JP5912752B2 - Sample holding plate and image acquisition method - Google Patents

Description

  The present invention relates to a sample holding plate having a recess capable of holding a liquid and an image acquisition method for acquiring an image of the recess.

  In medical and biological science experiments, for example, a liquid or gel-like fluid (for example, a culture solution or a medium) is injected into each well of a plate-like instrument provided with a large number of depressions, also called wells. Here, observation and measurement using a cultured cell or the like as a sample is performed (see, for example, Patent Document 1). Such an instrument is called, for example, a microplate or a microtiter plate. In recent years, a sample is imaged with a CCD camera or the like and converted into data, and the image data is subjected to various image processing and used for observation and analysis.

  One aspect of observation performed using this type of instrument is time-lapse observation. In this method, in order to observe temporal changes of cultured living cells or the like, the same sample is imaged a plurality of times at a predetermined time interval, and these images are comparatively observed. In this case, since the culture is continued in a certain environment, the sample may be stored in a thermostat called an incubator except for the imaging period.

JP 2010-032487 A

  When comparing a plurality of individually captured images, precise alignment between them is necessary. However, when the plate is placed on the imaging apparatus, it is inevitable that the plate placement position varies. Further, it is conceivable to perform positioning by abutting a plate against an abutting member provided in the imaging apparatus, but there is a concern about the influence caused by an impact applied to cells to be cultured.

  In order to avoid such a problem, there is a demand for a technique capable of performing alignment even from an image captured with position variations included, but such a technique has not been established. In particular, the cross-sectional shape of the well is generally a circular cross-section, and even if the plate was imaged in a state of rotating from its original position, it was impossible to grasp it later from the image. .

  This invention is made in view of the said subject, The sample holding plate which can acquire the information for grasping | ascertaining the attitude | position of a hollow part from the image imaged including position shift later, and the hollow part of this plate An object of the present invention is to provide an image acquisition method for acquiring the image.

One aspect of the present invention is a sample holding plate provided with a plurality of depressions capable of holding a liquid on the upper surface of a plate-like member, and in order to achieve the above object, for each of the depressions , inside of the recess, the bottom surface of the recess, and, adjacent region adjacent to the recess of the main surface of the plate-like member, the at least one location of an optically detectable alignment marks are provided, The said alignment mark provided with respect to the said one recessed part has an asymmetry with respect to rotation of the said recessed part around the axis | shaft orthogonal to the upper surface of the said plate-shaped member, It is characterized by the above-mentioned.

In the invention configured as described above, when the recess is imaged, the alignment mark can be included in the image along with the contents of the recess . Therefore, this alignment mark can be used as information for position reference when grasping the posture of the recess. For example, the same recess in comparing overlapping a plurality of images captured, by matching the A line instrument marks each other, it is possible to align the two images.

Here, alignment marks provided for each of the plurality of recesses are those having an asymmetry with respect to the rotation of the recess around the axis perpendicular to the upper surface of Plate-like member. In this way, even when an image tilt due to the rotation of the sample holding plate occurs, it can be detected later from the position and tilt of the alignment mark.

  In addition, for example, the alignment mark may include a plurality of graphic patterns having different shapes, which are provided in at least two locations apart from each other in the interior of the recess, the bottom surface of the recess, or the adjacent region. Good. By configuring the alignment mark with a graphic pattern provided at a plurality of locations, alignment can be performed even when one graphic pattern is shielded by the contents of a recess such as a cultured cell. Further, when the graphic patterns are made different, alignment between the images is automatically performed by performing alignment between the same graphic patterns among the plurality of images.

  In this case, a plurality of depressions may be provided on the upper surface of the plate-like member, and the combination of the shape of the plurality of graphic patterns and the arrangement thereof may be different between the plurality of depressions. In this way, since the pattern of the alignment mark is different between images obtained by imaging each of the plurality of depressions, it is possible to easily determine which depression is the image from only the image.

  For example, the cross-sectional shape of the recess in the cut surface parallel to the upper surface of the plate-like member may be a circle, an ellipse, or a regular polygon. In the concave portion having such rotational symmetry, it may be difficult to uniquely detect the amount of inclination of the image due to the rotation of the sample holding plate from its outline. By providing, reliable information as a position reference can be obtained, so that the amount of inclination of the image can be uniquely detected.

