Patent Publication Number: US-2023152566-A1

Title: Specimen observation apparatus and specimen observation method

Description:
TECHNICAL FIELD 
     The disclosure relates to a specimen observation apparatus that observes a specimen contained in a specimen container. 
     BACKGROUND ART 
     In a specimen observation apparatus that images a specimen contained in a specimen container, in order to accurately observe the specimen, it is useful to reliably image the specimen by determining a specimen observation area in the specimen container or a specimen position in the specimen container. This is because correct information on the specimen cannot be obtained if the specimen is not included in the captured image. 
     The following PTL 1 discloses a specimen observation apparatus. In PTL 1, cells to be measured are detected using captured images of a plurality of wide viewing angles bonded in a tile shape (see 0113 in PTL 1). 
     CITATION LIST 
     Patent Literature 
     PTL 1: JP-A-2015-227940 
     SUMMARY OF INVENTION 
     Technical Problem 
     For example, an apparatus that captures a microscope image of a specimen contained in a specimen container including a well-like specimen containing portion determines a relative position in the horizontal direction between a specimen observation area in the specimen container and an imaging field of view, and automatically performs focus adjustment and imaging. In such a specimen observation apparatus, when an observation image is obtained according to a preset imaging position, the specimen observation area and the imaging field of view may be deviated from each other. The specimen container, which is a consumable product, is generally a resin molded product, and has a manufacturing error larger than that of a machined product. Therefore, it is difficult to completely eliminate positional deviation caused by the manufacturing error of the specimen container no matter how accurately an apparatus mechanism holds the specimen container. Such deviation leads to a decrease in accuracy of the captured image and an amount of information, and may lead to erroneous determination during post-processing such as image observation and image analysis. In addition, if the deviation is large, the deviation may be excluded from determination targets of focus adjustment, and thus imaging may become impossible. For example, in a laser auto-focus system, focus adjustment cannot be performed when a laser irradiation position is out of a specimen observation area. 
     The disclosure has been made to solve the above problems, and an object thereof is to provide a technology whereby relative positioning in the horizontal direction between a specimen observation area in a specimen container and an imaging field of view can be reliably performed, even prior to adjusting the focal position in the vertical direction using an auto-focus system. 
     Solution to Problem 
     A specimen observation apparatus according to the disclosure obtains a luminance value for an image at a plurality of locations in a specimen container, prior to performing auto-focus, and uses the number of high-luminance regions and the widths of those regions to identify a central position of the specimen container in a horizontal direction, or uses the number of low-luminance regions and the widths of those regions to identify the central position of the specimen container in the horizontal direction. 
     Advantageous Effects of Invention 
     According to the specimen observation apparatus of the disclosure, the relative positioning in the horizontal direction between the specimen observation area in the specimen container and the imaging field of view can be reliably performed, even prior to adjusting the focal position in the vertical direction using an auto-focus system. 
    
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         FIG.  1    is a configuration diagram of a specimen observation apparatus  100  according to Embodiment 1. 
         FIG.  2    is an example of a shape of a specimen containing portion of a specimen container  101 . 
         FIG.  3    is a diagram illustrating refraction of light in the vicinity of an inclined portion of the specimen container  101 . 
         FIG.  4    is an example of a defocus image in a case where a periphery of the specimen container  101  is actually imaged at a wide viewing angle. 
         FIG.  5    is a schematic diagram showing a state in which a design value of a central position of a bottom surface of the specimen container  101  is different from an actual value. 
         FIG.  6    is a flowchart illustrating a procedure of identifying an actual well central position. 
         FIG.  7    is a diagram showing a positional relationship when a controller  200  obtains an observation image in step S 601 . 
         FIG.  8 A  is a diagram showing a state in which an imaging field of view is moved by bx in an X direction in step S 602 . 
         FIG.  8 B  is a graph showing a sum of luminance values of partial images obtained in  FIG.  8 A . 
         FIG.  8 C  is a diagram showing a modification of step S 602 . 
         FIG.  9 A  is a diagram showing a state in which the controller  200  moves the imaging field of view by ΔY in a Y direction in step S 604 . 
         FIG.  9 B  is a diagram showing a state in which S 602  is performed following  FIG.  9 A . 
