Patent Publication Number: US-11397313-B2

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/JP2018/033019 filed on Sep. 6, 2018, which claims priority under 35 U.S.C § 119(a) to Japanese Patent Application No. 2017-191004 filed on Sep. 29, 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, an imaging position (for example, coordinates) at which scanning and measurement are performed is 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 imaging position. 
     SUMMARY OF THE INVENTION 
     Even in a case where imaging is performed along the decided scanning trajectory, an unintended image may be captured. For example, in a case where a distance to the cultivation container is measured by a laser displacement meter before imaging and a focal length at the imaging position is specified based on a measurement result, the measurement result of the laser displacement meter may not be obtained in a range sufficient for specifying the focal length. Specifically, in a case where a field of view of the microscope is smaller than an accommodation part that accommodates the cell or the like in the cultivation container, a center of the accommodation part is not imaged, and an end of the accommodation part is imaged depending on the imaging position. Before the imaging of the end of the accommodation part, the end of the accommodation part that enters the field of view is measured by the laser displacement meter that is present before the end of the accommodation part. However, in a case where only the end of the accommodation part enters the field of view, a range (effective range) of the accommodation part that can be measured by the laser displacement meter within the field of view is narrower than the effective range in a case where the accommodation part enters most of the field of view. In a case where the effective range is narrow, the measurement result is significantly affected by disturbance when disturbance such as vibration occurs within the effective range. In a case where a focus is adjusted based on the measurement result significantly affected by disturbance, the focus may deviate from the cell or the like, and an unintended image may be captured. 
     The technology of the disclosure is conceived in view of the above point. An object of the technology of the disclosure is to provide an observation apparatus, an observation method, and an observation program capable of capturing an image by appropriately adjusting a focus regardless of a size of an effective range in which a distance to a cultivation container can be measured within a field of view. 
     An observation apparatus according to the technology of the disclosure comprises an imaging unit that images an observation target accommodated in an accommodation part of a container in a field of view smaller than the accommodation part at a series of predetermined imaging positions and acquires a series of partial images, a measurement unit that measures a distance from the imaging unit to the accommodation part before each imaging performed by the imaging unit at the series of imaging positions, a storage unit that stores shape information representing a shape of the container and imaging position information representing the series of imaging positions, a calculation unit that calculates effective range information indicating an effective range based on the shape information and the imaging position information, the effective range being a range in which the distance is measurable by the measurement unit before imaging within a range of the field of view of the imaging unit at the imaging positions, and a control unit that compares the effective range with a predetermined threshold value based on the effective range information and controls a focus of imaging using a measurement result measured by the measurement unit in the effective range and a measurement result of the measurement unit in a field of view adjacent to the field of view including the effective range in a case where the effective range is smaller than or equal to the threshold value. 
     The partial image is an image that is obtained by imaging by the imaging unit in a field of view of the imaging unit at each of a plurality of predetermined imaging positions. The shape information is information that is acquired in advance before capturing of the partial image, and is, for example, information representing a size of the accommodation part and a position of the accommodation part in the entire container. The effective range is a range in a scanning direction of the measurement unit and is a range in which the measurement unit can measure the distance from the imaging unit to the accommodation part within the range of the field of view at a time of imaging by the imaging unit from the imaging position. For example, the effective range information as information indicating the effective range indicates coordinate information of the effective range in a movement direction of the measurement unit or a length of the effective range in the movement direction of the measurement unit calculated from the coordinate information. The predetermined threshold value is a value that can be randomly decided by a user and, for example, is set to a length, in a movement direction of the imaging unit, of a range in which desired accuracy is secured even with disturbance as a scanning range in which the measurement unit measures the distance to the accommodation part. 
     In the observation apparatus, in a case where the effective range is smaller than or equal to the threshold value, the control unit may use the measurement result of the measurement unit in the adjacent field of view in a larger range as the effective range is smaller. 
     In the observation apparatus, the threshold value may be a length of half of a width of the field of view of the imaging unit. 
     In the observation apparatus, in a case where the effective range is smaller than or equal to the threshold value, the control unit may control the focus using the measurement result of the measurement unit in the adjacent field of view to an extent of an insufficient length of the effective range with respect to the threshold value. 
