spring that presses the bottom surface of the lower lid 428 to press the bottom surface of the specimen container 420 against the top surface of the housing recess 402. It has. The specimen container 420 and the lower lid 428 covered with the opening / closing lid 422 are installed in a state of being pressed by the spring member 404 in the accommodation recess 402 of the specimen tray 400, and the upper lid 426 is attached to the lower lid 428 around the hinge pin 424. On the other hand, it can be opened and closed.

  Here, each sample container 420 is positioned and held on the sample tray 400 so that the hinge pin 424 side is the inner peripheral side and the upper lid 426 is opened and closed toward the outer peripheral side. Therefore, the sample tray 400 includes a pin guide 406 on the inner peripheral side of each accommodating recess 402, and the outer peripheral surface 406a of the pin guide 406 on the sample container 420 side is used when the sample container 420 is placed on the sample tray 400. The abutment surface (positioning surface) of the opening / closing lid 422 is used. By abutting the opening / closing lid 422 against the outer peripheral surface 406a, it is possible to accurately set the distance in the radial direction from the center of the rotation axis with respect to the rotating sample tray 400. Further, in the pin guide 406, the two left and right surfaces 406 b that are orthogonal to the outer peripheral surface 406 a that is the abutting surface and parallel to the optical axis direction do not cause the open / close lid 422 to be greatly rattled in the rotation direction of the sample tray 400. The dimensional difference between the lower cover 428 and the notch 428b formed between the hinge pins 424 is about 0.5 mm. By configuring the pin guide 406 and the opening / closing lid 422 in such a relationship, the sample container 420 can be positioned and installed on the sample tray 400 easily and accurately.

  In addition, the culture observation apparatus 100 according to the present embodiment includes a lid opening / closing mechanism 440 for automatically opening and closing the upper lid 426 of the specimen container 420. The lid opening / closing mechanism 440 includes a pin 442 provided for each pin guide 406 corresponding to each upper lid 426, a protrusion 444 provided corresponding to each pin 442 on the back surface of the sample tray 400 in a predetermined positional relationship, It consists of a transmission member 446 having a link structure provided at one specific position on the surface of the sub-housing 106.

  First, the pin 442 has a cylindrical shape whose upper and lower ends are processed into a spherical shape, and is provided to be slidable in the vertical direction by a through hole 450 formed in the pin guide 406 in parallel with the optical axis. Note that both ends of the pin 442 are prevented from falling off by a retaining ring 448 so that the pin 442 does not fall or fall off due to its own weight. Such a pin 442 is disposed between the central axis of the hinge pin 424 and the specimen container 420 at a position where it contacts the base side receiving portion 426b on the bottom surface side of the upper lid 426 when the pin 442 is raised. The upper lid 426 is configured to be opened and closed by moving it up and down. That is, the pin 442 which is normally lowered by its own weight is raised and the upper end of the pin 442 is brought into contact with the base side receiving portion 426b of the upper lid 426. When the pin 442 is further raised, an external force is applied in the direction of lifting the base side receiving portion 426b of the upper lid 426, the upper lid 426 starts to rotate upward about the hinge pin 424, the upper lid 426 is opened, and the specimen container 420 is opened. The interior is open to the surrounding atmosphere. At this time, even if the contact position of the upper end of the pin 424 with the base side receiving portion 426b changes with the change of the rotation angle, it can be pushed up smoothly because it is formed in a spherical shape.

  Note that the push-up amount (lift amount) of the pin 442 is regulated so that the angle at which the upper lid 426 rotates and opens is less than 90 degrees, and the upper lid 426 is configured not to remain open. A stopper may be provided near the hinge pin 424 of the upper lid 426 to restrict the maximum opening angle. When the opening angle is less than 90 degrees, the weight of the opened upper lid 426 is always applied to the pin 424. When the pin 424 is lowered, the upper lid 426 is closed by its own weight while the base side receiving portion 426b is in contact with the upper end of the pin 424. Rotate in the direction.

