Patent Publication Number: US-2020278523-A1

Title: Observation apparatus, observation method, and observation program

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
     This application is a Continuation of PCT International Application No. PCT/JP2018/041976 filed on Nov. 13, 2018, which claims priority under 35 U.S.C § 119(a) to Japanese Patent Application No. 2017-240539 filed on Dec. 15, 2017. Each of the above application(s) is hereby expressly incorporated by reference, in its entirety, into the present application. 
    
    
     BACKGROUND OF THE INVENTION 
     1. Field of the Invention 
     The technology of the disclosure relates to an observation apparatus, an observation method, and an observation program for observing an observation target by relatively moving at least one of a container accommodating the observation target or an imaging unit with respect to the other. 
     2. Description of the Related Art 
     In recent years, various technologies for imaging an observation target such as various cells and analyzing an acquired image have been suggested. For example, a method of imaging a pluripotent stem cell such as an embryonic stem (ES) cell and an induced pluripotent stem (iPS) cell, a differentiation-induced cell, or the like using a microscope and determining a differentiation state or the like of the cell by recognizing a feature of the image has been suggested. For example, JP2016-149998A discloses an identification apparatus that can suitably specify an observation target even in a case where an imaging condition or the like changes. In addition, JP2016-099592A discloses a microscope that adjusts refraction of illumination light caused by a liquid surface shape of a solution for culturing a cell. In addition, WO2008/146474A discloses an observation apparatus that can recognize an amount of a culture medium for culturing a cell. 
     The pluripotent stem cell such as the ES cell and the iPS cell has a capability to differentiate into cells of various tissues and has drawn attention for its applicability in regenerative medicine, development of medication, identification of diseases, and the like. 
     In a case where the cell is imaged as described above, it is known that the cell as the observation target is scanned and measured by the microscope and is determined from an obtained image. In order to implement industrialization of regenerative medicine or multilevel experiment in drug discovery research, it is important to perform imaging at a high speed and determine quality at a high speed. 
     In a case where a cell that is cultured using a cultivation container such as a well plate, a Petri dish, and a flask is observed, coordinates at which scanning and measurement are performed are decided in accordance with a shape and a dimension of the cultivation container. The microscope is moved along a scanning trajectory that passes through the decided coordinates. 
     SUMMARY OF THE INVENTION 
     In a case where imaging is performed along the decided scanning trajectory, an unintended image may be captured. For example, in a case where the cultivation container is not correctly placed on the placing stand, the scanning trajectory with respect to the cultivation container is shifted in imaging using the coordinates which are decided by regarding the cultivation container as being at a correct position, and the accommodated cell cannot be appropriately imaged. 
     The technology of the disclosure is conceived in view of the above point, and an object thereof is to provide an observation apparatus, an observation method, and an observation program capable of capturing an image close to an appropriate imaging position even in a case where a scanning trajectory is shifted. 
     An observation apparatus according to the technology of the disclosure comprises an imaging unit that images an observation target accommodated in a container, a movement unit that scans the observation target using an observation region of the imaging unit by relatively moving at least one of the container or the imaging unit with respect to the other in accordance with a predetermined scheduled trajectory, a measurement unit that acquires measured shape information of the container by measuring a shape of the container along a scanning trajectory of the observation target using the observation region, a storage unit that stores reference shape information acquired by measuring the shape of the container using the measurement unit along a reference trajectory which is a reference in a case where the shape of the container is measured in accordance with the scheduled trajectory, a calculation unit that calculates a shift of the scanning trajectory with respect to the reference trajectory based on the reference shape information and the measured shape information, and a control unit that corrects the scheduled trajectory based on the shift and scans the observation target using the observation region by controlling the movement unit based on the corrected scheduled trajectory. 
     The observation region is a region that can be observed by the imaging unit in a range of a field of view. The scheduled trajectory is a predetermined trajectory and represents a trajectory on which at least one of the container or the imaging unit is scheduled to be relatively moved with respect to the other. The scanning trajectory is a trajectory on which the observation region is actually relatively moved with respect to the container in a case where the observation target is scanned using the observation region by relatively moving at least one of the container or the imaging unit with respect to the other in accordance with the scheduled trajectory. The reference trajectory is a trajectory on which the observation region is relatively moved with respect to the container in a case where the observation target is scanned in the container as assumed using the observation region by relatively moving at least one of the container or the imaging unit with respect to the other in accordance with the scheduled trajectory. For example, the container as assumed is a container in a state where the container is appropriately placed at a position for measurement without inclination. 
     In the observation apparatus, an accommodation part that accommodates the observation target is formed in the container, and the measurement unit acquires the measured shape information by measuring a shape of a bottom surface of the accommodation part along the scanning trajectory. 
     In the observation apparatus, the calculation unit may calculate an inclination of the scanning trajectory with respect to the reference trajectory as the shift based on the reference shape information and the measured shape information. 
     In the observation apparatus, the control unit may determine a direction in which the scanning trajectory is inclined with respect to a proceeding direction of the reference trajectory based on an image in which the observation region is imaged by the imaging unit, and correct the scheduled trajectory based on the direction in which the scanning trajectory is inclined and the shift calculated by the calculation unit. 
     An observation method according to the technology of the disclosure comprises an imaging step of imaging an observation target accommodated in a container using an observation region, a measurement step of acquiring measured shape information of the container by measuring a shape of the container along a scanning trajectory of the observation target using the observation region in the imaging step in accordance with a predetermined scheduled trajectory, a storage step of storing reference shape information acquired by measuring the shape of the container in the measurement step along a reference trajectory which is a reference in a case where the shape of the container is measured in accordance with the scheduled trajectory, a calculation step of calculating a shift of the scanning trajectory with respect to the reference trajectory based on the reference shape information and the measured shape information, and a control step of correcting the scheduled trajectory based on the shift and performing imaging close to the reference trajectory in the imaging step based on the corrected scheduled trajectory. 