     An observation method according to the technology of the disclosure comprises a measurement step of, in a case where an observation target accommodated in an accommodation part of a container is imaged by an imaging unit having a field of view smaller than the accommodation part at a series of predetermined imaging positions and a series of partial images are acquired, measuring a distance from the imaging unit to the accommodation part before acquisition of each of the series of partial images, a storage step of storing shape information representing a shape of the container and imaging position information representing the series of imaging positions, a calculation step of calculating effective range information indicating an effective range based on the shape information and the imaging position information, the effective range being a range in which the distance is measurable in the measurement step before imaging within a range of the field of view at the imaging positions, and a control step of comparing the effective range with a predetermined threshold value based on the effective range information calculated in the calculation step and controlling a focus of imaging using a measurement result measured in the measurement step in the effective range and a measurement result of the measurement step in a field of view adjacent to the field of view including the effective range in a case where the effective range is smaller than or equal to the threshold value. 
     An observation program according to the technology of the disclosure causes a computer to execute a measurement step of, in a case where an observation target accommodated in an accommodation part of a container is imaged by an imaging unit having a field of view smaller than the accommodation part at a series of predetermined imaging positions and a series of partial images are acquired, measuring a distance from the imaging unit to the accommodation part before acquisition of each of the series of partial images, a storage step of storing shape information representing a shape of the container and imaging position information representing the series of imaging positions, a calculation step of calculating effective range information indicating an effective range based on the shape information and the imaging position information, the effective range being a range in which the distance is measurable in the measurement step before imaging within a range of the field of view at the imaging positions, and a control step of comparing the effective range with a predetermined threshold value based on the effective range information calculated in the calculation step and controlling a focus of imaging using a measurement result measured in the measurement step in the effective range and a measurement result of the measurement step in a field of view adjacent to the field of view including the effective range in a case where the effective range is smaller than or equal to the threshold value. 
     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 a measurement step of, in a case where an observation target accommodated in an accommodation part of a container is imaged by an imaging unit having a field of view smaller than the accommodation part at a series of predetermined imaging positions and a series of partial images are acquired, measuring a distance from the imaging unit to the accommodation part before acquisition of each of the series of partial images, a storage step of storing shape information representing a shape of the container and imaging position information representing the series of imaging positions, a calculation step of calculating effective range information indicating an effective range based on the shape information and the imaging position information, the effective range being a range in which the distance is measurable in the measurement step before imaging within a range of the field of view at the imaging positions, and a control step of comparing the effective range with a predetermined threshold value based on the effective range information calculated in the calculation step and controlling a focus of imaging using a measurement result measured in the measurement step in the effective range and a measurement result of the measurement step in a field of view adjacent to the field of view including the effective range in a case where the effective range is smaller than or equal to the threshold value. 
     According to the technology of the disclosure, in a case where the effective range in which the measurement unit can perform measurement before imaging within the range of the field of view is smaller than or equal to the threshold value, the focus is controlled using the measurement result in the effective range and also the measurement result of the measurement unit in the adjacent field of view. Accordingly, an image can be captured by appropriately adjusting the focus regardless of the size of the effective range in which the distance to the cultivation container can be measured within the field of view. 
    
    
     
       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 a field of view 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 field of view is present at any position in the cultivation container. 
         FIG. 7  is a diagram for describing a state where a measurement unit measures an accommodation part. 
         FIG. 8  is a flowchart illustrating a flow of observation method executed by the observation apparatus. 
         FIG. 9  is a diagram illustrating an example in which a range extended from an effective range within the field of view is set as a range in which a measurement result of the measurement unit is used for autofocus control. 
     
    
    
     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  in a field of view 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 partial image is an image that is obtained by imaging by the microscope device  30  in a field of view of the microscope device  30  at each of a plurality of predetermined imaging positions. 
     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  is fixedly attached to the measurement unit  38  and 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. 
     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  consecutively detects a Z-directional position of the cultivation container  20  installed on the placing stand  10  along the scanning trajectory accompanied by the relative movement of at least one of the cultivation container  20  or the imaging unit  37 . 
     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 accommodation part  22  of the cultivation container  20  by irradiating the cultivation container  20  with laser light and detecting reflected light, and measures a distance from the imaging unit  37  to the bottom surface of the accommodation part  22 . The bottom surface of the accommodation part  22  is a boundary surface between a bottom portion of the accommodation part  22  and the cell which is the observation target, that is, an observation target installation surface. 