  In the present embodiment, the position near the specimen container 420 on the front side closest to the inner door 108 is set as the chemical solution operation position P2, which is approximately 90 degrees different from the observation position P1 on the optical axis. This chemical solution operation position P2 is set outside the range of the trajectory that can be observed by the microscope 300. The transmission member 446 is provided at a specific position in the vicinity of such a chemical liquid operation position P2 and is rotatably provided around a rotation shaft 452 on a plane parallel to the surface of the sub-housing 106. Here, a convex portion 454 is formed at one end of the transmission member 446 formed in a substantially L shape in the direction of the rotation axis of the sample tray 400. The convex portion 454 has inclined surfaces 454a and 454b on both sides. Further, the cylindrical protrusion 444 on the back surface of the sample tray 400 can contact the inclined surface 454a of the convex portion 454 of the transmission member 446 when the sample tray 400 is positioned and rotated at the chemical solution operation position P2, and during normal observation. Even if the sample tray 400 rotates, it is provided at a specific position that does not interfere with the convex portion 454 of the transmission member 446. Further, on the upper surface side of the other end of the transmission member 446, a slope 456 that can contact the spherical lower end of the pin 442 is formed along an arc centering on the rotation shaft 452. The pin 442 supported by the pin guide 406 can contact the inclined surface 456 when the sample tray 400 is positioned at the chemical solution operation position P2 and the transmission member 446 is rotated by the projection 444, and the sample tray 400 rotates during normal observation. Even so, the position is set so as not to interfere with the slope 456.

  When sequentially observing a plurality of sample containers 420 on the sample tray 400 including time-lapse observation described later, the protrusions 444 and the pins 442 may move on the maximum observable locus. That is, in the case of the present embodiment, referring to FIG. 3, the sample tray 400 includes a circumferential trajectory that rotates about the rotation axis P0, and the rotation axis deviates ± 5 mm in the X direction from P0. The range of the circumferential trajectory rotating around the position is within the range of the maximum observable trajectory, and the above-described chemical solution operation position P2 is set to the front front position outside the range of such observable trajectory. ing. Specifically, the sample tray 400 is set to a position where the sample tray 400 is linearly moved, for example, 7 mm in the X direction by the linear motor 504 in a state where the desired sample container 420 is positioned at the front side front position. The rotation axis is set to the position P0 ′.

  That is, when the sample tray 400 rotates around the rotational axis P0 and a position shifted by ± 5 mm in the X direction, the positional relationship is as shown in FIG. 3B, and the protrusion 444 is the convex portion 454 of the transmission member 446. 3 and the pin 442 does not interfere with the inclined surface 456. However, when the rotation axis of the sample tray 400 is shifted to the position of P0 ′ for the liquid chemical operation, the protrusions as shown in FIG. The position 444 and the pin 442 also have a positional relationship shifted by d = 7 mm in the X direction from the normal position. The protrusion 444 interferes with the convex portion 454 of the transmission member 446, and the pin 442 interferes with the inclined surface 456. .

  When the sample tray 400 is further linearly moved by the linear motor 504 at the interfering position as shown in FIG. 3B, the protrusion 444 contacts one inclined surface 454a of the transmission member 446, and the sample tray 400 is further linearly moved. Then, the projection 444 pushes the inclined surface 454a, and a force that rotates the transmission member 446 around the rotation shaft 452 in the counterclockwise direction works. At this time, the inclined surface 456 formed at the other end starts to contact the lower end of the pin 442 as the transmission member 446 rotates, and the inclined surface 456 is moved to the pin 442 by the further rotation of the transmission member 446 accompanying the further linear movement of the sample tray 400. The pin 442 is guided by the pin guide 406 and rises. Thereby, as described above, the upper lid 426 is automatically opened. When the sample tray 400 further moves linearly and the protrusion 444 is in contact with the convex portion 454, the transmission member 446 is at the maximum rotated position, and the interlocking inclined surface 456 pushes the pin 442 to the upper end point. When the sample tray 400 further moves linearly, the protrusion 444 starts to contact the other inclined surface 454b, the transmission member 446 rotates in a direction in which the inclined surface 456 is separated from the pin 442, and the pin 442 starts to descend. When the sample tray 400 moves linearly to a position where the protrusion 444 moves away from the inclined surface 454b, the pin 442 is lowered to the lowest point and the upper lid 426 is closed. Note that the sample tray 400 may be linearly moved in the opposite direction, and the pins 442 may be lowered using the inclined surface 454a.

  As described above, the lid opening / closing mechanism 440 according to the present embodiment is positioned horizontally for positioning an arbitrary specimen container 420 designated for a chemical operation at a chemical operation position P2 set outside the range of the trajectory that can be observed by the microscope 300. The upper lid 426 of the target specimen container 420 is automatically opened and closed in conjunction with the movement of the specimen tray 400 beyond the observable trajectory range by the moving mechanism 500.