         FIG.  9 C  is an example of a profile obtained in  FIG.  9 B . 
         FIG.  10 A  is an example of a scanning line satisfying conditions (1) and (2). 
         FIG.  10 B  is an example of a profile obtained in  FIG.  10 A . 
         FIG.  11    is a diagram showing a state in which the controller  200  obtains a coordinate Xc of an actual center in step S 605 . 
         FIG.  12 A  is a diagram showing a state in which a profile is obtained along a scanning line in the Y direction in S 607 . 
         FIG.  12 B  is an example of the profile obtained in  FIG.  12 A . 
         FIG.  13    is a plan view corresponding to a dimension example. 
         FIG.  14    is a diagram illustrating conditions (1) and (2) in Embodiment 2. 
         FIG.  15    shows an example of a case where a well bottom surface is directly detected when there is only one high-luminance region. 
     
    
    
     DESCRIPTION OF EMBODIMENTS 
     Embodiment 1 
       FIG.  1    is a configuration diagram of a specimen observation apparatus  100  according to Embodiment 1 of the disclosure. The specimen observation apparatus  100  is an apparatus that observes a specimen by obtaining an image of the specimen contained in a specimen container  101 . The specimen observation apparatus  100  includes an objective lens  102 , an objective lens actuator  103 , a dichroic mirror  104 , an optical pickup  105 , an imaging element  106 , an illumination  107  (light source), a specimen container holder  108 , an XY stage  109 , and a controller  200 . The controller  200  controls each unit provided in the specimen observation apparatus  100 . 
     The specimen container holder  108  holds one or more specimen containers  101 . Although each specimen container  101  in  FIG.  1    is assumed to be a multi-well plate, the type of the container is not limited thereto. The container may have any shape as long as the shape has one or more specimen containing portions and a determination method to be described later can be applied thereto. Hereinafter, a well of the multi-well plate corresponds to the specimen containing portion, and a well bottom surface is a specimen observation area, which is a target to be imaged. 
     The specimen container holder  108  is connected to the XY stage  109 . The XY stage  109  is a drive mechanism capable of moving the specimen container holder  108  in an X direction and a Y direction (two directions along the horizontal direction). Since an imaging field of view and the specimen container  101  may move relative to each other, an imaging system including the objective lens  102  and the imaging element  106  may move instead of moving the specimen container holder  108 . In the operation schematic diagram used in Embodiment 1, for convenience of description, the imaging field of view is shown as moving relative to the position of the fixed specimen container  101 . The imaging element  106  has 5 million pixels (2500×2000). 
     After a user of the apparatus or another automatic transport apparatus places the specimen container  101  on the specimen container holder  108 , the controller  200  performs an imaging operation. The controller  200  drives the XY stage  109  to adjust the position of the specimen container holder  108  in the X direction and the Y direction, aligns the imaging field of view with the well bottom surface, and positions a focal point of the objective lens  102  in the vicinity of the well bottom surface by the objective lens actuator  103  that is driven in a Z direction. By the above procedure, an image of the well bottom surface can be obtained. 
     The optical pickup  105  is incorporated with a laser diode and a photodiode, and drives the objective lens actuator  103  such that the focal point of the objective lens  102  is positioned in the vicinity of the well bottom surface when the photodiode in the optical pickup  105  detects reflected light of laser irradiated to the well bottom surface. As a result, the optical pickup  105  and the objective lens actuator  103  operate as an auto-focus mechanism of the objective lens  102 . Since auto-focus cannot be performed unless an XY position of the imaging field of view is aligned with the well bottom surface, an image that can be captured before auto-focus is executed is defocused, in principle. The auto-focus method is not limited to a method using the laser, and may also be a method of evaluating image contrast or a method using a phase difference. It is assumed that a height of the specimen in the Z direction is about several microns, and a target position of the auto-focus is on the well bottom surface or above the well bottom surface by several microns. 
       FIG.  2    is an example of a shape of the specimen containing portion of the specimen container  101 . An upper part of  FIG.  2    is a side cross-sectional view of the specimen container  101 . A lower part of  FIG.  2    shows a distribution of illumination transmitted light from above the specimen container  101 . The specimen container  101  includes an inclined portion such that a planar region perpendicular to an optical axis (hereinafter, this region is referred to as the well bottom surface or the bottom surface) is surrounded. Transmitted light is reduced by refraction at a boundary between the well bottom surface portion and the inclined portion. As a result, an annular low-luminance region S is generated. 