  In these cases, the alignment mark may be configured to be transparent to visible light, but opaque to light having a specific wavelength component outside the visible light region. According to such a configuration, the image of the alignment mark does not appear in the image of the contents of the depression imaged in the visible light range, so the alignment mark does not affect the image observation. On the other hand, since the alignment mark can be imaged with light outside the visible light region, there is no problem in the function of the alignment mark as a position reference.

  Further, for example, the sample holding plate according to the present invention includes a plate-like member having a through-hole penetrating from the upper surface to the lower surface, and a sheet-like member that contacts the lower surface of the plate-like member and closes the lower surface side opening of the through-hole. The space surrounded by the side surface of the through hole and the portion of the upper surface of the sheet-like member that faces the through hole may form a recess. By adopting such a configuration, it is possible to industrially efficiently produce a sample holding plate suitable for observing cells and the like. Further, the material can be selected independently when the wall surface and the bottom surface of the recess are formed.

  In addition, the alignment mark may be formed as a through-hole penetrating between the upper surface and the lower surface of the plate-like member, for example. According to such a configuration, it is possible to cope with imaging from either the upper surface side or the lower surface side of the plate-like member.

The image acquisition method according to the present invention, for achieving the above object, a plurality of recesses are provided on the upper surface of the plate-like member, for each of the recess, the inside of the recess, the recess portion the bottom, and, adjacent region adjacent to the recess of the main surface of the plate-like member, the at least one location of the sample holding plate optically detectable alignment marks are provided, in a substantially horizontal position A holding step for holding, and an imaging step for capturing an image of the contents of the recess by capturing the recess while including the alignment mark in an imaging range, and provided for the one recess The alignment mark thus formed is asymmetric with respect to the rotation of the recess around an axis orthogonal to the upper surface of the plate-like member .

  In the invention configured as described above, an image of a recess including an alignment mark serving as a position reference is obtained. Then, by using this alignment mark as information for alignment, for example, the inclination of the image can be detected, or alignment between a plurality of images captured at different timings can be performed afterwards. It becomes possible. In addition, since the image shift is grasped in this way, the requirement for positioning accuracy in holding the sample holding plate can be relaxed. Therefore, the apparatus cost for performing imaging can be suppressed.

  Here, in the imaging process, for example, the visible light component light emitted from the depression is received to image the contents of the depression, and the wavelength component light outside the visible light region emitted from the depression is received. Then, the alignment mark may be imaged. This eliminates the need for the alignment marks provided on the sample holding plate to be visible with visible light. Therefore, for example, the alignment mark can be made transparent to visible light so that it is not reflected in the image to be observed.

  In addition, a series of processes including a holding process and an imaging process are performed a plurality of times on the same sample holding plate to acquire a plurality of images of the same depression, and relative positional deviations between the plurality of images are obtained. You may make it further provide the correction process correct | amended based on the alignment mark detected from the image.

  When holding and imaging the sample holding plate repeatedly, it is extremely difficult to make the holding position of the sample holding plate completely the same. On the other hand, by performing imaging including the alignment mark, the relative displacement of the image due to the variation in the holding position can be corrected afterwards using the alignment mark as a position reference. By performing such correction, comparative observation of a plurality of images can be performed efficiently.

  According to this invention, an image including an alignment mark can be taken together with the contents of the recess, and the alignment mark can be used as information indicating the position reference of the image. For this reason, from the obtained image, it is possible to grasp the inclination of the image and the relative positional deviation between the plurality of images afterwards.

It is a figure which shows schematic structure of the one aspect | mode of the imaging device which can apply this invention suitably. It is a figure which shows the structure of a microplate in detail. It is a figure which shows the example of the alignment mark seen from the plate upper surface. 3 is a flowchart illustrating image acquisition processing in the imaging apparatus of FIG. 1. It is a figure which shows the comparative example of a time lapse image set. It is a figure which shows the example of the time lapse image set by this embodiment.

  FIG. 1 is a diagram showing a schematic configuration of an aspect of an imaging apparatus to which the present invention can be preferably applied. The imaging apparatus 1 is brought into contact with the peripheral edge of the lower surface of a sample (microplate) 2 in which a plurality of, for example, 96 (12 × 8 matrix arrangement) wells W are formed, so that the microplate 2 is in a substantially horizontal state. A holder 11 to be held, a light source 12 provided on the upper part of the holder 11, an imaging unit 13 provided on the lower part of the holder 11, and a control unit 10 for controlling these to execute a predetermined operation. Yes. For the following description, coordinate axes are set as shown in FIG. The XY plane is a horizontal plane, and the Z axis is a vertical axis.

