JP6580242B2 - Imaging apparatus and method, and imaging control program - Google Patents

Description

  The present invention relates to an imaging apparatus and method and an imaging control program for moving an imaging target range within an imaging region including cells by moving a moving stage and capturing an image for each imaging target range.

  Conventionally, pluripotent stem cells such as ES (Embryonic Stem) cells and iPS (Induced Pluripotent Stem) cells and differentiation-induced cells are imaged with a microscope, etc. A determination method has been proposed.

  Pluripotent stem cells such as ES cells and iPS cells have the ability to differentiate into cells of various tissues, and are attracting attention as being applicable in regenerative medicine, drug development, disease elucidation, etc. Yes.

  On the other hand, when a cell is imaged with a microscope as described above, in order to obtain a high-power wide-field image, for example, an imaging region such as a well is divided into a plurality of imaging target ranges, and each of the imaging target ranges is set at a high magnification. It has been proposed to perform so-called tiling photography that combines images for each imaging target range after imaging (see Patent Documents 1 to 4).

JP 2014-89410 A JP 2008-052227 A International Publication No. 2013/105373 Pamphlet JP 2005-514589 A

  Here, as a method of tiling photography as described above, a method of bidirectional scanning of an imaging target range in an imaging region has been proposed. In the bidirectional scanning, as shown in FIG. 13, in the imaging region R, the imaging target range IR is moved in the positive X-axis direction (rightward on the paper surface) and in the negative X-axis direction (moving leftward on the paper surface). This is a scanning method that alternately repeats the movement of.

  However, the movement of the imaging target range IR as described above is performed by moving a moving stage on which a well or the like is installed. However, a liquid liquid such as a culture solution accommodated in the well by the movement of the moving stage. Fluctuation occurs on the surface.

  In recent years, it has been desired to reduce the total imaging time, and the time required for imaging one imaging target range tends to be shortened. Therefore, the moving stage moves before the liquid level fluctuation generated in the well is subsided, and the liquid level fluctuation increases every time the moving stage is moved.

  On the other hand, so-called time-lapse imaging, in which cells are imaged multiple times over time, has been conventionally performed in order to observe changes in cells over time. In this time-lapse photography, dead cells float with the passage of time, but these dead cells move with the fluctuation of the liquid level accompanying the movement of the moving stage. When the above-described bidirectional scanning is performed, the movement stage is moved in the same direction continuously, for example, about 15 times, so that the side wall surface of the well W is indicated by the arrow in FIG. It was confirmed that dead cells gathered in the center of the well W where the wave front reflected in the direction, and the waves canceled each other. The direction of the arrow shown in FIG. 16 is the wavefront reflection direction. Moreover, the left figure of FIG. 17 is a schematic diagram showing the dispersion state of dead cells D before tiling photography, and the right figure of FIG. 17 shows the assembled state of dead cells D after tiling photography by bidirectional scanning. It is a schematic diagram shown.

  As shown in the right diagram of FIG. 17, when dead cells D gather at the center of the well W (imaging region), the appearance of the image is impaired, and the cell group S below the location where dead cells D gather is formed. There is a problem of disappearing. This phenomenon is a problem that appears more prominently as the time-lapse shooting period is extended.

  In view of the above problems, an object of the present invention is to provide an imaging apparatus and method, and an imaging control program that can prevent dead cells from gathering in the center of an imaging area as the moving stage moves. To do.

  An imaging apparatus according to the present invention moves an imaging target range within an imaging region including cells by moving an imaging unit that images cells, a moving stage on which a container containing cells is installed, and a moving stage. And an imaging control unit that captures an image for each imaging target range by controlling the imaging unit, and the imaging control unit does not continuously move the moving stage in the same direction three or more times.

  In the imaging apparatus of the present invention, a control switching condition acquisition unit that acquires a control switching condition of the moving stage can be provided, and the imaging control unit moves the moving stage in the same direction based on the control switching condition. Can be switched between the first stage control that does not continue three times or more and the second stage control that allows the movement stage to move continuously in the same direction three or more times.

  In the imaging apparatus of the present invention, the control switching condition is related to a condition related to the number of movements of the moving stage, a condition related to the imaging interval of the imaging target range, and a perturbation of the liquid level of the liquid stored in the container. It is preferable to use conditions or cell culture periods.

  In the imaging apparatus of the present invention, the conditions related to the number of movements of the moving stage are the imaging magnification, the size of the imaging area, the number of times of imaging when the imaging area is imaged a plurality of times, and the imaging area. It is preferable to include at least one of the period from the start to the end of imaging in the case of performing multiple times.

  In the imaging apparatus of the present invention, it is preferable that the condition related to the imaging interval of the imaging target range includes at least one of an autofocus method and an exposure time of the imaging target range.

  In the imaging device of the present invention, the conditions related to the peristaltic movement of the liquid level in the container may include at least one of the type of liquid, the amount of liquid, the viscosity of the liquid, and the size of the container. preferable.

  In the imaging apparatus of the present invention, a dead cell measuring unit that measures information related to the number of dead cells in the container can be provided, and the imaging control unit can control the movement stage based on the information related to the number of dead cells. It is possible to switch between the first stage control in which the movement in the same direction is not continued three times or more and the second stage control in which the movement of the moving stage can be continued in the same direction three or more times.

  In the imaging apparatus of the present invention, the imaging control unit can move the imaging target range spirally with respect to the imaging region in the container.