     An observation program according to the technology of the disclosure causes a computer to execute an imaging step of imaging an observation target accommodated in a container using an observation region, a measurement step of acquiring measured shape information of the container by measuring a shape of the container along a scanning trajectory of the observation target using the observation region in the imaging step in accordance with a predetermined scheduled trajectory, a storage step of storing reference shape information acquired by measuring the shape of the container in the measurement step along a reference trajectory which is a reference in a case where the shape of the container is measured in accordance with the scheduled trajectory, a calculation step of calculating a shift of the scanning trajectory with respect to the reference trajectory based on the reference shape information and the measured shape information, and a control step of correcting the scheduled trajectory based on the shift and performing imaging close to the reference trajectory in the imaging step based on the corrected scheduled trajectory. 
     Another observation apparatus according to the technology of the disclosure comprises a memory that stores an instruction to be executed by a computer, and a processor configured to execute the stored instruction. The processor executes an imaging step of imaging an observation target accommodated in a container using an observation region, a measurement step of acquiring measured shape information of the container by measuring a shape of the container along a scanning trajectory of the observation target using the observation region in the imaging step in accordance with a predetermined scheduled trajectory, a storage step of storing reference shape information acquired by measuring the shape of the container in the measurement step along a reference trajectory which is a reference in a case where the shape of the container is measured in accordance with the scheduled trajectory, a calculation step of calculating a shift of the scanning trajectory with respect to the reference trajectory based on the reference shape information and the measured shape information, and a control step of correcting the scheduled trajectory based on the shift and performing imaging close to the reference trajectory in the imaging step based on the corrected scheduled trajectory. 
     According to the technology of the disclosure, the shift of the scanning trajectory with respect to the reference trajectory is calculated from the reference shape information indicating the shape of the container acquired along the reference trajectory in accordance with the scheduled trajectory and the measured shape information obtained by actually measuring the container along the scanning trajectory, and the scheduled trajectory is corrected. Imaging is performed close to the reference trajectory controlling the movement unit and the imaging unit based on the corrected scheduled trajectory. Accordingly, even in a case where the scanning trajectory is shifted from the reference trajectory, the image can be appropriately captured using the observation region of the imaging unit by correcting the scheduled trajectory. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         FIG. 1  is a diagram illustrating a schematic configuration of an observation apparatus according to an embodiment of the technology of the disclosure. 
         FIG. 2  is a diagram illustrating one example of a placing stand. 
         FIG. 3  is a block diagram illustrating a configuration of a control unit according to the embodiment of the technology of the disclosure. 
         FIG. 4  is a diagram illustrating a scanning trajectory by a solid line M in a cultivation container. 
         FIG. 5  is a diagram illustrating a positional relationship among a first displacement sensor, a second displacement sensor, and the cultivation container in a case where an observation region is present at any position in the cultivation container. 
         FIG. 6  is a diagram illustrating a positional relationship among the first displacement sensor, the second displacement sensor, and the cultivation container in a case where the observation region is present at any position in the cultivation container. 
         FIG. 7  is a diagram illustrating an example of a two-dimensional shape of a container detected in an autofocus detection unit on a forward path. 
         FIG. 8  is a diagram illustrating an example of inclination of the scanning trajectory with respect to the cultivation container. 
         FIG. 9  is a flowchart illustrating a flow of observation method executed by the observation apparatus. 
         FIG. 10  is a diagram illustrating a shift of the scanning trajectory with respect to a reference trajectory. 
         FIG. 11  is a partial enlarged view of  FIG. 10 . 
     
    
    
     DESCRIPTION OF THE PREFERRED EMBODIMENTS 
     Hereinafter, one example of an embodiment according to the technology of the disclosure will be described with reference to the drawings. The same or equivalent constituents and parts in each drawing will be designated by the same reference signs. Dimensional ratios in the drawings are exaggerated for convenience of description and may be different from the actual ratios. 
       FIG. 1  is a diagram illustrating a schematic configuration of an observation apparatus according to the embodiment of the technology of the disclosure.  FIG. 2  is a diagram illustrating one example of a placing stand. 
     The observation apparatus is an apparatus for observing an observation target accommodated in a cultivation container  20  placed on a placing stand  10  by a microscope device  30 . The placing stand  10  and the microscope device  30  are controlled by a control unit  40 . Each configuration will be described in order. 
     The placing stand  10  is a stage on which the cultivation container  20  can be placed. As illustrated in  FIG. 2 , a rectangular opening  11  is formed at the center of the placing stand  10 . It is configured that the cultivation container  20  is installed on a member forming the opening  11 , and light for observation by the microscope device  30  passes through the cultivation container  20 . 
     A movement unit  12  is attached to the placing stand  10 . The movement unit  12  can freely move the placing stand  10  in an X direction and a Y direction that are orthogonal to each other. The X direction and the Y direction are directions orthogonal to a Z direction and are directions orthogonal to each other in a horizontal plane. In the present embodiment, the X direction is set as a main scanning direction, and the Y direction is set as a sub-scanning direction. The movement unit  12  is configured with an actuator that includes a piezoelectric element or the like. Movement of the placing stand  10  in an X-Y plane is controlled by the control unit  40 . By moving the placing stand  10  in the X-Y plane, the cultivation container  20  on the placing stand  10  moves with respect to the microscope device  30 . 