  The light source 12 is controlled by a light source control unit 112 provided in the control unit 10 and is collectively applied to a plurality of wells W from above the microplate 2 held by the holder 11 in accordance with a control command from the light source control unit 112. Then, the light L is irradiated. The irradiated light is visible light, and white light is particularly preferable.

  The imaging unit 13 functions as a camera that captures an image of the microplate M by receiving transmitted light Lt emitted from the light source 12 and transmitted below the microplate 2 held by the holder 11. . The imaging unit 13 is connected to a camera driving mechanism 113 provided in the control unit 10, and the camera driving mechanism 113 scans and moves the imaging unit 13 in the horizontal plane along the lower surface of the microplate 2 held by the holder 11. Let

  That is, in this embodiment, the imaging unit 13 can be scanned and moved along the lower surface of the microplate 2. Here, the imaging unit 13 moves with respect to the microplate 2, but it is sufficient that the relative movement between the imaging unit 13 and the microplate 2 is realized. In this sense, the microplate 2 is moved with respect to the imaging unit 13. You may make it move.

  Image data picked up by the image pickup unit 13 is given to the image processing unit 114. The image processing unit 114 appropriately performs image processing on the image data from the imaging unit 13 or executes predetermined calculation processing based on the image data. Data before and after processing is stored and saved in the storage unit 115 as necessary. Further, the detection processing unit 116 performs a predetermined detection process based on the image data given from the image processing unit 114, and detects a characteristic part included in the image. This detection process is a process for detecting areas in which the optical characteristics are different from the surrounding area in the image by analyzing luminance data of the image, for example, and by calculating a feature amount for the area, It is possible to classify the origin and type of the area. Since various techniques are known for the process of identifying and detecting a part having a certain feature from the image and the feature amount suitable for such a process, detailed description thereof is omitted here.

  The detection result by the detection processing unit 116 is also stored in the storage unit 115. As will be described later, the image processing unit 114 may perform image processing based on the detection result of the detection processing unit 116 as necessary. Then, the image data subjected to appropriate image processing is given to a display unit 118 having display means such as a liquid crystal display, and the display unit 118 displays an image corresponding to the given image data and presents it to the user. . Further, the imaging apparatus 1 includes an input receiving unit 117 for receiving an operation instruction input from the user regarding the contents of image processing, the display mode, and the like. The input receiving unit 117 is an input receiving unit such as a keyboard, a mouse, and a touch pad, or a combination of them. The input receiving unit 117 receives an instruction input from the user, and the control unit 10 reflects this in the operation of the apparatus. The function desired by the user is realized.

  The imaging apparatus 1 is configured such that a fluid (in this specification, a liquid, a gel-like or semi-fluid solid, and a fluid having a fluidity such as soft agar is held in each well W. And an optical image of an object to be imaged such as cells contained therein, or having a predetermined optical characteristic from the optical image, more specifically, in the well W The present invention can be applied to an application in which a specific portion having an optical characteristic different from that of a retained liquid or the like is detected using a difference in the optical characteristic. For example, it can be suitably used for the purpose of imaging a cell or a cell clump (spheroid) in a culture solution or a medium as an imaging object, or automatically detecting such a cell or the like by image processing.

  FIG. 2 is a diagram showing the structure of the microplate in more detail. As shown in FIG. 2A, the microplate 2 according to an embodiment of the present invention has a substantially cylindrical side surface (more precisely, with a taper whose sectional area gradually decreases toward the bottom surface). It has the upper plate 21 in which the holes 211 are regularly arranged in a two-dimensional matrix at a constant pitch, and the lower surface sheet 22 affixed to the lower surface of the upper plate 21 so as to close each through hole 211.

  As shown in FIG. 2B, the lower surface sheet 22 is in close contact with the lower surface of the upper plate 21, and liquid is supplied to the space surrounded by the side surface of the through hole 211 of the upper plate 21 and the lower surface sheet 22. It is possible to hold. That is, this space functions as a well W for holding a fluid, and the side surface of the through hole 211 forms the side wall surface of the well W, and the lower surface sheet 22 forms the bottom surface of the well W. The lower surface sheet 22 is a sheet formed of a transparent resin, for example, a PET (polyethylene terephthalate) resin.