  In the imaging apparatus of the present invention, the imaging control unit can count the number of movements of the moving stage based on a control signal to a drive motor that drives the moving stage.

  The imaging method of the present invention moves an imaging target range in an imaging region including cells by moving a moving stage on which a container in which cells are stored is installed, and captures an image for each imaging target range. And the movement of the moving stage in the same direction is not continued three times or more.

  The imaging control program of the present invention moves an imaging target range in an imaging area including cells by moving a moving stage on which a container containing cells is installed, and controls an imaging unit to thereby acquire an imaging target. An imaging control program for causing a computer to function as an imaging control unit that captures an image for each range, wherein the imaging control unit does not continuously move the moving stage in the same direction three or more times.

  An imaging apparatus and method and an imaging control program according to the present invention move an imaging target range within an imaging area including cells by moving a moving stage on which a container in which cells are stored is installed. The image of is taken. At this time, since the imaging apparatus and method and the imaging control program of the present invention do not continuously move the moving stage in the same direction three or more times, the moving stage as in the case of the bidirectional scanning described above. With this movement, dead cells can be prevented from gathering at the center of the imaging region.

1 is a block diagram showing a schematic configuration of a microscope observation system using a first embodiment of an imaging apparatus of the present invention. The figure which shows schematic structure of the microscope apparatus of the microscope observation system shown in FIG. Schematic diagram showing an example of an imaging region including a cell group and an imaging target range in the imaging region The figure which shows the moving direction of the imaging target range within the imaging region by the movement of the moving stage Schematic diagram showing the dispersion state of dead cells before tiling photography and the dispersion state of dead cells after tiling photography by movement control of the movement stage of the present invention The figure which shows the phase difference image of the predetermined time at the time of time-lapse imaging | photography by the movement control of the movement stage of this invention The figure which shows the phase-contrast image of the predetermined time at the time of carrying out the time-lapse imaging | photography by performing bidirectional scanning The figure which shows other embodiment of the movement control method which does not continue the movement to the same direction of a movement stage 3 times or more. The figure which shows other embodiment of the movement control method which does not continue the movement to the same direction of a movement stage 3 times or more. The flowchart for demonstrating the effect | action of the microscope observation system using 1st Embodiment of the imaging device of this invention. The block diagram which shows schematic structure of the microscope observation system using 2nd Embodiment of the imaging device of this invention. The figure which shows other embodiment of the movement control method which does not continue the movement to the same direction of a movement stage 3 times or more. The figure which shows the movement direction of the imaging target range in the case of performing bidirectional scanning The figure which shows the moving direction in the case of moving the imaging object range spirally with respect to an imaging region The block diagram which shows schematic structure of the microscope observation system using 3rd Embodiment of the imaging device of this invention. Diagram showing wavefront reflection direction when bidirectional scanning is performed Schematic diagram showing the dispersal state of dead cells before tiling imaging and the aggregated state of dead cells after tiling imaging by bidirectional scanning

  Hereinafter, a microscope observation system using a first embodiment of an imaging apparatus and method and an imaging control program of the present invention will be described in detail with reference to the drawings. FIG. 1 is a block diagram showing a schematic configuration of a microscope observation system 1 of the present embodiment. FIG. 2 is a schematic configuration diagram of the microscope apparatus 10 of the microscope observation system 1 of the present embodiment. In FIG. 1, only the configuration related to the control device 20 is shown for the microscope device 10.

  As shown in FIG. 1, the microscope observation system 1 according to the present embodiment includes a microscope device 10, a control device 20, a display device 30, and an input device 40.

  The microscope apparatus 10 captures a phase difference image of cultured cells. The microscope apparatus 10 performs so-called time-lapse imaging, in which cells are imaged multiple times over time. Specifically, as illustrated in FIG. 2, the microscope apparatus 10 includes a phase difference measurement white light source 11 that emits white light and a ring-shaped slit, and is emitted from the phase difference measurement white light source 11. A slit plate 12 that emits ring-shaped illumination light L1 when white light is incident thereon, and a ring-shaped illumination light L1 emitted from the slit plate 12 is incident, and the incident ring-shaped illumination light L1 is converted into a cell group. A first objective lens 13 that irradiates S (also referred to as a cell colony).

  The slit plate 12 is provided with a ring-shaped slit that transmits white light to a light shielding plate that blocks white light emitted from the white light source 11 for phase difference measurement, and the white light passes through the slit. As a result, ring-shaped illumination light L1 is formed.

  In addition, the microscope apparatus 10 includes a phase difference lens 14, an imaging lens 15, and an image sensor 16.

  The phase difference lens 14 includes a second objective lens 14a and a phase plate 14b. The phase plate 14b is obtained by forming a phase ring on a transparent plate that is transparent with respect to the wavelength of the illumination light L1. The slit size of the slit plate 12 described above is in a conjugate relationship with the phase ring of the phase plate 14b.

  In the phase ring, a phase film that shifts the phase of incident light by ¼ wavelength and a neutral density filter that attenuates incident light are formed in a ring shape. When the direct light incident on the phase ring passes through the phase ring, the phase is shifted by a quarter wavelength, and the brightness is weakened. On the other hand, most of the diffracted light diffracted by the cell group S passes through the transparent plate of the phase plate 14b, and its phase and brightness do not change.