       FIG.  3    is a diagram illustrating refraction of light in the vicinity of the inclined portion of the specimen container  101 . When an inclination angle of the inclined portion is 30 degrees and a thickness of the bottom surface is 1.2 mm, a deviation (ΔS) of 0.5 mm occurs due to refraction of light at the inclined portion. A diameter of the annular low-luminance region S is (a diameter of the well bottom surface)+2×0.5 mm. This numerical example is a calculated value based on an actual object, and may be slightly different from a dimension in an actual defocus image. 
       FIG.  4    is an example of the defocus image in a case where a periphery of the specimen container  101  is actually imaged at a wide viewing angle. The annular low-luminance region S can be visually recognized. The low-luminance region S corresponds to a refracted light image obtained by imaging light refracted by the inclined portion. 
       FIG.  5    is a schematic diagram showing a state in which a design value of a central position of a bottom surface of the specimen container  101  is different from an actual value. In a manufacturing stage of the specimen observation apparatus  100 , coordinates are defined. In a coordinate system of the XY stage  109 , coordinates (X 0 +ax, Y 0 +ay) are set such that a center of the imaging field of view coincides with a design central position of each well. When a position at which a distance between the design central position and the center of the imaging field of view is (ax, ay) is defined as an operation start point, XY stage coordinates of the operation start point are (X 0 , Y 0 ). An operation region (X 0 +bx, Y 0 +by) is set so as to include at least an actual central position of the well. Specifically, when a design value of the diameter of the well bottom surface is 1.5 mm and the design central position of the well is (X 0 +ax±1 mm, Y 0 +ay±1 mm), values of ax and ay should be about 1 mm+(a radius of the well bottom surface)=1.75 mm. 
     An actual operation of the apparatus will be described. First, a focal position of the objective lens  102  is moved above the well bottom surface such that a defocus image of the well bottom surface can be reliably obtained. Alternatively, the apparatus is set in advance such that the focal position of the objective lens  102  is located above the well bottom surface in a state in which the auto-focus is not performed (at an origin of the objective lens actuator  103 ). Although the actual central position of the well can be subjected to image determination even if a captured image is an in-focus image, erroneous determination may occur due to an influence of minute damage on the well bottom surface or the like. Therefore, in Embodiment 1, the actual central position of the well is determined using the defocus image. 
     The XY stage  109  moves the imaging field of view within the operation region (X 0 +bx, Y 0 +by) with the operation start point (X 0 , Y 0 ) serving as a start point. The imaging element  106  performs imaging at regular intervals, and obtains images of the whole or a part of the imaging field of view. A series of operations of obtaining the images at regular intervals while driving the XY stage  109  is referred to as scanning. 
     The operation of the XY stage  109  may or may not be stopped during exposure. In the case of not stopping, in order to prevent image blur, it is desirable that the time for moving over a distance corresponding to one pixel is shorter than an exposure time for obtaining the image. As a specific example, when the exposure time is 500 μs and pixel resolution is 0.345 μm/pixel, it is considered that no image blur will occur if a moving speed of the XY stage  109  is 0.69 mm/s or less. However, in Embodiment 1, since the well central position is identified by the defocus image, there is a high possibility that image blur of several pixels will not cause any problem. 
     The controller  200  analyzes information on a luminance value of the obtained image (hereinafter, referred to as image information) to derive XY stage coordinates that allow the actual well central position and the center of the imaging field of view to coincide with each other. A specific procedure will be described later. The term “image information” as used herein refers to coordinates where the image is obtained, a sum of luminance values in the image, an average of the luminance values, a mode value of the luminance values, and the like. 
       FIG.  6    is a flowchart illustrating a procedure of identifying the actual well central position. In Embodiment 1, the actual well central position is identified using the fact that the well bottom surface is a region surrounded by the low-luminance region S. An image obtained during scanning is a part of the imaging field of view (for example, a square image of 900 pixels). This image is referred to as a partial image. Although the partial image may be captured at any part of all the pixels of the imaging element  106 , the partial image is assumed to be a central part of the imaging element here. In this flowchart, coordinates and a sum of luminance values are used as the image information. Hereinafter, each step of  FIG.  6    will be described. 
