Patent Description:
In the related art, a method for capturing an image of a multipotential stem cell such as an embryonic stem (ES) cell or an induced pluripotent stem (iPS) cell, a differentiated and induced cell, or the like using a microscope or the like, and capturing a feature of the image to decide a differentiation state of the cell, or the like has been proposed.

The multipotential stem cell such as an ES cell or an iPS cell is able to be differentiated into cells of various tissues, and may be applied to regenerative medicine, development of medicines, explanation of diseases, or the like.

On the other hand, as described above, in a case where cells are imaged with a microscope, a technique for performing so-called tiling imaging has been proposed in order to acquire a high-magnification wide view image. Specifically, for example, a range of a culture container such as a well plate is scanned by an imaging optical system, and an image at each observation position is captured, and then, the images at the respective observation positions are combined.

In a case where such tiling imaging is performed, it has been proposed to obtain a high-quality image with less blur by performing an auto-focus control at each observation position in the culture container (see <CIT>, <CIT>, <CIT>, or the like).

Here, as described above, in a case where the auto-focus control is performed in the above-mentioned tiling imaging, it is important to perform the auto-focus control at high speed and with high accuracy from the viewpoint of reducing an imaging time.

However, for example, in a case where a well plate having a plurality of wells is used as a culture container, the entire well plate is scanned by an imaging optical system, and the tiling imaging is performed while performing the auto-focus control for each observation or the like.

Accordingly, for example, in a case where the auto-focus control is performed by detecting a position of the bottom surface of the well (an observation target installation surface) to perform the auto-focus control, in a case where the thickness of the bottom portion differs greatly between adjacent wells, since the position of the bottom surface of the well differs greatly, there is a problem that the time for the auto-focus control becomes longer and the imaging time becomes longer.

<CIT> discloses techniques for real-time focusing in line scan imaging, more particularly systems and methods for capturing a digital image of a slide using an imaging line sensor and a focusing line sensor. In an embodiment, a beam-splitter is optically coupled to an objective lens and configured to receive one or more images of a portion of a sample through the objective lens. The beam-splitter simultaneously provides a first portion of the one or more images to the focusing sensor and a second portion of the one or more images to the imaging sensor. A processor controls the stage and/or objective lens such that each portion of the one or more images is received by the focusing sensor prior to it being received by the imaging sensor. In this manner, a focus of the objective lens can be controlled using data received from the focusing sensor prior to capturing an image of a portion of the sample using the imaging sensor.

<CIT> discloses a biological sample observation system and biological sample observation method. A biological sample observation system has: an imaging section which observes mutually different regions that are previously selected, among regions to be observed including a biological sample, through an object lens for observing the biological sample in a culturing container through a part of the culturing container; an autofocus section which detects the focusing of the object lens with respect to a predetermined region among the regions to be observed; and a focusing drive control section which controls the focusing of the object lens when the biological sample is observed using the imaging section, based on the detection result of the focusing previously performed by the autofocus section. After the focus detection is performed by the autofocus section, without being intervened by the focus detection using the autofocus section, and the different regions are continuously observed by the imaging section, with the focusing controlled by the focusing drive control section.

The present disclosure has been made in view of the above problems, and has an object to provide an observation device and method, and an observation device control program capable of efficiently performing an auto-focus control and reducing the imaging time.

According to an aspect of the present disclosure, there is provided an observation device according to claim <NUM> of the appended claims.

According to the above aspect of the present disclosure, the operation section may perform a plurality of operations among the first operation, the second operation, and the fourth operation.

Further, according to the above aspects of the present disclosure, the observation device may further comprise an imaging system having an imaging element configured to capture the image of the observation target formed by the imaging optical system; wherein the operation section is configured to perform at least one of the first operation, the second operation, a third operation of moving the imaging element in the optical axis direction, or the fourth operation.

Further, according to the above aspects of the present disclosure, in the observation device, the operation section may be configured to perform a plurality of operations among the first operation, the second operation, the third operation, and the fourth operation.

Further, according to the above aspects of the present disclosure, in the observation device, the imaging optical system may further include an objective lens that forms the image of the observation target in the container, and the first operation may include at least one of an operation of changing a focal length of the imaging lens or an operation of changing a focal length of the objective lens.

Further, according to the above aspects of the present disclosure, the observation device may further include a focal length changing optical system that changes the focal length of the imaging optical system, in which the imaging optical system may further include an objective lens that forms the image of the observation target in the container, and the first operation may include at least one of an operation of changing a focal length of the imaging lens, an operation of changing a focal length of the objective lens, or an operation of changing the focal length of the imaging optical system by the focal length changing optical system.