  The diameter and depth of each well W in the microplate 2 are typically about several millimeters, and each well W is injected with a liquid such as a culture solution, a medium, a reagent, etc. (only a part is shown). Yes. The number of wells and the size of the microplate targeted by the imaging apparatus 1 are not limited to these and are arbitrary. Further, as long as the imaging object can be imaged, for example, it may be a well plate integrally formed as a whole.

  At the four corners of the upper surface of the microplate 2, posture detection marks 212 for detecting the posture when the microplate 2 is placed on the holder 11 are provided. The approximate position of the microplate 2 on the holder 11 can be confirmed using the posture detection mark 212. When the position of the microplate 2 is confirmed by imaging, the surface on which the mark is provided depends on the imaging direction. That is, it is formed on the surface of the upper and lower main surfaces of the microplate 2 that faces the imaging unit 13. When formed on the lower surface of the microplate 2, a posture detection mark may be formed on the lower surface sheet 22 as indicated by reference numeral 222, and the lower surface of the upper plate 21 is provided that the lower surface sheet 22 has light transmissivity. May be provided. When the upper plate 21 is formed of a transparent material, it may be provided on either the upper surface or the lower surface. In the case of a through-hole type mark, which will be described later, the upper plate 21 may be opaque because the image can be taken from either the upper or lower direction.

  The top surface of the microplate 2 has a shape in which two adjacent vertices of the four vertices of the rectangle are cut out, and the cutout portion 213 causes the microplate 2 to be asymmetric with respect to rotation in the horizontal plane. It has an outer shape. Thereby, the attitude | position of the microplate 2 in a horizontal surface is uniquely defined.

  In the microplate 2, the peripheral region of each well W, more specifically, the adjacent region R 1 adjacent to the upper surface side opening of the through hole 211 on the upper surface of the upper plate 21, the through hole in the well W Any one of the side wall surface R2 of 211, the region R3 of the bottom surface of the well W near the peripheral edge of the well, and the adjacent region R4 on the lower surface of the upper plate 21 and adjacent to the opening on the lower surface side of the through hole 211; As a through hole H penetrating between the upper surface and the lower surface of the upper plate 21, an alignment mark described later is provided. This alignment mark is provided to detect the posture of each well W in the image picked up by the image pickup apparatus 1, particularly the amount of rotation around the vertical axis.

  In the image obtained by imaging the well W by the imaging device 1, images of cells or the like previously cultured in the well W are distributed in a substantially circular outline corresponding to the side wall surface of the well W. Since the outline is circular, for example, the state in which the well W is rotated by the inclination of the microplate 2 placed on the holder 11 in the horizontal plane (XY plane), that is, the rotation about the vertical axis (Z axis). However, it is virtually impossible to grasp that from the captured image.

  Therefore, in this embodiment, an alignment mark for posture confirmation is provided in advance in each well W, and when the well W is imaged, imaging is performed in a state where the alignment mark is within the same imaging range as the well W. . In this way, by detecting the position of the alignment mark from the captured image, it becomes possible to accurately and later grasp the posture of the well W in the image, particularly the rotation amount thereof.

  FIG. 3 is a view showing an example of alignment marks as seen from the top surface of the plate. FIG. 3A shows an example in which alignment marks are formed in the well peripheral region R 1 on the upper surface of the upper plate 21. In the example on the left side of FIG. 3A, four graphic patterns P11 to P14 are provided at equiangular intervals so as to surround the periphery of the well W1. Of these, the three patterns P12 to P14 have the same shape of “+” type, but only one pattern P11 has a different shape, that is, “Δ” type. In this way, by making the pattern arrangement of the alignment marks asymmetric with respect to the rotation of the well in the horizontal plane, the rotation amount of the well W1 can be uniquely determined by detecting the position of each graphic pattern. Is possible.

  On the other hand, in the example on the right side of FIG. 3A, two “+”-type patterns P21 and P22 are provided so as to sandwich the well W2 from the left and right, and a character pattern “A1” is provided above the well W2. ing. Even with such a pattern, it is possible to uniquely determine the rotation amount of the well W2 from the position of each graphic pattern. The character pattern “A1” is an identification code for identifying the well W2 from a plurality of wells provided on the microplate 2, and an identification code by a different character is assigned to each well W. By doing so, it becomes possible to easily determine which well the image corresponds to. Thereby, mistakes, such as a mix-up of a sample, can be prevented beforehand. In addition, for example, it is possible to display information relating to the sample such as the culture conditions of the well stored separately in association with the image.