     (FIG.  6 : Step S 601 ) 
     The controller  200  sets the center of the imaging field of view to the operation start point (X 0 , Y 0 ). A planar image of this step is shown in  FIG.  7    to be described later. 
     (FIG.  6 : Step S 602 ) 
     The controller  200  moves the imaging field of view by bx along a scanning line in the X direction. That is, the center of the imaging field of view is moved from (X 0 , Y 0 ) to (X 0 +bx, Y 0 ). A planar image after the movement is shown in  FIG.  8 A  to be described later. While the imaging field of view is moved, the controller  200  obtains the partial image for N times at an interval ΔX. The controller  200  stores the obtained partial image in a memory, analyzes the partial image immediately, and calculates a sum of luminance values of all pixels (here, 900 pixels) in the partial image. The controller  200  stores, in the memory, a correlation (referred to as a profile) between the sum of the luminance values and coordinates where the partial image is obtained. 
     (FIG.  6 : Step S 603 ) 
     The controller  200  analyzes the profile obtained in S 602  and determines whether the following two conditions are satisfied. However, condition (2) is determined only for a profile that satisfies condition (1). An example of a planar image in a case where the conditions are satisfied is shown in  FIG.  10 A  to be described later together with significance of each condition. When the conditions are satisfied, the process proceeds to S 605 , and when the conditions are not satisfied, the process proceeds to S 604 . 
     Condition (1): in the profile, there are two or more low-luminance regions below a determination threshold value. 
     Condition (2): in the profile, a width ΔH of a high-luminance region interposed between the two low-luminance regions is within a set allowable range. 
     (FIG.  6 : Step S 604 ) 
     The controller  200  moves the imaging field of view in the Y direction by ΔY, returns to S 602 , and repeats the same process. In order to shorten the scanning time, it is desirable to alternately switch a scanning direction between a +X direction and a −X direction each time when S 602  is performed. An example of scanning in the −X direction is shown in  FIG.  9 B  to be described later. 
     (FIG.  6 : Step S 605 ) 
     The controller  200  sets a center of the width ΔH of the high-luminance region interposed between the two low-luminance regions as a coordinate Xc of an actual center of the well in the X direction. Xc corresponds to an average value of X coordinate points in the high-luminance region. A specific example of this step is shown in  FIG.  11    to be described later. 
     (FIG.  6 : Steps S 606  to S 608 ) 
     The controller  200  moves the imaging field of view to (Xc, Y 0 ) (S 606 ). In the same manner as S 602 , the controller  200  obtains a profile by moving the imaging field of view by “by” along a scanning line in the Y direction (S 607 ). In the same manner as S 605 , the controller  200  sets the center of the width ΔH of the high-luminance region interposed between the two low-luminance regions as a coordinate Yc of the actual center of the well in the Y direction (S 608 ). An example of a state in which S 606  to S 608  are performed is shown in  FIGS.  12 A and  12 B  to be described later. 
     According to this flowchart, the actual central position and the center of the imaging field of view can coincide with each other. After this flowchart is performed, the controller  200  executes auto-focus, and images the specimen by the imaging element  106 . The obtained image is used for observation and analysis of the specimen. 
       FIG.  7    is a diagram showing a positional relationship when the controller  200  obtains an observation image in step S 601 . The center of the imaging field of view is set to (X 0 , Y 0 ). 
       FIG.  8 A  is a diagram showing a state in which the imaging field of view is moved by bx in the X direction in step S 602 . In  FIG.  8 A , partial images are obtained at 11 positions (N=11) including the operation start point. 
       FIG.  8 B  is a graph showing a sum of luminance values of the partial images obtained in  FIG.  8 A . The vertical axis represents the sum of the luminance values of the partial images, and the horizontal axis represents the X coordinates of the partial images. In  FIG.  8 A , since all the partial images are obtained at bright regions, the sum of the luminance values of the respective partial images is equal to or higher than the threshold value. It is necessary to set the X coordinate interval ΔX at which the partial images are obtained to be sufficiently small. Specifically, it is desirable to set ΔX to ½ to ⅓ of ΔS (the width of the low-luminance region) or less. 