  Here, the lid opening / closing mechanism 440 sequentially pushes up the pins 442 by the inclined surfaces 456 to sequentially open the upper lid 426, and the push-up amount of the pins 442, that is, the opening amount of the upper lid 426 depends on the rotation amount of the transmission member 446. Therefore, the opening amount of the upper lid 426 is configured to be variable according to the moving amount of the sample tray 400 exceeding the range of the observable locus. Further, the lid opening / closing mechanism 440 is configured so that the opening / closing speed of the upper lid 426 can be varied according to the moving speed of the sample tray 400 exceeding the range of the observable trajectory.

  Incidentally, in correspondence with such a chemical solution operation position P2, the inner door 108 has an obliquely upper position where the sample container 420 positioned at the chemical solution operation position P2 is easily operated as shown in FIG. An operation opening 108a of a size that allows the pipette 120 and the like to be inserted is formed, and is normally closed by a silicon stopper 112.

  In such a configuration, a case where a desired sample container 420 is designated and a chemical liquid operation is performed will be described. In the present invention, “chemical solution operation” is a summary of operations such as chemical solution administration, solution stirring, and medium exchange as described above. For example, drug solution administration refers to an operation of administering a desired drug solution (reagent) to cultured cells in the specimen container 420 using, for example, the pipette 120. Stirring refers to an operation of stirring the solution in the sample container 420 by lightly sucking or discharging the solution in the sample container 420 using, for example, a pipette 120 and is called pipetting. In the case of pipetting, depending on the amount of the solution in the specimen container 420, the position of the cultured cells, the type and amount of the reagent to be supplied, and the viscosity, the operator handles the pipetting method according to the pipette 120 owned by the operator. The technique is controlled to the optimum state. That is, the pipetting operation such as the position and angle of the tip of the pipette 120, the discharge amount and the speed is finely adjusted. The medium exchange refers to an operation in which a deteriorated medium is sucked using, for example, the pipette 120 and then a new medium is administered. Further, the chemical operation itself may be manual or automatic.

  In any case, as shown in FIG. 2, the case door 110 is opened, the stopper 112 is removed, the pipette 120 is inserted from the operation opening 108a, and the operation is performed through the glass inner door 108. Since it is possible to visually confirm that the upper lid 426 has been opened and look at the inside, it becomes easy to operate. Further, the position at which the upper lid 426 is opened is the chemical solution operation position P2 closest to the operation opening 108a, and the operability is good, and the specimen container 420 is not moved while the upper lid 426 is opened. And the possibility of moisture evaporation can be greatly reduced.

  Next, a chemical solution operation during time-lapse observation generally used in long-term culture will be described. Here, in the culture observation apparatus 100, for example, as shown in Patent Document 1 and the like, the origin setting process of the sample tray 400 is completed according to the observation preparation program for setting the observation conditions in the personal computer 610, and the origin position is set as a reference. Each specimen container 420 on the specimen tray 400 can be designated by a container number, for example. In setting the observation conditions prior to time-lapse observation, the selection of the LED to be used, the brightness, the magnification, the imaging conditions of the microscope 300 such as the exposure time of the CCD camera 308, the time schedule including the time-lapse interval time (standby period), The experiment period and the like are set in advance by the personal computer 610 and stored in the database of the personal computer 610. The time lapse observation in this case includes a multi-point time lapse in which observation of a plurality of positions of cultured cells in the same specimen container 420 is sequentially performed. Thereby, the microscope 300 according to the present embodiment performs time-lapse observation in which the cultured cells in the sample container 420 sequentially positioned at the observation position P1 on the optical axis are intermittently observed with the set interval time interposed therebetween. Is set to

  FIG. 6 is a schematic flowchart showing an example of interrupt control of a chemical operation performed by the controller 600 based on a command from the personal computer 610 during execution of the time lapse capture mode by the time lapse observation method. First, based on the set condition based on the start instruction of the time lapse capture mode from the personal computer 610 (step S100). During such an operation, it is determined whether or not there is an instruction for a chemical operation through the personal computer 610 (step S101). If there is no instruction | indication of chemical | medical solution operation (step S101: No), execution of time lapse capture mode is continued (step S100).