     In the present embodiment, an example in which a position at which the observation target is observed by the microscope device  30  is changed by moving the placing stand  10  with respect to the microscope device  30  is illustrated. However, the example is not for limitation purposes. The microscope device  30  may be moved with respect to the placing stand  10 , or both of the placing stand  10  and the microscope device  30  may be moved. Any aspect can be employed as long as at least one of the cultivation container  20  placed on the placing stand  10  or the microscope device  30  is relatively moved with respect to the other. In the present disclosure, for example, the “microscope device  30  is represented as relatively moving with respect to the cultivation container  20 ” even in a case where a position of the microscope device  30  is fixed and only the cultivation container  20  is moving. In addition, in the present disclosure, a trajectory accompanied by the relative movement is represented as a “scanning trajectory” even in a case where any of the microscope device  30  and the cultivation container  20  is actually moving. 
     Instead of placing the cultivation container  20  on the placing stand  10  and moving the cultivation container  20 , the cultivation container  20  may be moved in the X-Y plane using a holding unit that holds at least a part of the cultivation container  20  by moving the holding unit. 
     In the cultivation container  20 , a plurality of accommodation parts  22  are formed in a plate  21  having a flat plate shape. For example, a Petri dish, a dish, or a well plate can be used as the cultivation container  20 . For example, the accommodation part  22  is a recessed portion having a circular shape in a plan view and is referred to as a well. The accommodation part  22  accommodates the observation target such as various cells immersed in a cultivation liquid. Cells accommodated in the accommodation part  22  include pluripotent stem cells such as an iPS cell and an ES cell, cells of a nerve, skin, cardiac muscle, and a liver that are differentiation-induced from a stem cell, cells of skin, a retina, cardiac muscle, a blood cell, a nerve, and an organ extracted from a human body, and the like. 
     The microscope device  30  captures a phase difference image of the observation target. In order to obtain a high magnification image, the microscope device  30  captures partial images of the observation target and the cultivation container  20  using an observation region smaller than each accommodation part  22  of the cultivation container  20 . As described above, by moving the cultivation container  20  with respect to the microscope device  30 , the microscope device  30  scans the cultivation container  20 , and a series of partial images is obtained. 
     The microscope device  30  comprises a light source  31 , a slit  32 , a condenser lens  33 , an objective lens  34 , a focus adjustment mechanism  35 , an image forming lens  36 , an imaging unit  37 , and a measurement unit  38 . 
     The light source  31  emits white light. The slit  32  is formed by disposing a ring shaped slit through which the white light is transmitted in a light screen that blocks the white light emitted from the light source  31 . Illumination light L having a ring shape is formed by causing the white light to pass through the slit. The condenser lens  33  condenses the illumination light L having the ring shape on the observation target. 
     The objective lens  34  is arranged to face the condenser lens  33  through the cultivation container  20 . The objective lens  34  forms an image of the observation target in the cultivation container  20 . The focus adjustment mechanism  35  includes a phase difference lens that can be moved in an optical axis direction (Z direction). By moving the phase difference lens in the optical axis direction, autofocus control is performed, and contrast of the phase difference image captured by the imaging unit  37  is adjusted. For example, the movement of the phase difference lens in the optical axis direction can be implemented by driving an actuator such as a piezoelectric element based on a signal from the control unit  40 . However, the piezoelectric element is not for limitation purposes, and the phase difference lens can be driven using other known configurations as long as the phase difference lens can be moved in the Z direction. In addition, a magnification of the phase difference lens may be configured to be changeable. Specifically, a phase difference lens or the focus adjustment mechanism  35  having a different magnification may be configured to be replaceable. The replacement may be automatically performed or may be manually performed by a user. 
     The phase difference image that passes through the focus adjustment mechanism  35  is incident on the image forming lens  36 , and the image forming lens  36  forms the phase difference image on the imaging unit  37 . 
     The imaging unit  37  captures the phase difference image formed by the image forming lens  36 . For example, the imaging unit  37  is an imaging element such as a charge-coupled device (CCD) image sensor or a complementary metal-oxide semiconductor (CMOS) image sensor. As the imaging element, an imaging element in which color filters of red, green, blue (RGB) are disposed may be used, or a monochrome imaging element may be used. The imaging unit  37  images a region observable in the range of the field of view as the observation region. 
     Hereinafter, the objective lens  34 , the focus adjustment mechanism  35 , the image forming lens  36 , and the imaging unit  37  will be collectively referred to as an image forming optical system C. 
     The measurement unit  38  is fixedly attached with respect to the imaging unit  37  and detects a Z-directional position of the cultivation container  20  installed on the placing stand  10 . Furthermore, the measurement unit  38  measures the shape of the cultivation container  20  by scanning the cultivation container  20  using the imaging unit  37  and the measurement unit  38  by relatively moving at least one of the cultivation container  20  or the imaging unit  37  with respect to the other and continuously detecting the Z-directional position of the cultivation container  20 . In the present embodiment, the measurement unit  38  projects measurement light (laser light) toward the cultivation container  20  and detects reflection caused by a difference in refractive index between the cultivation container  20  and the cultivation liquid accommodated in the accommodation part  22 . Thus, the measurement unit  38  measures a shape of a bottom surface of the accommodation part  22  of the cultivation container  20 . The measurement unit  38  acquires measured shape information by relatively moving with respect to the observation region of the imaging unit  37  and the cultivation container  20  in accordance with a scheduled trajectory and measuring the shape of the cultivation container  20  along the scanning trajectory. 