According to the above aspects of the present disclosure, in the observation device, the operation section may perform a fifth operation of moving the objective lens in the optical axis direction.

Further, according to the above aspects of the present disclosure, the observation device may further include a focal length changing optical system that changes the focal length of the imaging optical system, in which the first operation may include an operation of changing the focal length of the imaging optical system by the focal length changing optical system.

Further, according to the above aspects of the present disclosure, in the observation device, the imaging optical system may further include an objective lens that forms the image of the observation target in the container, and the operation section may perform a fifth operation of moving the objective lens in the optical axis direction.

According to the above aspects of the present disclosure, in the observation device, the auto-focus controller may start the auto-focus control of the observation position immediately after the boundary portion from a time point when the auto-focus control of the observation position immediately before the boundary portion is terminated until before the imaging optical system reaches the observation position immediately after the boundary portion.

According to the above aspects of the present disclosure, in the observation device, the auto-focus controller may start, for an observation position other than the observation position immediately after the boundary portion, the auto-focus control from a time point when the imaging optical system reaches the observation position.

Further, according to the above aspects of the present disclosure, in the observation device, a time for the auto-focus control of the observation position immediately after the boundary portion may be longer than a time for the auto-focus control of the observation position other than the observation position immediately after the boundary portion.

In addition, according to the above aspects of the present disclosure, the observation device may further include a detection section that precedently detects a vertical position of the container at the observation position before the imaging optical system reaches the observation position, in which the auto-focus controller may perform the auto-focus control for each observation position on the basis of a detection signal of the detection section.

Further, according to the above aspects of the present disclosure, in the observation device, the detection section may include at least two displacement sensors that are provided in parallel in the scanning direction with the imaging optical system being interposed therebetween, and the displacement sensor to be used may be switched according to a directional change of the scanning direction.

According to the above aspects of the present disclosure, in the observation device, the detection section may detect a boundary portion of the container.

Further, according to the above aspects of the present disclosure, in the observation device, the auto-focus controller may perform, in a case where the detection signal detected by the detection section is abnormal, for an observation position where the abnormal detection signal is detected, the auto-focus control based on the detection signals of the detection section for previous and next observation positions in the scanning direction of the observation position.

In addition, according to the above aspects of the present disclosure, the observation device may further include a storage unit that stores position information of a boundary portion of the container, in which the auto-focus controller may switches a start timing of the auto-focus control on the basis of the position information of the boundary portion stored in the storage unit.

Furthermore, according to claim <NUM> of the appended claims, the container is each well of a well plate.

According to still another aspect of the present disclosure, there is provided an observation method according to claim <NUM> of the appended claims.

According to still another aspect of the present disclosure, there is provided a computer-readable recording medium according to claim <NUM> of the appended claims.

According to the observation device and method, and the observation device control program of the above aspects of the present disclosure, by moving at least one of a plurality of containers in which an observation target is contained, or an imaging optical system having an objective lens that forms an image of the observation target in each of the containers, each observation position in the container is scanned and the observation target is observed. Further, by performing at least one of a first operation of changing a focal length of the imaging optical system, a second operation of moving the imaging lens in an optical axis direction, a third operation of moving the imaging element in the optical axis direction, or a fourth operation of moving the container in the optical axis direction, in a case where the auto-focus control for each observation position is performed, a start timing of an auto-focus control for each observation position is switched on the basis of a boundary portion between the adjacent containers in a scanning direction of the observation position. Accordingly, it is possible to more efficiently perform the auto-focus control, and reduce the imaging time.

According to the observation device and method, and the observation device control program of the above aspects of the present disclosure, by moving at least one of a plurality of containers in which an observation target is contained, or an imaging optical system having an imaging lens that forms an image of the observation target in each of the containers, each observation position in the container is scanned and the observation target is observed. Further, by performing at least one of a first operation of changing a focal length of the imaging optical system, a second operation of moving the imaging lens in an optical axis direction, or a fourth operation of moving the container in the optical axis direction, in a case where the auto-focus control for each observation position is performed, a start timing of an auto-focus control for each observation position is switched on the basis of a boundary portion between the adjacent containers in a scanning direction of the observation position. Accordingly, it is possible to more efficiently perform the auto-focus control, and reduce the imaging time.

Hereinafter, a microscope observation system that uses an observation device and an observation method according to an embodiment of the present disclosure will be described in detail with reference to the accompanying drawings. <FIG> is a diagram showing a schematic configuration of a microscope device <NUM> in a microscope observation system of the embodiment.