     The distance detected by the measurement unit  38  from the imaging unit  37  to the bottom surface of the accommodation part  22  is output to the control unit  40 . The control unit  40  performs the autofocus control (focus control) by controlling the focus adjustment mechanism  35  based on the input distance. 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 , a storage unit  44 , a calculation unit  45 , an input unit  46 , and a display unit  47 . 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  44 . The microscope device control unit  41 , the scanning control unit  42 , the display control unit  43 , and the calculation unit  45  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 distance detected by the measurement unit  38  from the imaging unit  37  to the bottom surface of the accommodation part  22  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  44 . 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  47 . 
     The storage unit  44  stores the observation program that implements each function unit. In addition, the storage unit  44  stores shape information of the cultivation container  20  corresponding to container information of the cultivation container  20 . For example, the container information of the cultivation container  20  includes specifications (a size, a number, intervals, and the like of accommodation parts  22 ) of the cultivation container and a model number, a maker, and the like of the cultivation container. The shape information of the cultivation container  20  is information such as the number ( 6 ,  24 ,  96 , or the like) of accommodation parts  22  of the cultivation container  20 , the intervals of the accommodation parts  22 , a diameter of the accommodation part  22 , a thickness of the accommodation part  22 , and a position of the accommodation part  22  in the cultivation container  20 . The shape information may be information of the specifications of the cultivation container  20  published from a manufacturing maker or the like or may be information of a solid shape of the cultivation container  20  obtained by measurement in advance by a shape measurement device such as a laser length measurement device. 
     Trajectory information indicating the scanning trajectory of the microscope device  30  and imaging position information indicating a series of imaging positions (coordinates) are decided by the scanning control unit  42  based on the container information or the shape information including information of the solid shape of the cultivation container  20 , and are stored in the storage unit  44 . 
     Based on the shape information and the imaging position information stored in the storage unit  44 , the calculation unit  45  calculates effective range information indicating an effective range that is a range in which the measurement unit  38  can perform measurement before imaging within a range of a field of view of the imaging unit  37  at the imaging position. The effective range is a range in a scanning direction of the measurement unit  38  and is a range in which the measurement unit  38  can measure the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  within the range of the field of view at a time of imaging by the imaging unit  37  from the imaging position. As will be described later, the measurement unit  38  can relatively move before the imaging unit  37  and measure the effective range until the imaging unit  37  reaches the imaging position. For example, the effective range information as information indicating the effective range is coordinate information indicating the effective range in a movement direction of the imaging unit  37  or a length of the effective range in a movement direction of the measurement unit calculated from the coordinate information. 
     The input unit  46  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  46  of the present embodiment receives an input of data related to the container information of the cultivation container  20  and the imaging positions. 
     The display unit  47  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  47  may be configured with a touch panel and double as the input unit  46 . 
     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 field of view is present at any position in the cultivation container.  FIG. 7  is a diagram for describing a state where the measurement unit measures the accommodation part. 
     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  two-dimensionally scans the inside of the cultivation container  20 . During the scanning, partial images of the cultivation container  20  and the observation target are captured in each field of view 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 . 
     The microscope device control unit  41  reads out the imaging position and an imaging timing for imaging in each field of view R from the storage unit  44  and causes the microscope device  30  to image the inside of the cultivation container  20  in fields of view R 1  to R 54  as illustrated by surrounding dot-dashed lines in  FIG. 4 . Consequently, the field of view R 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 field of view R 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 ) of the X direction. Next, the field of view R moves in the Y direction again and is scanned in the positive direction of the X direction again. By repeating reciprocation of the field of view R in the X direction and movement of the field of view R in the Y direction, the inside of the cultivation container  20  is two-dimensionally scanned in an order of the fields of view R 1  to R 54 . 
     Before the microscope device  30  performs imaging in the field of view R, the measurement unit  38  detects the distance from the imaging unit  37  to the bottom surface of the accommodation part  22 . 