     Specifically, the measurement unit  38  comprises a first displacement sensor  38   a  and a second displacement sensor  38   b . The first displacement sensor  38   a  and the second displacement sensor  38   b  are arranged in the X direction illustrated in  FIG. 1  with the image forming optical system C interposed therebetween. The first displacement sensor  38   a  and the second displacement sensor  38   b  in the present embodiment are laser displacement meters and detect a Z-directional position of a bottom surface of the cultivation container  20  by irradiating the cultivation container  20  with laser light and detecting reflected light. The bottom surface of the cultivation container  20  is a boundary surface between a bottom portion of the cultivation container  20  and the cell which is the observation target, that is, an observation target installation surface. 
     Z-directional positional information of the cultivation container  20  detected by the measurement unit  38  is output to the control unit  40 . The control unit  40  performs the autofocus control by controlling the focus adjustment mechanism  35  based on the input positional information. The detection of the position of the cultivation container  20  by the first displacement sensor  38   a  and the second displacement sensor  38   b , and the autofocus control will be described in detail later. 
     Next, a configuration of the control unit  40  controlling the microscope device  30  will be described.  FIG. 3  is a block diagram illustrating a configuration of the control unit according to the embodiment of the technology of the disclosure. 
     The control unit  40  controls the entire microscope device  30  as described above and executes various processes. The control unit  40  includes a microscope device control unit  41 , a scanning control unit  42 , a display control unit  43 , an extraction unit  44 , a storage unit  45 , a calculation unit  46 , an input unit  47 , and a display unit  48 . The control unit  40  is configured with a computer that comprises a central processing unit (CPU), a semiconductor memory, and the like. In the control unit  40 , an observation program according to one embodiment of the present invention is installed in the storage unit  45 . The microscope device control unit  41 , the scanning control unit  42 , and the display control unit  43  illustrated in  FIG. 3  function by causing the CPU to execute the observation program. 
     The microscope device control unit  41  controls the focus adjustment mechanism  35  based on the Z-directional positional information of the cultivation container  20  detected by the measurement unit  38  as described above. By driving the focus adjustment mechanism  35 , the phase difference lens moves in the optical axis direction, and the autofocus control is performed. 
     In addition, the microscope device control unit  41  controls imaging performed by the imaging unit  37  in a case where the cultivation container  20  is scanned. Basically, a timing of imaging during scanning is stored in advance in the storage unit  45 . The microscope device control unit  41  performs imaging based on the stored timing. 
     The scanning control unit  42  controls driving of the movement unit  12  and moves the placing stand  10  in the X direction and the Y direction. 
     The display control unit  43  generates one composite image by combining the series of partial images captured by the microscope device  30  and displays the composite image on the display unit  48 . 
     The extraction unit  44  extracts the shape of the cultivation container  20  included in the image obtained by imaging performed by the imaging unit  37 . Particularly, in the present embodiment, the extraction unit  44  extracts the shape of the edge of the accommodation part  22  of the cultivation container  20  included in the image. 
     The storage unit  45  stores the observation program that implements each function unit. In addition, the storage unit  45  stores container information related to the cultivation container  20 . For example, the container information of the cultivation container  20  includes specifications (a position of the accommodation part  22 , a size of the accommodation part  22 , and the like) of the cultivation container and a model number, a maker, and the like of the cultivation container. Information of the number ( 6 ,  24 ,  96 ) of accommodation parts  22  of the currently used cultivation container  20 , intervals of the accommodation parts  22 , a diameter of the accommodation part  22 , a thickness of the accommodation part  22 , and the like is obtained from the container information. The storage unit  45  may store information indicating a solid shape of the cultivation container  20  obtained by measurement performed in advance by a shape measurement device such as a laser length measuring device. The information indicating the solid shape of the cultivation container  20  includes a shape of the bottom surface of the cultivation container  20 , the position of the accommodation part  22  in the cultivation container  20 , and the like. 
     Information indicating the scheduled trajectory of the microscope device  30  and the series of imaging positions (coordinates) on the scheduled trajectory is decided by the scanning control unit  42  based on the container information or the information indicating the solid shape of the cultivation container  20  and is stored in the storage unit  45 . Furthermore, the storage unit  45  stores reference shape information indicating the shape of the cultivation container  20  when the cultivation container  20  is measured by the measurement unit  38  in a case where the cultivation container  20  is appropriately placed on the placing stand  10  as assumed and at least one of the cultivation container  20  or the imaging unit  37  is relatively moved with respect to the other along the scheduled trajectory. As described above, in a case where at least one of the cultivation container  20  placed as assumed or the imaging unit  37  is relatively moved with respect to the other along the scheduled trajectory, a trajectory on which the imaging unit  37  relatively moves with respect to the cultivation container  20  will be referred to as a reference trajectory. 
     The calculation unit  46  calculates a shift of the trajectory (scanning trajectory) on which the observation region of the microscope device  30  scans the cultivation container  20  with respect to the reference trajectory based on the reference shape information of the cultivation container  20  stored in the storage unit  45  and the measured shape information which is a measurement result obtained by measurement performed by the measurement unit  38 . Details will be described later. 
     The input unit  47  comprises a mouse, a keyboard, and the like and receives various necessary data and various setting inputs from the user. For example, the input unit  47  of the present embodiment receives an input of data related to the reference shape information of the cultivation container  20  and the imaging positions. 
     The display unit  48  comprises, for example, a liquid crystal display and displays a composite phase difference image generated by the display control unit  43  as described above. The display unit  48  may be configured with a touch panel and double as the input unit  47 . 