  In the personal computer 610, an instruction to operate the chemical solution can be given at any time desired by the operator. When instructing this chemical liquid operation, a desired specimen container 420 to be subjected to the chemical liquid operation is designated by inputting the container number. Further, when instructing the chemical operation, the opening amount and opening / closing speed of the upper lid 426 of the opening / closing lid 422 can be arbitrarily specified within an allowable range. Unless otherwise specified, the opening amount and the opening / closing speed are default values set in advance. Further, when instructing the chemical operation, the chemical liquid to be operated, the type of operation, and the like are also input.

  If there is an instruction to operate the chemical solution through the personal computer 610 (step S101: Yes), it is determined whether or not time-lapse capturing by the microscope 300 is being executed (step S102). This determination is made with reference to a time-lapse time schedule stored in the personal computer 610. If not being executed (step S102: No), the process proceeds to step S105. If it is being executed (step S102: Yes), it is determined whether or not the current position being observed has been captured (step S103). That is, although only one point of observation may be used, in multi-point time lapse observation, regarding the cultured cells in the same sample container 420, determination is made regarding the position under observation when observation at a plurality of positions is designated. If the capture has not been completed (step S103: No), the process waits until the completion. When the capture of the current position is completed (step S103: Yes), the subsequent time lapse capture process is temporarily interrupted (pause) (step S104). That is, during execution of the time lapse capture mode, an interruption for the chemical operation is performed.

  In time-lapse observation, even if the observation is taken at the same position, for example, the objective lens 420 is changed in focus position, the magnification is changed, the illuminating light is applied to the cultured cells, and the like. Therefore, this process takes time. Therefore, in the present embodiment, the timing (for example, the timing of cell division) that the operator wants to administer the drug solution regarding the drug solution operation even if the capture of the remaining imaging positions in the multipoint time-lapse observation has not been completed. In order to give priority, such interrupt processing is performed. Further, the reason for waiting until the current position being observed is completed is to prevent the observation conditions at this position from being changed midway.

  In the interruption process for the chemical operation, first, the horizontal movement mechanism 500 is driven and controlled, and the designated sample container 420 is moved to the chemical operation position P2 (step S105). That is, the sample tray 420 is rotated by the motor 502 around the rotation axis P0 to move the designated sample container 420 to the vicinity of the chemical solution operation position P2, and further, the linear motor 504 moves the sample tray 400 in the X direction to the dimension d ( 7 mm), and the designated specimen container 420 is positioned at the chemical solution operation position P2. In this state, the projection 444 corresponding to the designated sample container 420 becomes a pin driving start position where the projection 444 contacts the inclined surface 454a of the transmission member 446.

  Therefore, the sample tray 400 is further linearly moved by a predetermined amount by the linear motor 504 around the rotation axis P0 ′, whereby the transmission member 446 is actuated by the projection 444, the pin 442 is raised by the slope 456, and the upper lid 426 is opened ( Step S106). In this case, the linear moving amount and moving speed of the sample tray 400 by the motor 502 are controlled so as to be the opening amount and opening / closing speed specified at the time of instructing the chemical liquid operation. When the opening amount and opening / closing speed are not designated, the linear movement amount and movement speed of the sample tray 400 are controlled to be default values.

  As a result, as shown in FIG. 2, the chemical liquid operation can be performed on the designated sample container 420 located at the chemical liquid operation position P2. Therefore, the operator performs necessary chemical operations such as chemical administration, solution agitation, and medium exchange using the pipette 120 with respect to the designated specimen container 420 through the operation opening 108a of the inner door 108. The pipette 120 may be loaded in the operation opening 108a so that the chemical operation is automatically performed.

  When it is notified through the personal computer 610 that the chemical liquid operation for the designated sample container 420 has been completed (step S107: Yes), it is determined whether or not the chemical liquid operation information is stored in the database on the personal computer 610 (step S108). . This determination is made based on an operator's instruction through the personal computer 610. When there is an instruction to store (step S108: Yes), chemical operation information such as operation time, specimen container number, chemical type, type of operation, etc. is transmitted to the personal computer 610 side and stored in the database ( Step S109). By storing such chemical solution operation information in the database together with the time-lapse image captured by time-lapse observation, the status (operation time, experimental conditions, etc.) of the chemical solution operation is grasped when reviewing the time-lapse image on the personal computer 610. It becomes possible to do.