  The phase difference lens 14 is moved in the Z direction (arrow A direction) by an optical system driving unit 18 (see FIG. 1). The autofocus control is performed by the movement of the phase difference lens 14 in the Z direction, and the contrast of the phase difference image captured by the image sensor 16 is adjusted.

  The imaging lens 15 receives a phase difference image that has passed through the phase difference lens 14 and forms an image on the imaging element 16.

  The image sensor 16 captures a phase difference image formed by the imaging lens 15. As the imaging element 16, a CCD (Charge-Coupled Device) image sensor, a CMOS (Complementary Metal-Oxide Semiconductor) image sensor, or the like is used. As the image sensor, an image sensor provided with an RGB (Red Green Blue) color filter may be used, or a monochrome image sensor may be used.

  In the present embodiment, the above-described white light source for phase difference measurement 11, the slit plate 12, the first objective lens 13, the phase difference lens 14, the imaging lens 15, and the imaging element 16 are included in the imaging unit of the present invention. It is equivalent. The imaging unit, the moving stage 51 and the imaging control unit 21, which will be described later, correspond to the imaging device of the present invention.

  And between the 1st objective lens 13 and the phase difference lens 14 which were mentioned above, the moving stage 51 in which the culture container 50 in which the cell group S was accommodated is installed.

  As the culture vessel 50, a petri dish, a dish, a well plate, or the like can be used. The cell group S accommodated in the culture vessel 50 includes pluripotent stem cells such as iPS cells and ES cells, nerves induced to differentiate from stem cells, skin, myocardium and liver cells, and skin taken from the human body, There are cells of the retina, heart muscle, blood cells, nerves and organs. In the culture vessel 50, a culture solution C for culturing the cell group S is also accommodated.

  The moving stage 51 is moved in the X and Y directions orthogonal to each other by the stage driving unit 19 (see FIG. 1). The X direction and the Y direction are directions orthogonal to each other on a plane parallel to the installation surface of the cell group S.

  The stage drive unit 19 moves the moving stage 51 in an oblique direction between the X direction, the Y direction, and the orthogonal XY directions in response to a control signal from the imaging control unit 21 of the control device 20. A motor 19a and a moving mechanism (not shown) driven by the drive motor 19a are provided.

  The imaging control unit 21 moves the moving stage 51 to move the imaging target range IR within the imaging region R including the cell group S and controls the microscope apparatus 10 as shown in FIG. A phase difference image for each target range IR is captured.

  The moving stage 51 is moved in the X direction, the Y direction, and the oblique direction by the stage driving unit 19, and the entire imaging region R is scanned by repeating the movement.

  Here, as described above, when the movable stage 51 is moved and the conventional bidirectional scanning is performed, dead cells are gathered at the center of the imaging region R, and the phase difference image of the cell group S is obtained. And the appearance of the phase difference image is also impaired.

  Therefore, the imaging control unit 21 of the present embodiment performs control so that the movement of the moving stage 51 in the same direction is not continued three times or more. A solid line L shown in FIG. 4 indicates the moving direction of the imaging target range IR in the imaging region R due to the movement of the moving stage 51. The imaging target range IR moves to a point where the end points S and G of the solid line L and the solid line L are bent, and a phase difference image is captured at that point. Note that when the imaging target range IR moves from the point P1 to the point P2 shown in FIG. 4, the imaging target range existing between the points P1 and P2 has not yet been imaged, and the point P1. Since the line segment connecting the point P2 passes through the center of the imaging target range, the moving stage 51 stops at the position of the imaging target range and imaging is performed. The imaging target range IR is initially set at the position of the end point S, then moves along the solid line L from the outside to the inside of the imaging region R, and moves to the position of the end point G. That is, the imaging target range IR moves spirally with respect to the imaging region R in the culture vessel 50 by the movement of the moving stage 51.

  The imaging control unit 21 counts the number of movements of the moving stage 51 based on a control signal to the drive motor 19a of the stage driving unit 19. Note that the movement stage 51 is counted as one movement after the movement stage 51 starts to stop moving. Therefore, specifically, the imaging control unit 21 counts the number of movements of the moving stage 51 by counting the combination of the operation start control signal output and the operation stop control signal output to the drive motor 19a.

  Further, the movement of the imaging target range IR in an oblique direction that is not parallel to the X direction and the Y direction may be realized by a combination of the movement of the moving stage 51 in the X direction and the movement in the Y direction. Specifically, for example, the movement from the point P1 to the point P2 shown in FIG. 4 includes two movements from the point P1 in the X axis positive direction (rightward on the paper surface) and Y axis negative direction (downward on the paper surface). This is done in combination with two movements. In addition, the movement from the point P3 to the point P4 shown in FIG. 4 is performed by moving once from the point P3 in the X axis negative direction (leftward on the paper surface) and twice in the Y axis positive direction (upward on the paper surface). It is done by the combination.

  However, in that case, since the imaging target range existing between the points P1 and P2 shown in FIG. 4 is not picked up, after moving to the point G shown in FIG. It is only necessary to move so as to image the imaging target range existing between the points P1 and P2.

  By moving the imaging target range IR in the movement direction indicated by the solid line L in FIG. 4, it is possible to prevent the movement stage 51 from moving continuously in the same direction three or more times, and almost the entire imaging region R. The phase difference image can be captured.