     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. The field of view R of the image forming optical system C two-dimensionally scans the inside of the cultivation container  20  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 field of view R than a position of the field of view R of the image forming optical system C with respect to the cultivation container  20 . Specifically, in a case where the field of view R is moving in an arrow direction (a rightward direction in  FIG. 5 ) illustrated in  FIG. 5 , the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  is detected by the first displacement sensor  38   a  that is further in the movement direction of the field of view R between the first displacement sensor  38   a  and the second displacement sensor  38   b . In a case where the field of view 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 field of view R is moving in an arrow direction (a leftward direction in  FIG. 6 ) in  FIG. 6 , the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  is detected by the second displacement sensor  38   b  that is further in the movement direction of the field of view R between the first displacement sensor  38   a  and the second displacement sensor  38   b . In a case where the field of view 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 field of view R. Accordingly, the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  at the position of the field of view R can be always acquired before the capturing of the phase difference images in the field of view 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 distance from the imaging unit  37  to the bottom surface of the accommodation part  22  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 distance from the imaging unit  37  to the bottom surface of the accommodation part  22  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, a focal length is set by moving the phase difference lens in the optical axis direction, and the autofocus control corresponding to the Z-directional position of the cultivation container  20  is performed. 
     As the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  for the autofocus control, for example, an average value of the distance from the imaging unit  37  to the bottom portion of the accommodation part  22  between distances measured by the first displacement sensor  38   a  or the second displacement sensor  38   b  from the imaging unit  37  to a bottom surface of the cultivation container  20  within the field of view R is used. 
     A state where the measurement unit  38  measures the Z-directional position will be described with focus on one accommodation part  22  (the accommodation part  22  in an upper left part in  FIG. 4 ) of the cultivation container  20 . 
       FIG. 7  is a diagram for describing a state where the measurement unit measures the accommodation part. In  FIG. 7 , a scanning trajectory M of the measurement unit  38  when the accommodation part  22  is seen in a plan view is illustrated in an upper part, and a measurement result of the measurement unit  38  in the scanning trajectory is illustrated in a lower part. 
     For example, the measurement of the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  that is measured by the first displacement sensor  38   a  before the imaging of the field of view R 1  is performed in a range that is the entire region of the field of view R 1  in  FIG. 7 . However, in a case where all measurement values of the field of view R 1  are used, a measurement value that is not related to the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  is also included. Accordingly, in order to perform the autofocus control on the observation target in the accommodation part  22 , it is necessary to use the measurement result in which the measurement value not related to the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  is excluded. In other words, it is necessary to use a range (effective range) in which the measurement unit  38  can measure the distance to the bottom surface of the accommodation part  22 . The effective range is illustrated by a bidirectional arrow FO 1  in  FIG. 7 . 
     As is apparent from comparison between the fields of view R 1  to R 3 , the effective range in which the distance to the bottom portion of the accommodation part  22  can be measured varies for each field of view R. The entire width of the field of view R 2  in the scanning direction is set as an effective range FO 2 . However, only the second half of the field of view R 1  in the scanning direction is set as the effective range FO 1 , and only the first half of the field of view R 3  in the scanning direction is set as an effective range FO 3 . 
     An abnormal value may be included in a part of the measurement values within the effective range due to disturbance. For example, the disturbance may occur due to a scratch or the like on the bottom portion of the accommodation part  22 . In a case where there is a scratch or the like on the bottom portion of the accommodation part  22 , the laser light of the irradiation from the measurement unit  38  is subjected to diffuse reflection on the bottom portion of the accommodation part  22 , and the measurement value may be obtained as an abnormal value in a case where accurate detection cannot be performed in the measurement unit  38 . In addition, for example, the disturbance may occur due to vibration. As a result of vibration, a distance from the measurement unit  38  to the bottom surface of the accommodation part  22  instantaneously changes. In a case where the distance at the moment is measured by the measurement unit  38 , the measurement value is obtained as an abnormal value. In a case where an effect of the abnormal value is strong, consequently, the autofocus control cannot be appropriately performed. In a case where a distance of the effective range in the scanning direction in the field of view R as a target is sufficiently long, the effect of the abnormal value is weak even in a case where the abnormal value is included in a part of the measurement values within the effective range due to the disturbance. By averaging the measurement result using only the measurement result within the effective range, the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  for the autofocus control can be obtained. However, in a case where the distance of the effective range in the scanning direction in the field of view R as a target of the autofocus control is not sufficient, the effect of the abnormal value is strong in a case where the abnormal value is included in a part of the measurement values within the effective range due to the disturbance, and an appropriate value is not obtained when the distance from the imaging unit  37  to the bottom surface of the cultivation container  20  is obtained by averaging the measurement value within the effective range. Consequently, the autofocus control cannot be appropriately performed. The effect of the abnormal value is increased. 