The microscope device <NUM> captures a phase difference image of a cultured cell that is an observation target. Specifically, as shown in <FIG>, the microscope device <NUM> includes a white light source <NUM> that emits white light, a condenser lens <NUM>, a slit plate <NUM>, an imaging optical system <NUM>, an operation section <NUM>, an imaging element <NUM>, and a detection section <NUM>. Further, the microscope device <NUM> includes a focal length changing optical system <NUM>.

The operation section <NUM> includes a first operation section 15A, a second operation section 15B, a third operation section 15C, a fourth operation section 15D, a fifth operation section 15E, a sixth operation section 15F, and a seventh operation section <NUM>. Operations of the first to seventh operation sections 15A to <NUM> will be described later.

Further, a stage <NUM> is provided between the slit plate <NUM>, and the imaging optical system <NUM> and the detection section <NUM>. A culture container <NUM> in which cells that are observation targets are contained is installed on the stage <NUM>. <FIG> is a diagram showing an example of the stage <NUM>. At the center of the stage <NUM>, a rectangular opening 51a is formed. The culture container <NUM> is provided on a member that is formed with the opening 51a, and in this configuration, a phase difference image of a cell in the culture container <NUM> passes through the opening 51a.

In the present embodiment, as the culture container <NUM>, a well plate provided with a plurality of well plates in which cells are contained (in which one well corresponds to the container of the present disclosure) is used. In addition, as cells contained in the culture container <NUM>, multipotential stem cells such as induced pluripotent stem (iPS) cells and embryonic stem (ES) cells, cells of nerves, the skin, the myocardium and the liver, which are differentiated and induced from a stem cell, cells of the skin, the retina, the myocardium, blood corpuscles, nerves, and organs extracted from a human body, and the like, may be used.

The stage <NUM> is configured to be moved in an X direction and a Y direction that are orthogonal to each other by a horizontal driving section <NUM> (see <FIG>). The X direction and the Y direction are directions that are orthogonal to a Z direction, and are directions that are orthogonal to each other in a horizontal plane.

The slit plate <NUM> has a configuration in which a ring-shaped slit through which white light passes is formed in a light-shielding plate that shields white light emitted from the white light source <NUM>, and ring-shaped illumination light L is formed as the white light passes through the slit.

<FIG> is a diagram showing a detailed configuration of the imaging optical system <NUM>. The imaging optical system <NUM> includes a phase difference lens 14a and an imaging lens 14d, as shown in <FIG>. The phase difference lens 14a includes an objective lens 14b and a phase plate 14c. The phase plate 14c has a configuration in which a phase ring is formed in a transparent plate that is transparent with respect to a wavelength of the illumination light L. The size of the slit of the above-described slit plate <NUM> is in a cooperative relation with the phase ring of the phase plate 14c.

The phase ring has a configuration in which a phase membrane that shifts a phase of incident light by <NUM>/<NUM> of a wavelength and a dimmer filter that dims incident light are formed in a ring shape. The phase of direct light incident onto the phase ring shifts by <NUM>/<NUM> of a wavelength after passing through the phase ring, and its brightness is weakened. On the other hand, most of diffracted light diffracted by an observation target passes through the transparent plate of the phase plate 14c, and its phase and brightness are not changed.

The phase difference lens 14a having the objective lens 14b is moved in the optical axis direction of the objective lens 14b by the fifth operation section 15E of the operation section <NUM> shown in <FIG>. The fifth operation section 15E includes an actuator such as a piezoelectric element, for example. In this embodiment, the optical axis direction of the objective lens 14b and a Z direction (vertical direction) are the same direction. As the objective lens 14b is moved in the Z direction, an auto-focus control is performed, and contrast of a phase difference image captured by the imaging element <NUM> is adjusted.

Further, a configuration in which a magnification of the phase difference lens 14a is changeable may be used. Specifically, a configuration in which the phase difference lenses 14a or the imaging optical systems <NUM> having different magnifications are interchangeable may be used. The interchange between the phase difference lens 14a and the imaging optical systems <NUM> may be automatically performed, or may be manually performed by a user.

Further, the objective lens 14b is formed of a liquid lens whose focal length can be changed. As long as the focal length can be changed, the objective lens 14b is not limited to the liquid lens, and any other lens such as a liquid crystal lens or a shape deformable lens may be used. In the objective lens 14b, an applied voltage is changed by the sixth operation section 15F in the operation section <NUM> shown in <FIG>, and thus, the focal length is changed. Thus, the focal length of the imaging optical system <NUM> is changed. Due to the change of the focal length of the objective lens 14b, similarly, the auto-focus control is performed, and the contrast of the phase difference image captured by the imaging element <NUM> is adjusted.