       FIG.  8 C  is a diagram showing a modification of step S 602 . In S 602 , as shown in  FIG.  8 C , the partial images may be obtained simultaneously on a plurality of scanning lines extending in the same direction. As a result, the scanning time can be shortened. 
       FIG.  9 A  is a diagram showing a state in which the controller  200  moves the imaging field of view by ΔY in the Y direction in step S 604 . This step is for moving to the next scanning line adjacent in the Y direction when the conditions (1) and (2) are not satisfied on the current scanning line in S 603 . 
       FIG.  9 B  is a diagram showing a state in which S 602  is performed following  FIG.  9 A . Here, an example in which the imaging field of view is scanned in the −X direction is shown. As a result, it is not necessary to scan again in the +X direction after the imaging field of view is returned to X 0 , and thus the scanning time can be reduced. 
       FIG.  9 C  is an example of a profile obtained in  FIG.  9 B . In the profile of  FIG.  9 C , since there is only one low-luminance region, the condition (1) is not satisfied. Therefore, the controller  200  moves the imaging field of view by ΔY in the Y direction again in S 603  and S 604 . Such an operation is repeated, and a region of bx×by is scanned until the condition is satisfied. In order to reliably detect the well bottom surface, ΔY is preferably set to be equal to or less than the diameter of the well bottom surface. 
       FIG.  10 A  is an example of a scanning line satisfying the conditions (1) and (2). The conditions (1) and (2) have the significance of identifying the scanning line that intersect the annular low-luminance region S and the bottom surface portion inside the low-luminance region S, respectively. When luminance values are obtained along the scanning line of  FIG.  10 A , a luminance value of the bottom surface portion is high while luminance values of the low-luminance regions S on both sides thereof are low. Therefore, on the profile, the high-luminance region is interposed between the two low-luminance regions. In other words, there is one high-luminance region interposed between the two low-luminance regions. 
       FIG.  10 B  is an example of the profile obtained in  FIG.  10 A . The condition (1) can be determined by distinguishing the high-luminance region and the low-luminance region by a determination threshold value. As the determination threshold value, an appropriate value that can distinguish the well bottom surface and the region S from each other based on an actual image is set in advance. In  FIG.  10 B , there are two low-luminance regions. For the condition (2), the width ΔH of the high-luminance region interposed between the low-luminance regions is calculated, and it is confirmed that the width ΔH is within the allowable range. 
     Here, the allowable range of ΔH is set to ΔX≤ΔH≤(the diameter of the well bottom surface). That is, it is confirmed that the high-luminance region in the profile is continuous at two or more points, and the width is equal to or shorter than the diameter of the well bottom surface. By excluding cases where the high-luminance region includes only one point, an effect of reducing erroneous determination and increasing calculation accuracy of the central position can be achieved. If a lower limit value of the allowable range is further increased, the calculation accuracy can be expected to be improved, while the time taken until the determination is completed increases. 
       FIG.  11    is a diagram showing a state in which the controller  200  obtains the coordinate Xc of the actual center in step S 605 . Here, the same profile as that of  FIG.  10 B  is shown. Since the high-luminance region on the profile includes two coordinate points, the controller  200  calculates the midpoint as Xc. 
       FIG.  12 A  is a diagram showing a state in which a profile is obtained along the scanning line in the Y direction in S 607 . An interval at which the images are obtained is assumed to be ΔY. When scanning is performed in the Y direction at a location where the X coordinate is Xc, the scanning line passes through the well bottom surface interposed between the low-luminance regions S. Therefore, the scanning line at this time satisfies the conditions (1) and (2). 
       FIG.  12 B  is an example of the profile obtained in  FIG.  12 A . In the same manner as in  FIG.  10 B , the width ΔH of the high-luminance region interposed between the two low-luminance regions is calculated, and it is confirmed that the width ΔH is within the allowable range. The allowable range of ΔH is set to ΔY≤ΔH≤(the diameter of the well bottom surface). As in  FIG.  10 B , the cases where the high-luminance region includes only one point are excluded. The center point coordinate Yc can be obtained by an average value of coordinate points of the high-luminance region. 