     In order to prevent such a case, the observation apparatus of the present embodiment measures the distance to the bottom surface of the accommodation part  22  in a sufficient effective range in any field of view R and performs imaging by appropriate autofocus control. Hereinafter, an observation method of the observation apparatus will be described. An algorithm illustrated below is implemented by causing the CPU to execute the program stored in the storage unit  44 . 
       FIG. 8  is a flowchart illustrating a flow of observation method executed by the observation apparatus. Each step is executed by the control unit  40 .  FIG. 9  is a diagram illustrating an example in which a range extended from the effective range within the field of view is set as a range in which the measurement result of the measurement unit is used for the autofocus control. 
     First, the control unit  40  receives an input of the container information of the cultivation container  20  from the user in the input unit  46  and acquires the shape information of the cultivation container  20  stored in the storage unit  44  based on the input container information of the cultivation container  20  (step S 101 ). 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 shape information. 
     Next, the control unit  40  specifies the imaging positions from the shape information of the cultivation container  20  obtained in step S 101  (step S 102 ). The imaging positions are specified as coordinate positions of the X-Y plane of the placing stand  10  at which the observation target accommodated in the cultivation container  20  can be observed. For example, the control unit  40  specifies XY coordinates of the image forming optical system C for performing imaging in the field of view R 1  to the field of view R 54  illustrated in  FIG. 4  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 scanning trajectory M. The control unit  40  may first decide the scanning trajectory based on the shape information of the cultivation container  20  and specify the coordinates of the imaging positions on the scanning trajectory. The scanning trajectory is preferably configured with straight lines as far as possible except for a time when the direction is changed. 
     Next, the control unit  40  substitutes i with 1 as an initial value in all fields of view Ri (i=1 to 54) in order to perform a subsequent process (step S 103 ). 
     The control unit  40  calculates the effective range information indicating the effective range of the measurement unit  38  in the field of view Ri (step S 104 ). 
     The control unit  40  determines whether or not the effective range of the measurement unit  38  in the field of view Ri is smaller than or equal to a predetermined threshold value (step S 105 ). The threshold value is a value that can be randomly decided by the user, and is a value that indicates a length so as to be compared with the effective range. For example, the threshold value is set to a length of a range in which desired accuracy is secured even with the disturbance as a scanning range in which the measurement unit  38  measures the distance from the imaging unit  37  to the bottom surface of the accommodation part  22 . In the present embodiment, the threshold value is described as half of a width of the field of view Ri of the imaging unit  37  along the scanning trajectory. However, as described above, the threshold value can be randomly decided by the user considering measurement accuracy and is not limited to half of the width of the field of view Ri. In a case of the field of view R 1  in  FIG. 9 , the control unit  40  determines that the effective range FO 1  is smaller than or equal to half of the width of the field of view in the scanning direction. In a case of the field of view R 2  in  FIG. 9 , the control unit  40  determines that the effective range FO 2  is larger than half of the width of the field of view in the scanning direction. 
     In a case where the effective range is not smaller than or equal to half of the width of the field of view (step S 105 : NO), the control unit  40  stores only the effective range within the field of view Ri in the storage unit  44  as the effective range in which the measurement result of the measurement unit  38  is used for the autofocus control (step S 106 ). The reason is that a size of the effective range is regarded as a sufficient size for use in the autofocus control in the present embodiment. 
     In a case where the effective range is smaller than or equal to half of the width of the field of view (step S 105 : YES), the control unit  40  stores not only the effective range within the field of view Ri but also a range extended from the effective range within the field of view Ri in the storage unit  44  as a measurement range in which the measurement result of the measurement unit  38  is used for the autofocus control (step S 107 ). The reason is that the size of the effective range is insufficient for use in the autofocus control. 