  The pattern shown in FIG. 3A is formed on the upper and lower main surfaces of the upper plate 21 in the direction in which imaging is performed. That is, the well W is formed on the upper surface of the upper plate 21 in order to correspond to the case of imaging from the upper side, and is formed on the lower surface of the upper plate 21 in order to correspond to the case of imaging from the lower side. In order to be compatible with both cases, it may be formed on both upper and lower surfaces.

  FIG. 3B shows an example in which alignment marks are formed on the well side wall surface R2. In the example on the left side of FIG. 3B, three protruding portions P31 to P33 protruding outward in the radial direction are provided on the peripheral edge of the well W3. On the other hand, in the example on the right side of FIG. 3B, three protruding portions P41 to P43 protruding radially inward are provided on the peripheral edge of the well W4. By providing such a protruding portion on the side wall surface of the well, the rotation of the well in the image can be detected. By making the arrangement of the protruding portions asymmetric with respect to the rotation of the well, the amount of rotation of the well can be uniquely determined.

  Since these alignment marks are all formed on the upper plate 21, for example, when the upper plate 21 is a resin molded product, an unevenness corresponding to the alignment mark is previously formed on a mold for manufacturing the upper plate 21. Just keep it. Further, the upper plate 21 and the lower surface sheet 22 may be processed by, for example, printing by an inkjet method, marking by a laser beam, or a method of attaching a seal having an alignment mark.

  On the other hand, FIG. 3C shows an example in which an alignment mark is formed in the well bottom region R3. In the example on the left side of FIG. 3 (c), graphic patterns P51 to P54 are provided at four positions on the peripheral edge of the bottom surface of the well W5, and two adjacent patterns P51 and P52 are “+” type and the remaining two P53. , P54 are "-" type graphic patterns. Further, in the example on the right side of FIG. 3C, “△” -shaped pattern P61 is formed on the upper part of the bottom peripheral edge of well W6, and “+”-shaped and “□” -shaped graphic patterns P62 and P63 are formed on the left and right, respectively. Has been.

  Further, in the example shown in FIG. 3D, through holes P71 and P72 penetrating between adjacent regions R1 and R4 on the upper and lower main surfaces of the upper plate 21 are formed as alignment marks. In this case, it is possible to pick up an image from any direction. By forming at least the surface of the upper plate 21 from an opaque or colored material, the image contrast between the surface of the upper plate 21 and the alignment marks P71 and P72 may be increased to facilitate recognition. Such a through-hole type alignment mark needs to penetrate at least in the upper plate 21, but if the lower surface sheet 22 has optical transparency, it is necessary to penetrate through the lower surface sheet 22. Not necessarily. That is, a configuration in which the through-hole provided in the upper plate 21 is closed by the light-transmitting lower surface sheet 22 may be used.

  These patterns are also configured asymmetrically with respect to the rotation of the well, and it is possible to uniquely detect the amount of rotation of the well in the image even with such an alignment mark. Therefore, these alignment marks can be used as information for grasping the posture of the well.

  Further, in the example on the right side of FIG. 3C, it is possible to provide a function for identifying a plurality of wells W provided in the microplate 2 by making the three graphic patterns different in shape. is there. In other words, it is possible to easily identify which well the captured image belongs to by changing the combination pattern composed of the shape of the figure pattern, its arrangement order, position, etc. for each well. .

  When the alignment mark is provided on the bottom surface of the well, the alignment mark may be shielded by the contents of the well, for example, cells cultured in the medium injected into the well. In this sense, the alignment mark is preferably composed of as many graphic patterns as possible, preferably three or more graphic patterns. Further, in order to accurately determine the rotation amount of the well by detecting the position of the alignment mark, it is desirable that a graphic pattern be arranged in the region R3 near the peripheral portion of the well bottom surface.

  Note that the graphic pattern provided on the bottom surface of the well is either inside the well (that is, the upper surface of the lower surface sheet 22) or outside the well (that is, the lower surface of the lower surface sheet 22) as long as it can be accommodated in the imaging visual field from above or below the well. May be formed. When the lower surface sheet 22 is opaque, it is natural that an alignment mark needs to be formed on the surface on which the imaging unit 13 is disposed. The graphic pattern can be formed on the lower surface sheet 22 by, for example, an appropriate printing technique or imprinting technique.