     Embodiment 1: Calculation Example 
     Specific numerical values are used to estimate the time required for the series of operations. The diameter of the well bottom surface is 1.5 mm, ΔS is 0.5 mm, tolerance of the position of the well is±1 mm in the X and Y directions, ax is 1.8 mm, ay is 1.7 mm, bx is 3.6 mm, by is 3.6 mm, ΔX is 0.2 mm, ΔYs is 1.2 mm, and ΔY is 0.2 mm. Assuming that an expected value of the actual central position of the well bottom surface coincides with a design central position, an average processing time is calculated. 
       FIG.  13    is a plan view corresponding to the dimension example described above. An X-direction moving distance until Xc is calculated is 3.6 mm (bx)×2=7.2 mm. In the case of moving at an average speed of 0.69 mm/s, it takes 10.4 seconds for each movement in the X direction. Calculation of the sum of the luminance values is performed for 0.29 seconds during the ΔX movement. While moving by 1.2 mm (ΔY) in the Y direction, the moving may be performed at a relatively high speed during this time, and if it is assumed that moving is performed at an average of 20 mm/s, the time required is 0.06 seconds. After Xc is determined, a moving time from coordinates (X 0 , Y 0 +1.2) to coordinates (X 0 +1.8, Y 0 ) is 0.09 seconds when X and Y simultaneously start moving at an average of 20 mm/s. Scanning in the Y direction takes 5.2 seconds, assuming that 3.6 mm is moved at an average of 0.69 mm/s. These taken times are summed up to obtain the required time, which is 10.4+0.06+0.09+5.2=15.75 seconds. 
     Embodiment 1: Summary 
     The specimen observation apparatus  100  according to Embodiment 1 identifies the central position (Xc, Yc) of the bottom surface of the specimen container  101  by using the number of the high-luminance regions interposed between the low-luminance regions and the width ΔH thereof, prior to performing the auto-focus of the objective lens  102 . Since it is not necessary to align the focal position with a specimen surface when the central position is identified, the central position can be identified even if the specimen observation area and the imaging field of view are deviated to such an extent that the auto-focus becomes impossible. Since the defocus image is evaluated, the image can be analyzed without being affected by disturbance elements (for example, damage, scratches, or micro-cracks generated at the time of manufacturing the specimen container) present on an actual specimen observation surface. In addition, since it is not necessary to detect the shape of the bottom surface of the specimen container  101 , image blur of about several pixels can be allowed. Therefore, the operation speed of the XY stage  109  can be increased relative to the exposure time, and the time required for the determination can be shortened. 
     Since the specimen observation apparatus  100  according to Embodiment 1 analyzes only a part of the imaging field of view as the obtained image, even if the scanning range (bx x by) is widened, an amount of data to be subjected to image processing is small, and thus an image processing time can be shortened. Therefore, image processing capacity can be kept small, which is advantageous in terms of cost. 
     Embodiment 2 
     In Embodiment 2 of the disclosure, a method of identifying the central position of the well bottom surface by detecting a position and a width of the annular low-luminance region S will be described. In Embodiment 2, the following conditions are used as the determination conditions (1) and (2). Regardless of the condition (1), the central position is an average of coordinate points in the low-luminance region. Other items such as the configuration and the operation flow of the specimen observation apparatus  100  are the same as those of Embodiment 1. 
     Condition (1): in the profile, there is one or two low-luminance regions below the determination threshold value. 
     Condition (2): Widths ΔL of all the low-luminance regions in the profile are within a set allowable range. 
       FIG.  14    is a diagram illustrating the conditions (1) and (2) in Embodiment 2. Hereinafter, the conditions (1) and (2) and the allowable range in Embodiment 2 will be described using two scanning lines shown in  FIG.  14   . 
     When there is only one low-luminance region on the scanning line (condition (1)), the scanning line passes through the low-luminance region without crossing the well bottom surface. For example, the scanning line on an upper side of  FIG.  14    corresponds to this case. The allowable range of ΔL (condition (2)) is suitably equal to or higher than ΔX and equal to or less than a maximum length of the low-luminance region along the scanning line. This is because, in Embodiment 2, since the low-luminance region is detected, as the allowable range, it is necessary to set a range that is appropriate as the width of the low-luminance region. Specifically, as indicated by the scanning line on the upper side of  FIG.  14   , when the scanning line is in contact with an image of the well bottom surface, the length of the low-luminance region is maximized. ΔL is equal to or less than the maximum length. 