  Subsequently, the horizontal movement mechanism 500 is driven to move the sample tray 400 in the reverse direction, and the pin 442 is lowered to close the upper cover 426 of the designated sample container 420 with its own weight (step S110). That is, the sample tray 400 is linearly moved in the reverse direction by the amount of linear movement of the sample tray 400 in the X direction by the step motor 502, and the sample tray 400 is returned to the position of the rotation axis P0. In this case, the return movement speed of the sample tray 400 by the motor 502 is controlled to be the opening / closing speed designated at the time of instructing the chemical liquid operation. When the opening / closing speed is not designated, the return moving speed of the sample tray 400 is controlled to be a default value.

  Then, it is determined whether or not there is another sample container 420 for which the chemical liquid operation is designated (step S111), and if there is (step S111: Yes), the interruption process after step S105 is continued. If there is no other designated specimen container 420 (step S111: No), the time-lapse capture that has been temporarily suspended is resumed (step S112). The resumed time lapse capture is continued until the processing of the time lapse capture mode is completed (step S113).

  As described above, according to the culture observation apparatus 100 of the present embodiment, the specimen tray 400 holding the plurality of specimen containers 420 including the hinged opening / closing lid 422 is designated when performing a chemical operation on the desired specimen container 420. The desired sample container 420 is moved beyond the observable trajectory range by the horizontal movement mechanism 500 so that the desired sample container 420 is positioned at the chemical solution operation position P2, and the lid opening / closing mechanism 440 linked to the movement of the sample tray 400 is opened and closed. It is only necessary to automatically open and close the upper lid 426 of the 422 and perform the chemical liquid operation from the operation opening 108a at the chemical liquid operation position P2, so that only the movement range of the sample tray 400 by the horizontal movement mechanism 500 used for time-lapse observation is switched. It is cultivated by automatically opening and closing the upper lid 426 of the desired specimen container 420 with a simple configuration and control. While minimizing the environmental change in the space 202 can be in combination with the time lapse observation method performing chemical operations for the desired sample container 420.

  Further, according to the present embodiment, it is possible to specify the opening amount and opening / closing speed of the upper lid 426 at the time of instructing the chemical liquid operation, so that it is possible to cope with the operation content, the operator's request, and the like. For example, in the case where the chemical liquid operation is only to administer the chemical liquid, by specifying a small opening amount of the upper lid 426, the chemical liquid administration can be performed without hindrance, and germs in the sample container 420 with the upper lid 426 opened can be obtained. As a result, the environmental change in the sample container 420 can be reduced. On the other hand, if the chemical operation is pipetting to stir the solution or changing the medium, the operability is improved by increasing the open space when the upper lid 426 is opened by specifying a larger opening amount of the upper lid 426. Can be made. Such designation of the open amount can be made different depending on the timing even when the chemical solution operation is instructed to the same specimen container 420. This is because even when a chemical liquid operation is performed on the same specimen container 420, for example, the first time is a simple chemical liquid administration, but the medium may be changed after a long period of time. In addition, when it is not desired to give vibration to the culture medium in the specimen container 420 when the upper lid 426 is opened and closed, the upper lid 426 can be opened and closed with little impact by specifying a slower opening and closing speed of the upper lid 426. On the other hand, when it is desired to suppress the environmental change in the specimen container 420, it can be dealt with by opening and closing the upper lid 426 in a short time and shortening the opening time by specifying a higher opening / closing speed of the upper lid 426.

  The present invention is not limited to the above-described embodiment, and various modifications can be made without departing from the spirit of the present invention. For example, the lid opening / closing mechanism is not limited to the lid opening / closing mechanism 440 using the transmission member 446 of the illustrated example, but may be a lid opening / closing mechanism 460 as shown in FIG. The lid opening / closing mechanism 460 has pins 442, plate spring members 462 attached to the back surface of the sample tray 400 corresponding to the pins 442, and a specific position near the chemical operation position P 2 on the surface of the sub-housing 106. It is composed of one protruding member 464 provided. The leaf spring member 462 is disposed so that its longitudinal direction coincides with the rotational circumferential direction of the sample tray 400 that rotates and moves, and one end is fixed to the back side of the sample tray 400 and the other end is the lower end of the pin 442. In a normal state, the pin 442 is provided with a slope 462a so as to descend by its own weight. The protruding member 464 does not interfere with the leaf spring member 462 when moved within the range of the observable trajectory of the sample tray 400 during normal observation, but the chemical solution operation position P2 by rotating and moving the designated sample container 420 linearly. As shown in FIG. 7A, it is set at a position where it can interfere with the fixed side of the leaf spring member 426. And after positioning the designated sample container 420 in the chemical | medical solution operation position P2, the protrusion-like member 464 pushes up the slope 422a of the leaf | plate spring member 462 sequentially by moving the sample tray 400 linearly further in the X direction, As shown in FIG. 7B, the pin 442 is pushed up to open the upper lid 426.