  The above alignment marks are merely examples, and the shape, number, and arrangement of the graphic patterns are not limited to those described above, and various types can be used as long as they do not contradict the spirit of the present invention. It is.

  Next, the image acquisition process by the combination of the imaging device 1 and the microplate 2 configured as described above will be described. This image acquisition process is a process for acquiring an image for so-called time-lapse observation, in which images obtained by capturing the same sample at a predetermined time interval are compared and observed.

  A medium is injected into each well W of the microplate 2 to culture cells and the like. In order to maintain a predetermined temperature / humidity environment during culture, the microplate 2 is stored in a suitable incubator (not shown). Then, at every predetermined time interval, the microplate 2 is moved from the incubator to the imaging device 1 and imaging by the imaging device 1 is performed each time.

  In addition to this, the image acquisition process described below is also applied when an image is acquired for the purpose of observing changes in the well before and after the operation such as injecting a chemical solution into the well, for example. It is possible. This image acquisition process is realized by the control unit 10 executing a processing program set in advance according to a user execution instruction to control each unit of the apparatus.

  FIG. 4 is a flowchart showing image acquisition processing in the imaging apparatus 1 of FIG. First, it waits for the microplate 2 holding the sample to be imaged in the well W to be set in the imaging device 1 in a substantially horizontal posture (step S101). At this time, it is preferable to allow a certain degree of variation in the position where the microplate 2 is placed on the holder 11 so that the internal cells or the like are not affected by the impact on the sample. Variations in well position and inclination due to this can be corrected by image processing to be described later.

  Subsequently, the imaging unit 13 is scanned and moved with respect to the microplate 2 in this state, thereby imaging the entire microplate 2 and obtaining an image including all the wells W (step S102). Steps S101 and S102 described above are executed again at predetermined time intervals or with an operation on the well, and this is repeated until a necessary number of images are acquired (step S103).

  When the required number of images is acquired, a well image is created by cutting out a partial region including only one well region from an image including a plurality of wells (step S104). At this time, the well image is cut out from the entire image so as to include the alignment mark. When the alignment mark is formed in the well region (side wall surface R2 and bottom surface R3), the alignment image is automatically included in the well image by cutting out a partial region in which the entire contour of the well is contained. On the other hand, when the alignment mark is formed in the well adjacent region R1, the partial region is cut out from the entire image so as to include the alignment mark located outside the well outline. Subsequent processing is executed for each well image.

  From each well image thus obtained, an alignment mark included in the image is detected (step S105). Then, the positional relationship of the alignment marks in a plurality of well images obtained by imaging the same well at time intervals is compared, and the relative well inclination amount between them is calculated (step S106).

  The inclination of each well image can be obtained from the positions of a plurality of detected graphic patterns. For example, the absolute inclination amount of the well depends on how much the inclination in the image of the line segment connecting the centroids of the two detected graphic patterns is different from the angle in the well image having no inclination assumed in advance. Can be grasped. Further, it is possible to grasp the relative amount of inclination between the plurality of images depending on how much the inclination of the line segment differs between the plurality of images.

  Subsequently, rotation correction of the well image is performed based on the calculated inclination amounts (step S107). The purpose is to equalize the amount of well inclination between a plurality of images obtained by imaging the same well. Accordingly, the posture of the well in each image may be aligned in a predetermined reference direction (for example, the vertical direction of the image), or the posture of the well of other images may be adjusted to that based on one of the plurality of images. It may be. Regarding the rotation of the image, a circle corresponding to the outline of the well is identified in the image by image analysis, and a process of rotating the pixels belonging to the well region inside the circle by a predetermined angle may be performed.

  As for images after rotation correction, images obtained by imaging the same well as one set are arranged in the order of imaging and stored as a time-lapse image set for the well (step S108). By doing so, it becomes possible to use a series of images representing how the cells and the like have changed in each well as needed.

  5 and 6 are diagrams showing examples of time-lapse image sets. More specifically, FIG. 5 shows an example of a time-lapse image set created without performing the above-described rotation correction, and FIG. 6 shows an example of the time-lapse image set of the present embodiment created by performing rotation correction. Is shown. Here, an image of a well provided with an alignment mark on the right side of FIG. 3C is illustrated, but the same applies when other alignment mark patterns are used.