     Next, movement control of the placing stand  10  by the scanning control unit  42  and control of the microscope device  30  by the microscope device control unit  41  will be described in detail. 
       FIG. 4  is a diagram illustrating the scanning trajectory by a solid line M in the cultivation container.  FIG. 5  and  FIG. 6  are diagrams illustrating a positional relationship among the first displacement sensor, the second displacement sensor, and the cultivation container in a case where the observation region is present at any position in the cultivation container.  FIG. 7  is a diagram illustrating an example of a two-dimensional shape of a container detected in an autofocus detection unit on a forward path. 
     In the present embodiment, the placing stand  10  is moved in the X direction and the Y direction under control of the scanning control unit  42 , and the microscope device  30  scans the inside of the cultivation container  20  in two dimensions. During the scanning, partial images of the cultivation container  20  and the observation target are captured in each observation region of the microscope device  30 . In the present embodiment, a well plate that includes six accommodation parts  22  is used as the cultivation container  20 . 
     As illustrated in  FIG. 4 , the observation region of the microscope device  30  moves along the solid line M from a scanning start point S to a scanning end point E. That is, the observation region is scanned in a positive direction (a rightward direction in  FIG. 4 ) of the X direction and then, moves in the Y direction (a downward direction in  FIG. 4 ) and is scanned in the opposite negative direction (a leftward direction in  FIG. 4 ). Next, the observation region moves in the Y direction again and is scanned in the positive direction again. By repeating reciprocation of the observation region in the X direction and movement of the observation region in the Y direction, the inside of the cultivation container  20  is scanned in two dimensions. 
     In the present embodiment, as illustrated in  FIG. 5  and  FIG. 6 , the first displacement sensor  38   a  and the second displacement sensor  38   b  are arranged in the X direction with the image forming optical system C interposed therebetween. An observation region R of the image forming optical system C scans the inside of the cultivation container  20  in two dimensions as described above. At this point, the Z-directional position of the cultivation container  20  is detected at a position that is further in a movement direction of the observation region R than a position of the observation region R of the image forming optical system C with respect to the cultivation container  20 . Specifically, in a case where the observation region R is moving in an arrow direction (a rightward direction in  FIG. 5 ) illustrated in  FIG. 5 , the Z-directional position of the cultivation container  20  is detected by the first displacement sensor  38   a  that is further in the movement direction of the observation region R between the first displacement sensor  38   a  and the second displacement sensor  38   b . In a case where the observation region R moves from the position illustrated in  FIG. 5  to a position of the first displacement sensor  38   a , the autofocus control is performed using the previously detected Z-directional positional information of the cultivation container  20 , and the partial images are captured. 
     In a case where the observation region R is moving in an arrow direction (a leftward direction in  FIG. 6 ) in  FIG. 6 , the Z-directional position of the cultivation container  20  is detected by the second displacement sensor  38   b  that is further in the movement direction of the observation region R between the first displacement sensor  38   a  and the second displacement sensor  38   b . In a case where the observation region R moves from the position illustrated in  FIG. 6  to a position of the second displacement sensor  38   b , the autofocus control is performed using the previously detected Z-directional positional information of the cultivation container  20 , and the phase difference images are captured. 
     The detection of the cultivation container  20  using the first displacement sensor  38   a  and the detection of the cultivation container  20  using the second displacement sensor  38   b  are switched depending on the movement direction of the observation region R. Accordingly, the Z-directional positional information of the cultivation container  20  at the position of the observation region R can be acquired at all times prior to capturing of the phase difference image of the observation region R. 
     Based on the Z-directional positional information of the cultivation container  20  detected beforehand as described above, the microscope device control unit  41  performs the autofocus control by controlling driving of the focus adjustment mechanism  35 . Specifically, a relationship between the Z-directional positional information of the cultivation container  20  and a movement amount of the image forming optical system C in the optical axis direction is set in advance in the microscope device control unit  41 . The microscope device control unit  41  obtains the movement amount of the image forming optical system C in the optical axis direction based on the input Z-directional positional information of the cultivation container  20  and outputs a control signal corresponding to the movement amount to the focus adjustment mechanism  35 . The focus adjustment mechanism  35  is driven based on the input control signal. Accordingly, the phase difference lens is moved in the optical axis direction, and focus adjustment corresponding to the Z-directional position of the cultivation container  20  is performed. 
     The measurement performed by the measurement unit  38  is also used for other than the focus adjustment. The two-dimensional shape of the cultivation container  20  illustrated in a lower part of  FIG. 7  is obtained by causing the measurement unit  38  to perform scanning along the scanning trajectory M and measure the Z-directional position of the cultivation container  20 . In an upper part of  FIG. 7 , a part of the cultivation container  20  in a plan view is illustrated, and the scanning trajectory M in a case where the measurement unit  38  scans three linearly arranged accommodation parts  22 . In the lower part of  FIG. 7 , the shape of the cultivation container  20  along the scanning trajectory M is illustrated by signal strength received by the measurement unit  38 . As illustrated in  FIG. 7 , at a position at which the accommodation part  22  of the cultivation container  20  is present, the measurement light is reflected by the accommodation part  22 . Thus, the signal strength is high. At a position at which the accommodation part  22  is not present, reflection of the measurement light is low, and the signal strength is low. That is, the shape of the cultivation container  20  measured as a waveform in which the signal strength is high at a part corresponding to the accommodation part  22  and the signal strength is low at the other part. The measurement result of the measurement unit  38  is sequentially stored in the storage unit  45 . 