     When there are two low-luminance regions on the scanning line (condition (2)), the scanning line passes through the low-luminance region=&gt;the high-luminance region (well bottom surface)=&gt;the low-luminance region in this order. For example, the scanning line on a lower side of  FIG.  14    corresponds to this case. The allowable range of ΔL (condition (2)) is suitably equal to or higher than ΔX and equal to or less than the width of the low-luminance region S (0.5 mm in the example of  FIG.  4   ). This is because, in Embodiment 2, since the low-luminance regions are detected, as the allowable range, it is necessary to set a range that is appropriate as the width of each low-luminance region. 
     Embodiment 2: Summary 
     The specimen observation apparatus  100  according to Embodiment 2 can identify the well central position particularly even if there is one low-luminance region under the condition (1). That is, the central position can be identified at a relatively early stage when scanning the range (bx, by) to be scanned. As a result, a distance by which the imaging field of view is moved in order to identify the central position can be shortened, which is advantageous since the central position can be identified more quickly. 
     Embodiment 3 
     In Embodiment 3 of the disclosure, a method of directly detecting the well bottom surface will be described. In Embodiment 2, the following conditions are used as the determination conditions (1) and (2). Regardless of the condition (1), the central position is an average of coordinate points in the high-luminance region. Other items such as the configuration and the operation flow of the specimen observation apparatus  100  are the same as those of Embodiment 1. 
     Condition (1): in the profile, there are N high-luminance regions exceeding the determination threshold value (N≥1). 
     Condition (2): The width ΔH of a high-luminance region at one location on the profile is within a set allowable range, while the widths ΔH of high-luminance regions at (N−1) locations are not within the set allowable range. 
     In the examples of the upper scanning lines in  FIGS.  10 A and  14   , there are three high-luminance regions (corresponding to a case where N=3 under the condition (1)). It is assumed that the width ΔH of the high-luminance region corresponding to the well bottom surface is within a design tolerance range of the well bottom surface. Therefore, it is appropriate that the allowable range of ΔH is (the design value of the diameter of the well bottom surface−a lower limit tolerance of the diameter of the well bottom surface)≤ΔH≤(the design value of the diameter of the well bottom surface+an upper limit tolerance of the diameter of the well bottom surface). Meanwhile, it is considered that the widths of the high-luminance regions located on an outer side relative to the low-luminance regions do not fall within the design tolerance range of the well bottom surface. Therefore, by setting the allowable range as described above, only the high-luminance region corresponding to the well bottom surface can be detected. When the diameter of the well bottom surface is 1.5 mm±0.1 mm, 1.4≤ΔH≤1.6. 
       FIG.  15    shows an example of a case where the well bottom surface is directly detected when there is only one high-luminance region. Depending on the size of the range (bx, by) to be scanned, there may be a case where only one high-luminance region corresponding to the well bottom surface is included even if the entire range is scanned.  FIG.  15    shows an example thereof. In this case, even if N=1 under the condition (1), if ΔH falls within the design tolerance range, the high-luminance region can be regarded as the well bottom surface. 
     However, this method is suitable for a case where a well position error is relatively small and a possibility of erroneous detection is small even if the range (bx, by) to be scanned is set to be small to some extent. This is because, when the well position error is large, depending on the size of the range (bx, by) to be scanned and an initial position, there is a possibility that a high-luminance region located on the outer side of the low-luminance regions is erroneously recognized as the well bottom surface. 
     Embodiment 3: Summary 
     The specimen observation apparatus  100  according to Embodiment 3 detects the high-luminance region, and distinguishes whether the high-luminance region is the well bottom surface according to the preset allowable range. As a result, the well bottom surface to be searched can be directly found, and thus a determination algorithm can become relatively simple. 
     Embodiment 4 
     In Embodiment 4 of the disclosure, an example in which the entire imaging field of view is set as an image to be obtained will be described. The configuration of the specimen observation apparatus  100  is the same as that of  FIG.  1   . In Embodiment 4, the conditions (1) and (2) described in Embodiments 1 to 3 are not used, and the central position of the well bottom surface is identified by the following procedure. 