  First, FIG. 5 will be described. Two well images Iw1 and Iw2 shown in FIG. 5A are sets of images obtained by imaging the same well at different times. Here, the case where there are two images obtained by imaging the same well is taken, but the concept is the same when there are three or more images. It is assumed that there is a difference in the angle θ between the inclinations of the wells in the two well images due to the positional deviation when the microplate 2 is attached to and detached from the imaging device 1.

  Consider a case in which these images are displayed while being switched with time on the display unit 118 of the imaging device 1 or an appropriate display device. Such a display method is useful for identifying changes in position and size of cells distributed in a well over time, and cells corresponding to each other between different images. . As shown in FIG. 5B, when two images Iw1 and Iw2 of the time-lapse image set S1 created while including a difference in inclination are switched and displayed, cells or the like displayed at a certain position in the previous image Iw1 Are displayed at different positions in the subsequent image Iw2.

  For this reason, the correspondence between the image objects in the two images cannot be understood. If there is no change in the image object between the two images, it is not impossible to recognize the tilt, but in observation in the field of biological science, the image object itself changes with time due to increase or decrease of cells, so the correspondence between images Is particularly difficult to grasp. In the prior art, at the stage of the well image, it is impossible to grasp afterwards how much the well has been imaged, and this problem cannot be solved.

  On the other hand, in the time lapse image set of the present embodiment shown in FIG. 6, the relative inclination difference θ between the two images Iw1 and Iw2 is eliminated by rotation correction, as shown in FIG. 6A. Here, with respect to the well image Iw2, the well region Iw2c whose tilt is corrected is created by rotating the well region as indicated by the arrow.

  By constructing the time-lapse image set S2 with the rotation-corrected images in this way, as shown in FIG. 6B, the well inclinations match between the images switched and displayed with time, and the same image object is As long as there is no movement of cells etc., they are displayed at the same position in the images before and after switching. Moreover, when a cell etc. move, the movement amount can be easily calculated | required from an image. As a result, it becomes easier to grasp the occurrence / disappearance of cells and the change in size, and the observation by the user is efficiently supported.

  As described above, in this embodiment, the microplate 2 corresponds to the “sample holding plate” of the present invention, and the well W corresponds to the “concave portion” of the present invention. Further, the upper plate 21 and the lower sheet 22 function as a “plate member” and a “sheet member” of the present invention, respectively.

  In the image acquisition process of FIG. 4, step S <b> 101 corresponds to the “holding process” of the present invention, and step S <b> 102 corresponds to the “imaging process” of the present invention. Steps S105 to S107 correspond to the “correction step” of the present invention.

  The present invention is not limited to the above-described embodiment, and various modifications other than those described above can be made without departing from the spirit of the present invention. For example, in the above-described embodiment, the well contents and the alignment mark are simultaneously imaged with visible light (white light). However, in the configuration in which the inclination of the well image is automatically corrected, the alignment mark may be optically It is sufficient if it is detected by the means, and it is not always necessary to be visible by humans. Moreover, it can be said that it is preferable that it is not visually recognized from the viewpoint of observing the contents of the well.

  Therefore, for example, it is assumed that the alignment mark is transparent to visible light but can be imaged by light outside the visible region (for example, infrared rays), and the visible image and infrared image of the same well are individually captured. It may be. In this case, the well may be irradiated with visible light and infrared rays separately, and imaging may be performed each time, or imaging is performed under irradiation light including both wavelength components, and a visible image and an infrared image are separated by spectroscopy. May be obtained at the same time. The well tilt amount based on the detection result of the alignment mark imaged with infrared rays is naturally the same in the visible image. Therefore, the well tilt is corrected by correcting the visible image based on the infrared image. Is possible. In some cases, when the well contents and the alignment mark are imaged based on different wavelength components, they can be separated when the well contents overlap the alignment mark.

  Further, in the above embodiment, the rotation correction of the well region in the image is performed based on the position detection result of the imaged alignment mark. However, as the image shift due to the mounting position shift of the microplate 2, Not only the well rotation direction (tilt) but also displacement in the direction along the image plane may occur. Such misregistration can also be grasped from the coordinate position of the alignment mark in the image. Therefore, correction processing for the misalignment may be executed together. In short, it is possible to eliminate both in-plane misalignment and inclination if correction is performed so that the alignment marks are exactly matched when the well images forming the time-lapse image set are overlapped.