     As illustrated in  FIG. 7 , in a case where an arrangement direction of the accommodation parts  22  of the cultivation container  20  coincides with a direction of the scanning trajectory M, a waveform in which repetition of roughness is regular is measured. The arrangement direction of the accommodation parts  22  of the cultivation container  20  may not coincide with the direction of the scanning trajectory M. For example, a case where the cultivation container  20  is arranged at an inclination with respect to the placing stand  10  and the scanning trajectory M is inclined with respect to the arrangement direction of the accommodation parts  22  is present. 
       FIG. 8  is a diagram illustrating an example of inclination of the scanning trajectory with respect to the arrangement direction of the accommodation parts  22  of the cultivation container  20 . In the case illustrated in  FIG. 8 , even in a case where imaging is performed using the image forming optical system C along the scanning trajectory, a scheduled part of the accommodation part  22  cannot be imaged, and the observation target also cannot be appropriately imaged. The observation apparatus according to the embodiment of the present disclosure performs the following control such that the scanning trajectory M is corrected using the shape of the cultivation container  20  measured by the measurement unit  38  even in a case where the scanning trajectory M is inclined with respect to the arrangement direction of the accommodation parts  22  of the cultivation container  20 . An algorithm illustrated below is implemented by causing the CPU to execute the program stored in the storage unit  45 . 
       FIG. 9  is a flowchart illustrating a flow of observation method executed by the observation apparatus. Each step is particularly executed by the control unit  40 . 
     First, the control unit  40  receives an input of the container information related to the cultivation container  20  in the input unit  47  (step S 101 ). For example, the container information of the cultivation container  20  includes specifications (the size of the accommodation part  22 , the number of accommodation parts  22 , and the like) of the cultivation container and the model number, the maker, and the like of the cultivation container. Information of the number of accommodation parts  22  of the currently used cultivation container  20 , the intervals of the accommodation parts  22 , the diameter of the accommodation part  22 , and the like is obtained from the container information. Even with the same specifications, arrangement positions and the like of the accommodation parts  22  may vary depending on the maker. Thus, information on the maker is also useful for specifying the shape of the cultivation container  20 . 
     Next, the control unit  40  specifies the imaging positions and the scheduled trajectory from the container information obtained in step S 101  (step S 102 ). The imaging positions are specified as coordinate positions at which the observation target accommodated in the cultivation container  20  can be observed. For example, the control unit  40  specifies coordinates of the image forming optical system C at which the observation target in the accommodation part  22  can be observed as the imaging positions by assuming a state where the cultivation container  20  of which the shape is specified in step S 101  is appropriately placed on the placing stand  10 . In addition, the control unit  40  specifies a trajectory connecting the imaging positions as the scheduled trajectory representing a trajectory on which the imaging unit  37  is scheduled to be relatively moved with respect to the cultivation container  20 . The control unit  40  may first decide the scheduled trajectory based on the container information and specify the coordinates of the imaging positions on the scheduled trajectory. The scheduled trajectory is preferably configured with straight lines as far as possible except for a time when the direction is changed. 
     A case where scanning is performed on the cultivation container  20  as assumed in accordance with the scheduled trajectory is scanning that is a reference along the arrangement of the accommodation parts  22 . A trajectory of the reference with respect to the cultivation container  20  is the reference trajectory. For example, the cultivation container as assumed is a container in a state where the container is appropriately placed at a position for measurement without inclination. A trajectory on which the imaging unit  37  and the measurement unit  38  are actually relatively moved with respect to the cultivation container  20  along the scheduled trajectory will be referred to as the scanning trajectory below. 
     Even in a case where the cultivation container  20  is scanned in accordance with the scheduled trajectory, the actual scanning trajectory of the measurement unit  38  does not coincide with the reference trajectory with respect to the cultivation container  20  in a case where the cultivation container  20  is placed at an inclination on the placing stand  10 . In a case where the cultivation container  20  is appropriately placed on the placing stand  10  without inclination, the actual scanning trajectory coincides with the reference trajectory in a case where the cultivation container  20  is scanned in accordance with the scheduled trajectory. Thus, the reference trajectory may be a reference with respect to the scanning trajectory which is the actual scanning trajectory. The reference trajectory includes a part that is parallel to the arrangement direction of the accommodation parts  22  of the cultivation container  20 . 
     Next, as the reference shape information, the control unit  40  creates shape information of the cultivation container  20  that may be obtained in a case where it is assumed that the shape of the cultivation container  20  is measured by the measurement unit  38  along the reference trajectory decided in step S 102  by appropriately placing the cultivation container  20  based on the container information obtained in step S 101  (step S 103 ). In this step, the reference shape information may be obtained by actually measuring the correctly placed cultivation container  20  using the laser length measurement device or the measurement unit  38 , or the reference shape information may be calculated based on the container information and the reference trajectory without actual measurement. Step S 101  to step S 103  are pre-processing of observation performed by the observation apparatus. Accordingly, step S 101  to step S 103  may be executed in advance separately from step S 104  and subsequent steps. 
     The control unit  40  starts scanning the cultivation container  20  by moving the placing stand  10  in accordance with the scheduled trajectory predetermined in step S 102  using the scanning control unit  42  and starting imaging performed by the microscope device  30  (step S 104 ). The imaging performed by the microscope device  30  accompanies measurement of the shape of the cultivation container  20  performed by the measurement unit  38  as described above. 
     The control unit  40  measures the shape of the cultivation container  20  using the measurement unit  38  (step S 105 ) and images the observation target using the image forming optical system C at the imaging positions (step S 106 ). 