The imaging lens 14d receives a phase difference image passed through the phase difference lens 14a, so that an image is formed on the imaging element <NUM> from the phase difference image. In the present embodiment, the imaging lens 14d is formed of a liquid lens whose focal length can be changed. As long as the focal length can be changed, the objective lens 14b is not limited to the liquid lens, and any other lens such as a liquid crystal lens or a shape deformable lens may be used. In the imaging lens 14d, an applied voltage is changed by the first operation section 15A in the operation section <NUM> shown in <FIG>, and the focal length is changed. Thus, the focal length of the imaging optical system <NUM> is changed. Due to the change of the focal length of the imaging lens 14d, similarly, the auto-focus control is performed, and the contrast of the phase difference image captured by the imaging element <NUM> is adjusted.

The imaging lens 14d is moved in the optical axis direction of the imaging lens 14d by the second operation section 15B in the operation section <NUM> shown in <FIG>. The second operation section 15B includes an actuator such as a piezoelectric element, for example. In this embodiment, the optical axis direction of the imaging lens 14d and the Z direction (vertical direction) are the same direction. As the imaging lens 14d is moved in the Z direction, the auto-focus control is performed, and the contrast of the phase difference image captured by the imaging element <NUM> is adjusted.

The imaging element <NUM> captures an image on the basis of the phase difference image formed by the imaging lens 14d. As the imaging element <NUM>, a charge-coupled device (CCD) image sensor, a complementary metal-oxide semiconductor (CMOS) image sensor, or the like may be used. As the imaging element, an imaging element in which color filters of red, green, and blue (R, G, and B) are provided may be used, or a monochromic imaging element may be used.

Further, the imaging element <NUM> is moved in the Z direction by the third operation section 15C in the operation section <NUM> shown in <FIG>. The third operation section 15C includes an actuator such as a piezoelectric element, for example. In the present embodiment, a direction perpendicular to an imaging surface of the imaging element <NUM> and the Z direction are the same direction. As the imaging element <NUM> is moved in the Z direction, similarly, the auto-focus control is performed, and the contrast of the phase difference image captured by the imaging element <NUM> is adjusted.

Further, the stage <NUM> is moved in the Z direction by the fourth operation section 15D, and thus, the culture container <NUM> is moved in the Z direction. The fourth operation section 15D includes an actuator such as a piezoelectric element, for example. In the present embodiment, a direction perpendicular to a surface of the stage <NUM> on which the culture container <NUM> is provided and the Z direction are the same direction. As the stage <NUM> is moved in the Z direction, similarly, the auto-focus control is performed, and the contrast of the phase difference image captured by the imaging element <NUM> is adjusted.

<FIG> is a schematic diagram showing a configuration of the focal length changing optical system. As shown in <FIG>, the focal length changing optical system <NUM> includes a circular first wedge prism <NUM> and a circular second wedge prism <NUM>. The seventh operation section <NUM> moves the first wedge prism <NUM> and the second wedge prism <NUM> to be synchronized with each other in opposite directions. With this configuration, focal positions of the imaging optical system <NUM> are changed. The change of the focal position means that the focal length increases or decreases. Thus, as the focal position of the imaging optical system <NUM> is changed, the focal length of the imaging optical system <NUM> is changed. In the present embodiment, the change of the focal length of the imaging optical system <NUM> includes the change of the focal length of the imaging lens 14d by the first operation section 15A, and the change of the objective lens 14b by the sixth operation section 15F, and additionally, the change of the focal position of the imaging optical system <NUM> due to the change of the focal length of the imaging optical system <NUM> by the seventh operation section <NUM>.

The first and second wedge prisms <NUM> and <NUM> are prisms in which two surfaces that can be a light incident surface and a light emitting surface are not parallel, that is, one surface is inclined with respect to the other surface. In the following description, a surface arranged perpendicular to the optical axis is referred to as a right-angled surface, and a surface arranged inclined with respect to the optical axis is referred to as a wedge surface. The wedge prisms <NUM> and <NUM> are prisms that deflect light that is incident perpendicularly to the right-angle surfaces. The seventh operation section <NUM> includes an actuator such as a piezoelectric element, for example, and moves the first wedge prism <NUM> and the second wedge prism <NUM> to be synchronized with each other in opposite directions on the basis of control signals output from an auto-focus controller <NUM> (which will be described later), while maintaining the right-angled surfaces in parallel. That is, in a case where the first wedge prism <NUM> is moved rightward in <FIG>, the second wedge prism <NUM> is moved leftward. Conversely, in a case where the first wedge prism <NUM> is moved leftward in <FIG>, the second wedge prism <NUM> is moved rightward. As described above, by moving the first and second wedge prisms <NUM> and <NUM>, an optical path length of light emitted from the imaging optical system <NUM> is changed, so that the focal position of the imaging optical system <NUM> is changed, to thereby make it possible to change the focal length. Accordingly, the auto-focus control is performed, and contrast of a phase difference image captured by the imaging element <NUM> is adjusted.