  Moreover, although the microplate 2 of the said embodiment has the well W whose horizontal cross-sectional shape is substantially circular, the cross-sectional shape of a well is not limited to this, A various shape can be used. In particular, when a microplate having a well having a cross-sectional shape having rotational symmetry with respect to the vertical axis such as an ellipse or a regular polygon is used, the effect of the present invention is particularly remarkable.

  Moreover, in the imaging device 1 of the said embodiment, although necessary correction | amendment is performed to the some image imaged at different time, and the time lapse image set is produced, it is not limited to this. According to the present invention, since the information regarding the positional deviation of the well at the time of imaging is included in the image itself, it is possible to later extract the information regarding the positional deviation from the body of the image. Therefore, well imaging and image processing may be performed in different apparatuses.

  The present invention can be particularly suitably applied to a field that requires observation of a sample in which a fluid is injected into a recess, such as a well on a microplate used in the medical / biological science field. The application field is not limited to the medical / biological science field.

1 Imaging device 2 Microplate (sample holding plate)
DESCRIPTION OF SYMBOLS 10 Control part 11 Holder 13 Imaging unit 21 Upper plate (plate-shaped member)
22 Bottom sheet (sheet-like member)
S101 Holding process S102 Imaging process S105-S107 Correction process W Well (concave part)

Claims (10)

A sample holding plate having a plurality of recesses capable of holding liquid on the upper surface of a plate-shaped member,
For each of the recesses, optically detected in at least one of the interior of the recess, the bottom surface of the recess, and the adjacent area adjacent to the recess of the main surface of the plate-like member. Possible alignment marks,
The sample holding plate according to claim 1, wherein the alignment mark provided for one of the recesses is asymmetric with respect to rotation of the recess around an axis orthogonal to the upper surface of the plate-like member.   The alignment mark includes a plurality of graphic patterns having different shapes, which are provided in at least two locations apart from each other in the interior of the recess, the bottom surface of the recess, or the adjacent region. The sample holding plate as described.   3. The sample holding plate according to claim 2, wherein a plurality of the recesses are provided on an upper surface of the plate-like member, and a combination of a shape of the plurality of graphic patterns and an arrangement thereof are different between the plurality of recesses.   The sample holding plate according to any one of claims 1 to 3, wherein a cross-sectional shape of the recess in a cut surface parallel to the upper surface of the plate-like member is a circle, an ellipse, or a regular polygon. The plate-like member having a through-hole penetrating from the upper surface to the lower surface; and a sheet-like member that contacts the lower surface of the plate-like member and closes the lower surface side opening of the through-hole; The sample holding plate according to any one of claims 1 to 4, wherein a space surrounded by a portion of the upper surface of the sheet-like member facing the through hole constitutes the recess . The sample holding plate according to any one of claims 1 to 5, wherein the alignment mark is transparent to visible light, but opaque to light having a specific wavelength component outside the visible light region . The sample holding plate according to any one of claims 1 to 5 , wherein the alignment mark is formed as a through hole penetrating between an upper surface and a lower surface of the plate-like member. A plurality of recesses are provided on the upper surface of the plate-like member, and for each of the recesses, the inside of the recess, the bottom surface of the recess, and the recess of the main surface of the plate-like member A holding step of holding a sample holding plate provided with an optically detectable alignment mark in at least one of adjacent areas, in a substantially horizontal posture;
An imaging step of capturing the recess while including the alignment mark in an imaging range and obtaining an image of the contents of the recess,
The image acquisition method according to claim 1, wherein the alignment mark provided for one of the recesses is asymmetric with respect to rotation of the recess around an axis orthogonal to the upper surface of the plate-like member.   In the imaging step, light of a visible light component emitted from the depression is received to image the contents of the depression, and light of a wavelength component outside the visible light region emitted from the depression is received. The image acquisition method according to claim 8, wherein the alignment mark is imaged. Performing a series of processes including the holding step and the imaging step for the same sample holding plate a plurality of times, obtaining a plurality of images of the same recess,
The image acquisition method according to claim 8 or 9, further comprising a correction step of correcting a relative positional shift between the plurality of images based on the alignment marks detected from the respective images.

Images