     It is assumed that the imaging element  106  has 5 million pixels of 2500×2000, and the pixel resolution is 0.345 μm/pixel. An actual field of view is 0.86 mm×0.69 mm. Each of bx and by is an integer multiple of 0.86 mm and 0.69 mm, respectively, ΔX is 0.86 mm and ΔYs is 0.69 mm. A region of bx×by is scanned to stack images. The stacked images are combined as shown in  FIG.  4   , and a region having a high luminance value is determined based on a preset threshold value. The well bottom surface is determined by comparing an area of the determined high-luminance-value region with an allowable area value. When the diameter of the well bottom surface is 1.5 mm±0.1 mm, an allowable area range is 1.5 mm 2  to 2.0 mm 2 . 
     However, when the well position error is large, that is, when bx and by are large, a high-luminance region outside the well may be recognized as the well bottom surface. In this case, the well bottom surface can be determined by evaluating shape feature values such as an aspect ratio and circularity of the high-luminance-value region. A geometric center of the determined high-luminance region is calculated, and Xc and Yc are determined at the same time. 
     &lt;Modification of Disclosure&gt; 
     The disclosure is not limited to the embodiments described above, and has various modifications. For example, the embodiments described above have been described in detail for easy understanding of the disclosure, and the invention is not necessarily limited to those including all the configurations described above. In addition, a part of the configurations of one embodiment can be replaced with the configurations of another embodiment, and the configurations of the other embodiment can be added to the configurations of the one embodiment. In addition, a part of the configurations of each embodiment may be added, deleted, or replaced with other configurations. 
     In the embodiments described above, the controller  200  may be implemented by hardware such as a circuit device on which the functions are implemented, or may be implemented by executing software in which the functions are implemented by an arithmetic apparatus such as a central processing unit (CPU). 
     In the embodiments described above, the imaging element  106  may be disposed on a transmission side of the dichroic mirror  104 , and the optical pickup  105  may be disposed on a reflection side. In addition, an appropriate optical component such as an optical filter (not shown) may be disposed on the optical path. 
     In the embodiments described above, the focal position of the objective lens  102  is set above the well bottom surface (inside the specimen container  101 ). In a case where a central axis deviation between a well outer bottom surface and the well bottom surface (see  FIG.  2   ) is small and negligible, the focal position of the objective lens  102  may be set below the well bottom surface instead of being above the well bottom surface. In this case, a defocus image of the well outer bottom surface is obtained and analyzed. A procedure for identifying the central position is the same as that of the embodiments described above. 
     Although the central position coordinates are determined in the order of Xc and Yc in the embodiments described above, this order may be changed. 
     Although it has been described in Embodiment 4 that the threshold value for determining the high-luminance region is set in advance, the threshold value may be automatically set for each observation image by using any known method of automatically setting the threshold value. 
     In the embodiments described above, an example has been described in which the well bottom surface is circular, and the inclined portion around the well bottom surface is also arranged concentrically relative to the well bottom surface. The well shape whose central position can be identified by the invention is not limited thereto, and the invention can also be applied to other shapes in which the low-luminance region is formed around the well bottom surface by the inclined portion. For example, when the well bottom surface and the low-luminance region around the well bottom surface are line-symmetric along the X direction (relative to the Y axis), a coordinate average of the high-luminance region or the low-luminance region can be regarded as Xc. Similarly, in the case of being line-symmetric along the Y direction (relative to the X axis), the coordinate average of the high-luminance region or the low-luminance region can be regarded as Yc. 
     The embodiments described above may be used in combination. For example, it is conceivable to finally identify the central position by averaging central position coordinates identified in the case where there is only one low-luminance region under the condition (1) described in Embodiment 2 and central position coordinates identified in Embodiment 1. Alternatively, it is conceivable that a reliability coefficient may be determined in advance for each embodiment, and the central position may be finally identified by adding up results of multiplying the central position coordinates identified in each embodiment by the reliability coefficient. Other appropriate methods may be used in combination with the embodiments. 
     REFERENCE SIGNS LIST 
       100 : specimen observation apparatus 
       101 : specimen container 
       102 : objective lens 
       103 : objective lens actuator 
       104 : dichroic mirror 
       105 : optical pickup 
       106 : imaging element 
       107 : illumination 
       108 : specimen container holder 
       109 : XY stage