  Then, by not moving the moving stage 51 in the same direction three or more times, it is possible to prevent dead cells from gathering at the center of the imaging region R as in the conventional bidirectional scanning. That is, by moving the moving stage 51 in different directions without continuously moving the moving stage 51 in the same direction three or more times, waves can be generated in different directions on the liquid surface of the culture medium C. The dead cells floating on the liquid surface can be dispersed without collecting them. The left diagram of FIG. 5 is a diagram schematically showing the dispersion state of dead cells D before the movement stage 51 is moved, and the right diagram of FIG. 5 is an object to be imaged by movement control of the movement stage 51 of the present embodiment. It is the figure which showed typically the dispersion | distribution state of the dead cell D after moving the range IR and imaging the phase difference image.

  FIG. 6 shows a phase difference image at a predetermined time when the imaging target range IR is moved by the movement control of the moving stage 51 of the present embodiment and the entire imaging region R is time-lapse shot every hour. It is. As shown in FIG. 6, in this case, the assembled state of dead cells is not particularly confirmed. On the other hand, FIG. 7 shows a phase difference image at a predetermined time when the imaging target range IR is moved by conventional bidirectional scanning and the entire imaging region R is time-lapse shot every hour. As shown by an ellipse in FIG. 7, it can be seen that dead cells are gathered in the central portion of the imaging region R.

  Note that the movement control method for the moving stage 51 is not limited to the example shown in FIG. 4, and other movement control methods may be adopted as long as the moving stage 51 is not moved continuously in the same direction three or more times. It may be.

  Specifically, the moving stage 51 may be moved so that the imaging target range IR moves along the solid line L shown in FIGS. 8 and 9, the imaging target range IR is first set at the position of the end point S, and then moves along the solid line L and then moves to the position of the end point G. In the example shown in FIGS. 8 and 9, the phase difference image is picked up at the point where the end points S and G of the solid line L and the solid line L are bent, and further, while moving from one point to the next point, When the imaging target range IR exists, the imaging target range IR has not been captured yet, and the solid line L passes through the center of the imaging target range IR, the imaging target range IR is also imaged.

  Next, as described above, the microscope apparatus 10 according to the present embodiment performs autofocus control by moving the phase difference lens 14 in the Z direction, but can perform two types of autofocus control. It is. In one autofocus control method, the phase difference lens 14 is moved in the Z direction, and a plurality of phase difference images for each position of the phase difference lens 14 are picked up by the image sensor 16, and the contrast of the plurality of phase difference images is maximized. This is so-called contrast autofocus control in which the phase difference lens 14 is moved to a position where

  Another autofocus control method is pattern illumination light autofocus control by irradiation of pattern illumination light. The pattern illumination light autofocus control will be described in detail later.

  Contrast autofocus control and pattern illumination light autofocus control are switched by the user as needed. In general, in contrast autofocus control, a phase contrast image with clearer contrast can be captured. In pattern illumination light autofocus control, multiple times such as contrast autofocus control can be obtained. Since there is no need to capture a phase difference image, autofocus control can be performed at higher speed. The user switches these autofocus controls according to an instruction input from the input device 40.

  And the microscope apparatus 10 of this embodiment is provided with the pattern illumination light irradiation part 55 and the autofocus light detection part 60 as a structure for performing pattern illumination light autofocus control, as shown in FIG.

  The pattern illumination light irradiating unit 55 irradiates the imaging region R including the cell group S with the pattern illumination light L2. The pattern illumination light irradiation part 55 of this embodiment irradiates the illumination light which has a striped pattern as the pattern illumination light L2. Specifically, the pattern illumination light irradiation unit 55 includes an autofocus white light source 56 that emits white light, a linear portion that transmits white light emitted from the autofocus white light source 56, and a linear portion that blocks light. , A mirror 58 that reflects the pattern illumination light L2 having a striped bright and dark pattern emitted from the grid 57, and the pattern illumination light L2 that reflects the mirror 58 is directed toward the imaging region R. And a first half mirror 59 for reflection.

  The first half mirror 59 has an optical characteristic of transmitting a phase difference image of the cell group S and a stripe image for autofocus described later and reflecting the pattern illumination light L2 in the direction of the imaging region R. .

  The autofocus light detection unit 60 includes a second half mirror 61, an optical path difference prism 62, and a line sensor 63.

  The second half mirror 61 reflects the autofocus fringe image reflected from the bottom surface of the culture vessel 50 by irradiating the culture vessel 50 with the pattern illumination light L2 in the direction of the optical path difference prism 62, and the cell group S. The phase difference image is transmitted.

  The optical path difference prism 62 divides the incident fringe image into two optical paths and forms images at two different locations of the line sensor 63. The line sensor 63 outputs the image signal of the first fringe image and the image signal of the second fringe image captured at two locations to the imaging control unit 21. The imaging control unit 21 performs autofocus control by moving the phase difference lens 14 in the Z direction based on the image signal of the first fringe image and the image signal of the second fringe image. Specifically, the phase difference lens 14 is moved to a position where the contrast (waveform pattern) of the first fringe image and the contrast (waveform pattern) of the second fringe image are closest.

  Returning to FIG. 1, the control device 20 includes an imaging control unit 21 and a display control unit 22. The control device 20 is obtained by installing an embodiment of an imaging control program of the present invention on a computer.