     The control unit  40  calculates an inclination of the scanning trajectory with respect to the reference trajectory as a shift of the actual scanning trajectory with respect to the reference trajectory (step S 107 ). A specific method of calculating the inclination of the scanning trajectory will be described later with reference to  FIG. 10 . 
     The control unit  40  determines a direction in which the scanning trajectory is inclined with respect to a proceeding direction of the reference trajectory (step S 108 ). For example, in the example illustrated in  FIG. 8 , the scanning trajectory m is inclined to the right side in a view from a proceeding direction of a reference trajectory K illustrated by a dotted line. In order to determine an inclination side, for example, the control unit  40  checks the image in which the observation region is imaged by the imaging unit  37 . As illustrated in  FIG. 8 , in a case where the scanning trajectory M is inclined in a rightward direction (right side) of a scanning direction, the image captured by the imaging unit  37  is shifted in a positive Y direction (lower side in  FIG. 8 ). Such a shift can be determined in a case where the shape of the edge of the accommodation part  22  in the image is checked. Accordingly, an inclination direction of the scanning trajectory M can be determined by extracting the shape of the edge of the accommodation part  22  in the image captured by the imaging unit  37  using the extraction unit  44 . 
     The control unit  40  corrects the scheduled trajectory and the imaging positions based on the inclination and the inclination direction of the scanning trajectory M obtained in step S 107  and step S 108  (step S 109 ). 
     The control unit  40  relatively moves the imaging unit  37  to the corrected imaging positions by controlling the movement unit  12  based on the corrected scheduled trajectory and imaging positions and performs imaging using the imaging unit  37  (step S 110 ). 
     Next, the control unit  40  determines whether or not the scanning is completed to the end (step S 111 ) and returns to the process of step S 105  in a case where the scanning is not completed yet (step S 111 : NO). In a case where the scanning is completed to the end (step S 111 : YES), the control unit  40  finishes an observation process. 
     Next, calculation of the inclination of the scanning trajectory will be specifically described with reference to  FIG. 10  and  FIG. 11 . 
       FIG. 10  is a diagram illustrating a shift of the scanning trajectory with respect to the reference trajectory.  FIG. 11  is a partial enlarged view of  FIG. 10 . 
     In an upper part of  FIG. 10 , a part of the cultivation container  20  in a plan view is illustrated. The scanning trajectory M in a case where the measurement unit  38  actually performs scanning is illustrated by a solid line arrow, and the reference trajectory K is illustrated by a dotted line arrow. In a lower part of  FIG. 10 , the shape information (measured shape information) of the cultivation container  20  along the scanning trajectory M is illustrated by a solid line, and the shape information (reference shape information) of the cultivation container  20  along the reference trajectory K is illustrated by a dotted line. 
     As illustrated in the upper part of  FIG. 10 , the reference trajectory K intersects with the edges of the accommodation parts  22  at positions A 1  to A 6  in the arrangement direction of the accommodation parts  22 . In a case where the scanning trajectory M is inclined from the reference trajectory K, the scanning trajectory M is also inclined from the arrangement direction of the accommodation parts  22  and intersects with the edges of the accommodation parts  22  at positions B 1  to B 6 . 
     Consequently, as illustrated in the lower part of  FIG. 10 , a part in which the accommodation part  22  is present and a part in which the accommodation part  22  is not present regularly appear in the reference shape information of the cultivation container  20  measured by the measurement unit  38  along the reference trajectory K. That is, the reference shape information is a waveform in which a time in which the signal strength is high evenly appears. The part in which the accommodation part  22  is present and the part in which the accommodation part  22  is not present do not regularly appear in the measured shape information of the cultivation container  20  actually measured along the scanning trajectory M. In the measured shape information along the scanning trajectory M, a waveform in which a range in which the signal strength is high is decreased from the range in the shape information of the cultivation container along the reference trajectory K as the scanning proceeds is obtained. 
     For example, it is assumed that a time from the scanning start point S to A 1  on the reference trajectory K is denoted by TA 1 , a time from the scanning start point S to B 2  on the scanning trajectory M is denoted by TB 2 , and a relative movement speed of the placing stand  10  with respect to the imaging unit  37  is denoted by V. In addition, as illustrated in  FIG. 11 , it is assumed that a radius of the accommodation part  22  is denoted by R and an inclination angle θ of the scanning trajectory M with respect to the reference trajectory K is denoted by θ. An xy coordinate system in which the scanning start point S is an origin is considered. 
     In this case, the circular edge of the accommodation part  22  is represented by 
       { x −( V×TA 1+ R )} 2   +y   2   =R   (1).
 
     A straight line of the scanning trajectory M is represented by 
         y=x ×tan θ  (2).
 
     Substituting y of Expression (1) with x×tan θ of Expression (2) results in 
       { x −( V×TA 1+ R )} 2 +( x ×tan θ) 2   =R   (3).
 
     An x coordinate of the point B 2  is obtained as 
         x=V×TB 2×cos θ  (4).
 
     In a case where x of Expression (3) is substituted with V×TB 2 ×cos θ of Expression (4), an expression in which θ is the only variable is obtained, and a value of θ can be calculated. 