     As a specific example of a method of extending the effective range, the control unit  40  stores the effective range and a range extended to the field of view Ri+1 or the field of view Ri−1 adjacent to the field of view Ri in the storage unit  44  as the measurement range. For example, in the case of the field of view R 1  in  FIG. 9 , the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  can be sufficiently measured by the measurement unit  38  in the adjacent field of view R 2 . Therefore, the control unit  40  sets an effective range FM 1  extended from the effective range FO 1  within the field of view R 1  to the inside of the field of view R 2  as the effective range to be used in the autofocus control in a case where the field of view R 1  is imaged by the microscope device  30 . Accordingly, as illustrated in the lower part of  FIG. 9 , the measurement result of the measurement unit  38  in the extended effective range FM 1  is averaged and is used in the autofocus control of the field of view R 1 . As illustrated in  FIG. 9 , the extended effective range FM 1  preferably includes the effective range FO 1  and the measurement range within the field of view R 2  that is contiguous with the effective range FO 1 . For example, the extended effective range FM 1  is set to be equal to the length of the threshold value. In this case, the effective range of the adjacent field of view R 2  is included to an extent of an insufficient length of the effective range FO 1  with respect to half of the width of the field of view, which is the threshold value, and the extended effective range FM 1  is obtained. Alternatively, the extended effective range FM 1  may be set to be larger than or equal to the threshold value (half of the field of view R) and smaller than or equal to the entire length of the field of view R. 
     In addition, for example, in a case of the field of view R 3  in  FIG. 9 , the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  can be sufficiently measured by the measurement unit  38  in the adjacent field of view R 2 . The control unit  40  sets an effective range FM 3  extended from the effective range FO 3  within the field of view R 3  to the inside of the field of view R 2  as the measurement range to be used in the autofocus control in a case where the field of view R 3  is imaged by the microscope device  30 . Accordingly, as illustrated in the lower part of  FIG. 9 , the measurement result of the measurement unit  38  in the extended effective ranges FM 1  and FM 3  is averaged and is used in the autofocus control of the field of view R 1  and the field of view R 3 . 
     Next, the control unit  40  determines whether or not the determination of the effective range is completed for all fields of view (step S 108 ). In a case where the determination for all fields of view is not finished (step S 108 : NO), the control unit  40  repeats the process from step S 104  by increasing i by 1 (step S 109 ). In a case where the determination for all fields of view is finished (step S 108 : YES), the control unit  40  starts scanning the cultivation container  20  by starting imaging by the microscope device  30  while causing the scanning control unit  42  to move the placing stand  10  along the scanning trajectory (step S 110 ). 
     The control unit  40  measures the distance from the imaging unit  37  to the bottom surface of the accommodation part  22  by the measurement unit  38  before imaging and stores the distance in the storage unit  44  (step S 111 ). 
     The control unit  40  averages the measurement result of the measurement unit  38  in the effective range stored in step S 106  or step S 107  and uses the measurement result in the autofocus control, and performs imaging by the microscope device  30  at the imaging position in each field of view (step S 112 ). 
     The control unit  40  determines whether or not the scanning is completed, that is, whether or not the imaging is finished at all imaging positions (step S 113 ). In a case where the scanning is not completed (step S 113 : NO), a return is made to the process of step S 111 , and the measurement by the measurement unit  38  and subsequent imaging are performed. In a case where the scanning is completed (step S 113 : YES), the control unit  40  finishes an observation process. 
     As described thus far, even in a case where the effective range of the measurement unit  38  in the field of view R, that is, the length of the accommodation part  22  included in the field of view R along the scanning trajectory, is smaller than or equal to the threshold value (half of the width of the field of view), the observation apparatus of the embodiment uses the measurement result of the measurement unit  38  by setting the effective range extended to the effective range of the adjacent field of view. Accordingly, even in a case where the disturbance such as vibration occurs, the effect of the disturbance can be decreased compared to the effect of the disturbance in a case where the autofocus control is performed using the measurement result of only the effective range of the field of view R which is the target of the autofocus control. In other words, the observation apparatus can capture an image by appropriately performing the autofocus control regardless of the size of the effective range in which the distance to the accommodation part of the cultivation container  20  can be measured within the field of view R. 
     In the embodiment, the imaging performed by the microscope device  30  is started after the completion of the determination of the effective range for all fields of view. However, the imaging is not for limitation purposes. The determination of the effective range of each field of view may be performed in parallel with the imaging performed by the microscope device  30 . 
     The observation process that is executed by causing the CPU to read software (program) in the 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 the embodiment, an aspect in which the program of the observation process is stored (installed) in advance in the storage unit  44  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 : storage unit 
               45 : calculation unit 
               46 : input unit 
               47 : display unit 
             C: image forming optical system 
             E: scanning end point 
             FO 1  to FO 3 : effective range 
             FM 1 , FM 3 : extended range 
             L: illumination light 
             M: scanning trajectory 
             R, R 1  to R 54 : field of view 
             S: scanning start point