  The control device 20 includes a central processing unit, a semiconductor memory, a hard disk, and the like, and an embodiment of the imaging control program of the present invention is installed on the hard disk. Then, when this program is executed by the central processing unit, the imaging control unit 21 and the display control unit 22 shown in FIG. 1 operate.

  The imaging control unit 21 performs imaging control of the phase difference image in the microscope apparatus 10. Specifically, the imaging control unit 21 controls the imaging of the phase difference image by controlling the emission of white light from the phase difference measuring white light source 11 and the operation of the imaging element 16. The imaging control unit 21 controls the movement of the moving stage 51 by controlling the stage driving unit 19 as described above, and thereby controls the movement of the imaging target range IR in the imaging region R.

  Further, the imaging control unit 21 controls the movement of the phase difference lens 14 in the Z direction by controlling the optical system driving unit 18 before imaging of the phase difference image of each imaging target range IR, thereby auto- Focus control is performed. As described above, contrast autofocus control or pattern illumination light autofocus control is possible as autofocus control, and autofocus control designated by the user is performed.

  The display control unit 22 generates a single phase difference image of the entire imaging region R by combining the phase difference images for each imaging target range IR captured by the microscope apparatus 10, and displays the phase difference image on the display device. 30 is displayed.

  The display device 30 displays the phase difference image generated by the display control unit 22 as described above, and includes a liquid crystal display, for example. Further, the display device 30 may be configured by a touch panel and may also be used as the input device 40.

  The input device 40 includes a mouse, a keyboard, and the like, and accepts various setting inputs by the user. The input device 40 of the present embodiment receives, for example, the designation of the type of autofocus control described above.

  Next, the operation of the microscope observation system 1 of the present embodiment will be described with reference to the flowchart shown in FIG.

  First, the culture vessel 50 in which the cell group S and the culture medium C are accommodated is placed on the moving stage 51 (S10).

  Next, the moving stage 51 moves to a position where the first imaging target range IR in the imaging region R is imaged (S12). Then, autofocus control is performed on the imaging target range IR (S14).

  After the autofocus control is performed, the phase-difference image of the imaging target range IR is captured by the imaging element 16 by irradiating the imaging target range IR with the ring-shaped illumination light L1 (S16).

  Next, when the imaging of the phase difference images of all the imaging target ranges IR is not completed (S18, NO), the moving stage 51 moves to the position where the next imaging target range IR is imaged (S20). Then, after auto focus control is performed for the next imaging target range IR (S14), a phase difference image is captured (S16).

  The movement of the imaging target range IR, the auto focus control, and the imaging of the phase difference image are repeatedly performed until the imaging of the phase difference images of all the imaging target ranges IR is completed, and the phase difference image for each imaging target range IR is displayed. The information is sequentially stored in the control unit 22.

  Then, when the imaging of the phase difference images of all the imaging target ranges IR is completed (S18, YES), the display control unit 22 combines the phase difference images for each imaging target range, A phase difference image is generated (S22), and the generated phase difference image is displayed on the display device 30 (S24).

  Next, a microscope observation system using a second embodiment of the imaging apparatus and method and the imaging control program of the present invention will be described. FIG. 11 is a block diagram showing a schematic configuration of the microscope observation system 2 of the present embodiment. In addition, about the structure of the microscope apparatus 10, it is the same as that of the microscope observation system 1 of 1st Embodiment.

  In the microscope observation system 1 according to the first embodiment, control is performed so that the movement of the moving stage 51 in the same direction is not continued three times or more. Even if movement control is not performed, there may be no problem that dead cells gather in the center of the imaging region R. In such a case, it may be preferable to move the moving stage 51 continuously three times or more in the same direction because the imaging time of the entire imaging region is shortened. That is, for example, when the imaging target range IR is moved along the solid line L as shown in FIG. 12 from the end point S to the end point G, in order to prevent the movement in the same direction from continuing three times or more, As the movement increases, it becomes necessary to move back to the same position as in the example shown in FIG. 8, and the total movement distance is extended as compared with simple bidirectional scanning. Therefore, when the moving speed of the moving stage 51 is the same, the bidirectional scanning may be preferable to the moving control in which the movement in the same direction does not continue three times or more because the photographing time is shortened.

  Therefore, the microscope observation system 2 of the second embodiment is configured to switch the movement control method of the moving stage 51 according to conditions. Specifically, in the microscope observation system 2 of the second embodiment, the first stage control that does not continuously move the moving stage 51 in the same direction three times or more and the movement of the moving stage 51 in the same direction 3 The second stage control that can be continuously performed more than once can be switched.

  The control device 20 of the microscope observation system 2 of the second embodiment further includes a control switching condition acquisition unit 23 that acquires a control switching condition for switching between the first stage control and the second stage control described above, and imaging control. The unit 21 switches between the first stage control and the second stage control based on the control switching condition acquired by the control switching condition acquisition unit 23.

  In the present embodiment, as the second stage control, bidirectional scanning is performed in which the moving stage 51 is moved in both directions in the X direction as in the conventional case. FIG. 13 shows the moving direction of the imaging target range IR in the case of the second stage control by a solid line M. The imaging target range IR moves from the end point S to the end point G, and a phase difference image is captured in the imaging target range IR on the solid line M. However, the present invention is not limited to this, and the imaging target range is spirally moved with respect to the imaging area as in the first stage control. However, as indicated by the solid line N in FIG. You may make it control the movement stage 51 so that it may make it move spirally more than once.