  Further, in this embodiment, even in the case of multipoint time lapse observation, when the time lapse image capture at the current position under observation is completed, the opening operation is performed so as to start the interrupt process for the chemical operation. Although the start timing is determined, depending on the priority of the time lapse observation and the liquid chemical operation, the time lapse images are taken in all the observation positions set for the cultured cells in the sample container 420 positioned at the observation position P1. After completing the above, the timing of the opening operation may be determined so as to start the interruption process for the chemical liquid operation. Furthermore, when a chemical operation instruction is given, the time lapse image is not being captured, but the time lapse image capture is started immediately after referring to a preset time schedule for time lapse observation. If priority is given to time-lapse observation, the timing of the opening operation may be determined so that interruption processing for chemical operation is started after the capture of the time-lapse image is completed.

  Further, the moving means is not limited to the horizontal moving mechanism 500 that constitutes the Xθ stage, and may be a horizontal moving mechanism that constitutes an XY stage, for example.

It is a vertical front view which shows the outline of the whole structural example of the culture observation apparatus concerning embodiment of this invention. It is a schematic longitudinal side view which expands and shows a part of culture apparatus. It is a disassembled perspective view which shows the example of a structure around a sample tray. It is a perspective view which shows a cover obstruction | occlusion state. It is a perspective view which shows a cover open state. It is a schematic flowchart which shows the interruption control example of the chemical | medical solution operation in execution of the time lapse taking-in mode. It is a schematic perspective view which shows the modification of a lid | cover opening / closing mechanism.

Explanation of symbols

DESCRIPTION OF SYMBOLS 100 Culture observation apparatus 108a Operation opening 200 Culture apparatus 202 Culture chamber 300 Microscope 400 Specimen tray 420 Sample container 422 Opening / closing lid 424 Hinge pin 440 Lid opening / closing mechanism 460 Lid opening / closing mechanism 500 Horizontal movement mechanism 600 Controller

Claims (4)

A culture observation apparatus for observing cultured cells while culturing them,
A culture device in which the environment is controlled to form a culture space for culturing cultured cells, and an operation opening is formed in part;
A specimen tray that is accommodated in the culture space, contains cultured cells and culture medium, and positions and holds a plurality of specimen containers closed by a hinge-type opening and closing lid;
A microscope that intermittently observes the cultured cells in the specimen container that are sequentially positioned at the observation position on the optical axis across the waiting period;
Moving means for moving the sample tray two-dimensionally along a plane perpendicular to the optical axis of the microscope;
The moving means that exceeds the range of the observable trajectory for positioning any sample container designated for the chemical liquid operation at the chemical liquid operation position that is set outside the range of the observable trajectory by the microscope and can be operated from the operation opening. A lid opening and closing mechanism for opening and closing the opening and closing lid of the specimen container in conjunction with the movement of the specimen tray by
A lid opening / closing control means for controlling the moving means to operate the lid opening / closing mechanism when a chemical operation is instructed with the designation of the sample container;
A culture observation apparatus comprising:   The lid opening / closing control means determines the start timing of the opening operation by confirming the observation state by the microscope prior to opening the opening / closing lid by the lid opening / closing mechanism of any designated specimen container. The culture observation apparatus according to claim 1. The lid opening / closing mechanism is capable of varying the opening / closing amount of the opening / closing lid in accordance with the amount of movement of the sample tray that exceeds the range of the observable trajectory.
The lid opening / closing control means controls the amount of movement of the sample tray by the moving means that exceeds the range of the observable trajectory so as to be the opening amount of the opening / closing lid set at the time of designating the chemical liquid operation. The culture observation apparatus according to claim 1 or 2. The lid opening / closing mechanism is capable of varying the opening / closing speed of the opening / closing lid according to the speed of movement of the sample tray exceeding the range of the observable locus,
The lid opening / closing control means controls the moving speed of the sample tray by the moving means exceeding the range of the observable trajectory so as to be the opening / closing speed of the opening / closing lid set at the time of designating the chemical liquid operation. The culture observation apparatus according to any one of claims 1 to 3.

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