     In a case where the inclination θ is obtained and the inclination side of θ is found in step S 108 , the positions B 3 , B 4 , B 5 , and B 6  can be calculated without actually performing scanning to the positions B 3 , B 4 , B 5 , and B 6  of the scanning trajectory M. Accordingly, the control unit  40  can calculate correction amounts Δx and Δy for correcting the position B 3 , the position B 4 , the position B 5 , and the position B 6  to the position A 3 , the position A 4 , the position A 5 , and the position A 6 , respectively, and can correct the imaging positions based on the calculated correction amounts. The control unit  40  can also correct the scheduled trajectory in accordance with correction of the imaging positions. The control unit  40  preferably corrects sub subsequent imaging positions in accordance with the calculated correction amounts. However, in a case where the shift is large and correcting the imaging positions to completely remove the shift needs the movement unit  12  to be stopped or unreasonably driven or causes an unreasonable imaging timing of the imaging unit  37 , correction may not be necessarily performed in accordance with the calculated correction amounts. In this case, the control unit  40  corrects the scheduled trajectory to approach the reference trajectory K as far as possible without causing unreasonable driving of the movement unit  12  and an unreasonable imaging timing of the imaging unit  37 . 
     The method of calculating the inclination θ is one example, and θ may be calculated using any method. For example, while θ is calculated by actually performing measurement to the position B 2  in the calculation method, the inclination θ may be calculated after measurement is performed to more positions such as the position B 3  and the position B 4 . 
     Calculation of the actual shift of the scanning trajectory M with respect to the reference trajectory K may be performed only once immediately after imaging of one cultivation container  20  is started, or may be performed a plurality of times during imaging. 
     As described above, according to the embodiment of the present disclosure, the observation apparatus calculates the actual shift of the scanning trajectory M with respect to the reference trajectory K from the reference shape information indicating the shape of the cultivation container  20  along the reference trajectory K and the measured shape information obtained by measurement performed by the measurement unit  38 . The observation apparatus controls the imaging positions and the movement direction set by the movement unit  12  such that the scanning trajectory M approaches the reference trajectory K. Accordingly, even in a case where the cultivation container  20  is not installed in a correct posture on the placing stand  10  and the scanning trajectory M is shifted from the reference trajectory K, the observation apparatus can capture the image close to an appropriate imaging position. 
     The measurement unit  38  measures the shape of the cultivation container  20  as a waveform showing the part in which the accommodation part  22  is present and the part in which the accommodation part  22  is not present. Accordingly, an error of the scanning trajectory can be easily calculated using a tendency of the waveform. 
     The control unit  40  determines the direction in which the scanning trajectory is inclined with respect to the proceeding direction of the reference trajectory K based on the image in which the observation region is imaged by the imaging unit  37 . Accordingly, as illustrated in  FIG. 10 , even in a case where the reference trajectory K exactly passes through the diameter of the accommodation part  22 , which side (in the example in  FIG. 10 , an upper side or a lower side) the obtained inclination θ is present with respect to the reference trajectory K can be determined. In a case where the reference trajectory K does not pass through the diameter of the accommodation part  22 , the inclination direction of the inclination θ can be determined by only the waveform in  FIG. 10  without using the image captured by the imaging unit  37 . 
     In the embodiment, a case where the cultivation container  20  is not appropriately placed on the placing stand  10  has been described as a case where the scanning trajectory M is shifted from the reference trajectory K. However, the technology of the present disclosure can also be applied to a case where the scanning trajectory M is shifted from the reference trajectory K due to any other causes. For example, even in a case where the accommodation parts  22  are irregularly arranged due to a manufacturing error or the like in the cultivation container  20 , the scheduled trajectory can be corrected in accordance with the arrangement direction of the accommodation parts  22 , and scanning can be performed by the imaging unit  37  in the arrangement direction. 
     The observation process that is executed by causing the CPU to read software (program) in embodiment may be executed by various processors other than the CPU. In this case, the processors are illustrated by a programmable logic device (PLD) such as a field-programmable gate array (FPGA) of which a circuit configuration can be changed after manufacturing, a dedicated electric circuit such as an application specific integrated circuit (ASIC) that is a processor having a circuit configuration dedicatedly designed to execute a specific process, and the like. In addition, the observation process may be executed by one of the various processors or may be executed by a combination of two or more processors of the same type or different types (for example, a plurality of FPGAs and a combination of a CPU and an FPGA). In addition, a hardware structure of the various processors is more specifically an electric circuit in which circuit elements such as semiconductor elements are combined. 
     In embodiment, an aspect in which the program of the observation process is stored (installed) in advance in the storage unit  45  is described. However, the aspect is not for limitation purposes. The program may be provided in a form of a recording on a recording medium such as a compact disk read only memory (CD-ROM), a digital versatile disk read only memory (DVD-ROM), and a Universal Serial Bus (USB) memory. In addition, the program may be downloaded from an external device through a network. 
     EXPLANATION OF REFERENCES 
     
         
         
           
               10 : placing stand 
               11 : opening 
               12 : movement unit 
               20 : cultivation container 
               21 : plate 
               22 : accommodation part 
               30 : microscope device 
               31 : light source 
               32 : slit 
               33 : condenser lens 
               34 : objective lens 
               35 : focus adjustment mechanism 
               36 : image forming lens 
               37 : imaging unit 
               38 : measurement unit 
               38   a : first displacement sensor 
               38   b : second displacement sensor 
               40 : control unit 
               41 : microscope device control unit 
               42 : scanning control unit 
               43 : display control unit 
               44 : extraction unit 
               45 : storage unit 
               46 : calculation unit 
               47 : input unit 
               48 : display unit 
             A 1  to A 6 , B 1  to B 6 : position 
             C: image forming optical system 
             E: scanning end point 
             K: reference trajectory 
             L: illumination light 
             M: scanning trajectory 
             R: observation region 
             S: scanning start point