  As the control switching condition, a condition related to the number of movements of the moving stage 51, a condition related to the imaging interval of the imaging target range, a condition related to the peristalsis of the liquid level of the culture medium C stored in the culture vessel 50, and There are cell culture periods.

  Specifically, the conditions relating to the number of movements of the moving stage 51 include imaging magnification, the size of the imaging region R, the number of times of imaging when imaging of the imaging region R is performed a plurality of times, and imaging of the imaging region R. There is a period from the start to the end of imaging in the case of performing multiple times.

  For example, since the field of view is narrower when the magnification of imaging is higher than when the imaging magnification is low, the size of the imaging target range IR is small, and when imaging the imaging region R of the same size, the number of times of imaging of the imaging target range IR is large. Become. That is, as the imaging magnification is higher, the number of movements of the moving stage 51 increases, and dead cells tend to gather in the center of the imaging region R.

  Therefore, the first stage control may be performed when the imaging magnification is relatively high, and the second stage control may be performed when the imaging magnification is relatively low. The imaging magnification condition is set and input by the user using the input device 40. Note that the imaging magnification is performed by exchanging the phase difference lens 14 of the microscope apparatus 10 and may be automatically exchanged, or may be manually exchanged by the user.

  Next, regarding the size of the imaging region R, the number of times of imaging is larger than when the imaging region R is larger than the smaller one. That is, as the imaging region R is larger, the number of movements of the moving stage 51 increases, and dead cells tend to gather at the center of the imaging region R.

  Therefore, the first stage control may be performed when the imaging region R is relatively large, and the second stage control may be performed when the imaging region R is relatively small. The size of the imaging region is set and input by the user using the input device 40, and the imaging control unit 21 moves the moving stage 51 according to the information.

  Next, when imaging of the imaging region R is performed a plurality of times, that is, when time-lapse imaging is performed as in the present embodiment, the first stage control and the second stage control are switched according to the number of imaging of the time-lapse imaging. It may be. When the number of times of time lapse imaging is small, the number of times of movement of the moving stage 51 is small. Therefore, it is difficult for dead cells to collect in the center of the imaging region R. Since the number of times of movement increases, it becomes easy for dead cells to gather at the center of the imaging region R.

  Therefore, the first stage control may be performed when the number of times of imaging from the start of the type lapse shooting to the end of the time lapse shooting is large, and the second stage control may be performed when the number of times is small. The number of times of imaging from the start of the type-lapse shooting to the end of the time-lapse shooting is set and input by the user using the input device 40. Alternatively, the control switching condition acquisition unit 23 counts the number of times of imaging from the start of the type-lapse shooting, and when the number of times of imaging is less than a preset threshold value, the second stage control is performed and the number of times is equal to or greater than the threshold value. In this case, it may be switched to the first stage control.

  Alternatively, the first stage control and the second stage control may be switched according to the period from the start to the end of time-lapse shooting. That is, when the period from the start of the time lapse imaging to the end is short, the number of times of movement of the moving stage 51 is small. In this case, since the number of times of movement of the moving stage 51 is increased, dead cells are likely to gather at the center of the imaging region R.

  Therefore, the first stage control may be performed when the period from the start to the end of the type-lapse shooting is long, and the second stage control may be performed when the period is short. A period from the start to the end of the type-lapse shooting is set and input by the user using the input device 40. Alternatively, the control switching condition acquisition unit 23 measures the period from the start of the type-lapse shooting with a timer or the like, and if the period is less than a preset threshold, the second stage control is performed and the threshold is exceeded. In some cases, the first stage control may be switched.

  Next, conditions relating to the imaging interval of the imaging target range IR include an autofocus method and an exposure time for each imaging target range IR.

  As described above, the microscope apparatus 10 of the microscope observation system 2 of the present embodiment can switch between the contrast autofocus control and the pattern illumination light autofocus control, but as described above, the pattern illumination light is more effective than the contrast autofocus control. Autofocus control can be performed faster. Therefore, when the pattern illumination light autofocus control is performed, the phase difference image for each imaging target range can be captured at high speed, that is, the imaging interval of the imaging target range IR is controlled by contrast autofocus control. Shorter than the case.

  When the imaging interval of the imaging target range IR is long, the time from the time of moving to the current imaging target range IR to the time of moving to the next imaging target range IR becomes long. It is possible to secure a time until the wave generated on the liquid surface of the culture solution C is settled, that is, dead cells are unlikely to collect at the center of the imaging region R. On the other hand, when the imaging interval of the imaging target range IR is short, it is not possible to secure a time until the wave generated on the liquid surface of the culture medium C is stopped. It will be easy to gather in the department.

  Therefore, when performing contrast autofocus control in which the imaging interval in the imaging target range IR is increased, the second stage control is performed, and pattern illumination light autofocus control in which the imaging interval in the imaging target range IR is reduced is performed. Alternatively, the first stage control may be performed.

  Further, as for the exposure time for each imaging target range IR, as described above, when the exposure time for each imaging target range IR is long, the time until the wave generated on the liquid surface of the culture medium C is secured is secured. In other words, it is difficult for dead cells to collect at the center of the imaging region R. On the other hand, when the exposure time for each imaging target range IR is short, it is not possible to secure the time until the wave generated on the liquid surface of the culture medium C is stopped. It will be easy to gather in the department.

  Accordingly, the second stage control is performed when the exposure time for each imaging target range IR is relatively long, and the first stage control is performed when the exposure time for each imaging target range IR is relatively short. It may be. The exposure time for each imaging target range IR is set and input by the user using the input device 40.

  Next, conditions relating to the peristalsis of the liquid level of the culture solution C accommodated in the culture vessel 50 include the type of the culture solution C, the amount of the culture solution C, the viscosity of the culture solution C, the size of the culture vessel 50, and the like. There is.

  Depending on the type of the culture solution C, there are a culture solution C in which the viscosity and the like are different and a wave is hardly generated on the liquid surface, and conversely, a culture solution C in which a wave is easily generated on the liquid surface.

  Accordingly, the first stage control may be performed when the culture medium C is likely to generate waves, and the second stage control may be performed when the culture medium C is unlikely to generate waves. The relationship between the type of the culture medium C and the stage control method is stored in advance as a table. When the user sets and inputs the type of the culture medium C using the input device 40, the first stage control and the second stage are performed. Control can be switched. The relationship between the viscosity of the culture medium C and the stage control method is stored in advance as a table, and the user sets and inputs the viscosity of the culture medium C using the input device 40, so that the first stage control and the second stage control method are performed. The stage control may be switched.

  Further, when the same type of culture solution C is stored in the same culture vessel 50, the larger the amount of the culture solution C, the easier the wave is generated on the liquid surface, and conversely, the smaller the amount, the less the wave is generated on the liquid surface. .

  Therefore, the first stage control may be performed when the amount of the culture medium C is relatively large, and the second stage control may be performed when the amount of the culture medium C is relatively small. The amount of the culture medium C may be set and input by the user using the input device 40, or may be automatically measured by providing a weight sensor or the like on the moving stage 51.

  In addition, when using culture vessels 50 having different sizes, specifically, for example, when using well plates having different well sizes, the larger the well opening, the easier the wave is generated on the liquid surface. The smaller the well opening, the stronger the surface tension, so that waves are less likely to be generated on the liquid surface.

  Therefore, the first stage control may be performed when the well is relatively large, and the second stage control may be performed when the well is relatively small. Information on the size of the well is set and input by the user using the input device 40.

  Next, the first stage control and the second stage control may be switched based on the cell culture period. As the cell culture period is longer, dead cells are more likely to be generated, and dead cells are more likely to gather at the center of the imaging region R due to movement of the moving stage 51.

  Therefore, the first stage control may be performed when the cell culture period is relatively long, and the second stage control may be performed when the cell culture period is relatively short. The cell culture period is set and input by the user using the input device 40.

  Next, a microscope observation system using a third embodiment of the imaging apparatus and method and imaging control program of the present invention will be described. FIG. 15 is a block diagram showing a schematic configuration of the microscope observation system 3 of the present embodiment. In addition, about the structure of the microscope apparatus 10, it is the same as that of the microscope observation system 1 of 1st Embodiment.

  In the microscope observation system 2 of the second embodiment, the first stage control and the second stage control are switched based on the control switching condition acquired by the control switching condition acquisition unit 23. The microscope observation system 3 according to the third embodiment actually measures the number of dead cells existing in the imaging region R based on the phase difference image captured at each time point by time-lapse imaging, and performs the first measurement according to the number. The first stage control and the second stage control are switched.

  Specifically, the microscope observation system 3 of the third embodiment includes a dead cell measurement unit 24 as shown in FIG. In the microscope observation system 3 of the third embodiment, time-lapse imaging is performed in the same manner as in the above-described embodiment, but at each imaging time point, a phase difference image for dead cell measurement is captured before the phase difference image is captured. Pre-photographing is performed.

  The pre-photographing is preferably performed at a lower resolution than in the case of the main photographing. Specifically, in the pre-photographing, the magnification of the microscope apparatus 10 is set to be lower than that in the case of the main photographing. do it. The magnification can be changed by changing the magnification of the second objective lens 14a of the phase difference lens 14. The change of the magnification of the phase difference lens 14 may be performed automatically or manually. In addition, the magnification in the case of pre-photographing is desirably set to a magnification that can photograph the entire imaging region R by one photographing, for example. However, the present invention is not limited to this, and a phase difference image for each imaging target range may be captured with a smaller number of times than the actual capturing. In this case, the movement control of the moving stage 51 may be bidirectional scanning as in the past, or the movement in the same direction may not be continued three times or more.

  Then, the dead cell measurement phase difference image captured by the pre-photographing is input to the dead cell measurement unit 24, and the dead cell measurement unit 24 determines the position based on the input dead cell measurement phase difference image. The number of dead cells included in the phase difference image is counted.

  Dead cells are generally imaged as white, generally circular masses. Therefore, the dead cell measurement unit 24 detects dead cells by detecting a circular pattern with high luminance from the phase difference image. Specifically, a circular pattern may be detected by generalized Hough transform, or an area having a luminance equal to or higher than a preset luminance threshold is detected, and the circularity of the area is calculated and set in advance. You may make it detect the area | region which has circularity more than a circularity threshold value as a dead cell area | region.

  Further, as described above, when each circular pattern is detected by image processing, if the number of dead cells increases to some extent, the processing time may become longer. Therefore, a more simplified method may be adopted. Specifically, when the culture vessel 50 is suddenly moved, dead cells move. Therefore, the operation of moving the culture container 50 in the direction of the installation surface is performed automatically or manually, pre-photographing is performed before and after the operation, and the difference between the phase difference images captured by the two pre-photographing is calculated, You may make it measure the number of dead cells by dividing the half of the area of the part changed between two phase difference images by the reference dead cell area set beforehand.

  The imaging control unit 21 performs the first stage control when the number of dead cells measured by the dead cell measurement unit 24 is equal to or larger than a preset threshold value, and when the number is smaller than the threshold value, the first control is performed. 2 stage control is performed.

  In the present embodiment, the number of dead cells is measured as described above. However, the number of dead cells may not necessarily be counted directly. For example, the area occupied by dead cells in a phase difference image is calculated. The first stage control may be performed when the measured area of the dead cell is equal to or larger than a preset threshold value, and the second stage control may be performed when the area is less than the threshold value.

  In the microscope observation systems 1 to 3 of the first to third embodiments, the phase difference image is picked up by the microscope apparatus 10, but not limited to the phase difference image, for example, a bright field image, differential interference, and the like. An image or a fluorescence image may be taken as a cell image.

1-3 Microscope observation system 10 Microscope device 11 White light source for phase difference measurement 12 Slit plate 13 Objective lens 14 Phase difference lens 14a Objective lens 14b Phase plate 15 Imaging lens 16 Imaging element 18 Optical system drive unit 19 Stage drive unit 19a Drive Motor 20 Control device 21 Imaging control unit 22 Display control unit 23 Control switching condition acquisition unit 24 Dead cell measurement unit 30 Display device 40 Input device 50 Culture vessel 51 Moving stage 55 Pattern illumination light irradiation unit 56 White light source for autofocus 57 Grid 58 Mirror 59 Half mirror 60 Autofocus light detector 61 Half mirror 62 Optical path difference prism 63 Line sensor

Claims (8)

An imaging unit for imaging cells;
A moving stage on which a container containing the cells is installed;
By moving the moving stage, the imaging target range is moved within the imaging region including the cells, and by controlling the imaging unit, an image for each imaging target range is captured, and the moving stage An imaging control unit that switches between a first stage control that does not continue the movement in the same direction three or more times and a second stage control that enables the movement stage to move in the same direction three or more times. ,
The imaging control unit
Switch to the first stage control when the number of movements of the moving stage is large, switch to the second stage control when the number of movements is small,
When the imaging interval of the imaging target range is short, switch to the first stage control, and when the imaging interval is long, switch to the second stage control,
Switch to the first stage control when the liquid level of the liquid stored in the container is high, switch to the second stage control when the liquid level is low, and
An imaging apparatus that switches to the first stage control when the culture period of the cells is long, and switches to the second stage control when the culture period is short.   When the number of times of movement of the moving stage is large, when the magnification of the imaging is relatively high, when the size of the imaging area is relatively large, the number of times of imaging when imaging of the imaging area is performed a plurality of times The case of being at least one of a case where the threshold is equal to or greater than a preset threshold and a case where the period from imaging start to end when imaging of the imaging region is performed a plurality of times is equal to or greater than a preset threshold. The imaging device described.   The case where the imaging interval of the imaging target range is short is at least one of a case where pattern illumination light autofocus control is performed and an exposure time of the imaging target range is relatively short. Imaging device.   When the liquid level of the liquid stored in the container is large, when the liquid is liable to generate a wave, when preset based on the viscosity of the liquid, when the amount of liquid is relatively large The imaging device according to any one of claims 1 to 3, wherein the imaging device is at least one of cases where the size of the container is relatively large.   The imaging device according to any one of claims 1 to 4, wherein the imaging control unit moves the imaging target range in a spiral shape with respect to the imaging region in the container.   The imaging apparatus according to any one of claims 1 to 5, wherein the imaging control unit counts the number of movements of the movable stage based on a control signal to a drive motor that drives the movable stage. By moving a moving stage on which a container containing cells is moved, an imaging target range is moved within an imaging region including the cells, an image for each imaging target range is captured, and the moving stage An imaging method for switching between a first stage control that does not continuously move in the same direction three or more times and a second stage control that allows the movement stage to move in the same direction three or more times,
Switch to the first stage control when the number of movements of the moving stage is large, switch to the second stage control when the number of movements is small,
When the imaging interval of the imaging target range is short, switch to the first stage control, and when the imaging interval is long, switch to the second stage control,
Switch to the first stage control when the liquid level of the liquid stored in the container is high, switch to the second stage control when the liquid level is low, and
An imaging method for switching to the first stage control when the culture period of the cells is long and switching to the second stage control when the culture period is short. By moving a moving stage in which a container containing cells is moved, the imaging target range is moved within the imaging region including the cells, and an image is captured for each imaging target range by controlling the imaging unit. And a first stage control that does not continuously move the moving stage in the same direction three or more times, and a second stage control that enables the moving stage to move in the same direction three or more times. An imaging control program for causing a computer to function as an imaging control unit for switching,
The imaging control unit switches to the first stage control when the number of movements of the moving stage is large, and switches to the second stage control when the number of movements is small.
When the imaging interval of the imaging target range is short, switch to the first stage control, and when the imaging interval is long, switch to the second stage control,
Switch to the first stage control when the liquid level of the liquid stored in the container is high, switch to the second stage control when the liquid level is low, and
An imaging control program that switches to the first stage control when the culture period of the cells is long and switches to the second stage control when the culture period is short.