The detection section <NUM> detects a Z-directional (vertical direction) position of the culture container <NUM> installed on the stage <NUM>. Specifically, the detection section <NUM> includes a first auto-focus displacement sensor 18a and a second auto-focus displacement sensor 18b. The first and second auto-focus displacement sensors 18a and 18b correspond to displacement sensors of the present disclosure.

The first auto-focus displacement sensor 18a and the second auto-focus displacement sensor 18b are provided in parallel in the X direction as shown in <FIG> with the imaging optical system <NUM> being interposed therebetween. The first auto-focus displacement sensor 18a and the second auto-focus displacement sensor 18b in this embodiment are laser displacement meters, which irradiate the culture container <NUM> with laser light and detect its reflection light to detect a Z-directional position of a bottom surface of the culture container <NUM>. The bottom surface of the culture container <NUM> refers to a boundary surface between a bottom portion of the culture container <NUM> and cells that are observation targets, that is, a surface on which the observation targets are placed.

Information on the Z-directional position of the culture container <NUM> detected by the detection section <NUM> is output to the auto-focus controller <NUM>, and the auto-focus controller <NUM> controls the operation section <NUM> on the basis of the input position information to perform the auto-focus control.

More specifically, in the microscope device <NUM> according to the embodiment, before the imaging optical system <NUM> reaches a predetermined observation position in the culture container <NUM> on the stage <NUM>, information on the Z-directional position of the culture container <NUM> at the observation position is precedently detected by the first or second auto-focus displacement sensor 18a or 18b, and in a case where the imaging optical system <NUM> reaches the observation position, the operation section <NUM> is controlled on the basis of the position information detected by the first or second auto-focus displacement sensor 18a or 18b to perform the auto-focus control.

Here, in a case where a well plate having a plurality of wells is used as the culture container <NUM> as in the present embodiment, if the auto-focus control is performed from a time point when the imaging optical system <NUM> reaches each observation position for all observation positions in the well plate as in the related art, in performing the auto-focus control at an observation position immediately after a boundary portion between adjacent wells in the scanning direction of the observation position, that is, an initial observation position of a well on a front side in the scanning direction among adjacent wells is performed, it takes a long time to perform the auto-focus control due to a difference in thicknesses of bottom portions of the respective wells. The scanning direction of the observation position described above is a direction opposite to the movement direction of the stage <NUM>.

<FIG> is a schematic cross-sectional view of an example of the culture container <NUM> (well plate) provided with a plurality of wells <NUM>. "D" shown in <FIG> is a boundary portion between adjacent wells, and "52a" is a bottom portion of the well <NUM>. In addition, as shown in <FIG>, the thickness of the bottom portion 52a of each well <NUM> varies depending on manufacturing variations.

Further, in a case where the scanning is performed in a two-dimensional manner while reciprocating the stage <NUM> in the X direction and moving the stage <NUM> in the Y direction, since the imaging optical system <NUM> passes the boundary of the well <NUM> many times, the time loss of the auto-focus control when straddling the boundary portion D of the well <NUM> becomes large.

Accordingly, in the present embodiment, as shown in <FIG>, a timing when the auto-focus control is started is switched between an observation position R2 immediately after the boundary portion D between the adjacent wells <NUM> and an observation position other than the observation position R2 immediately after the boundary portion D. Specifically, in the present embodiment, the auto-focus control of the observation position R2 immediately after the boundary portion D is started from a time point when the auto-focus control of the observation positionR1 immediately before the boundary portion is terminated. A rectangular range indicated by a broken line in <FIG> indicates each observation position R.

That is, in the present embodiment, since it is not necessary to perform imaging by the auto-focus control for an observation position included in the boundary portion D between the adjacent wells <NUM>, the auto-focus control is not performed for the observation position included in the boundary portion D, and the auto-focus control for the observation position R2 immediately after the boundary portion D is performed using a scanning time for the observation position included in the boundary portion D. <FIG> is a diagram showing an example of a start timing and an end timing of the auto-focus control of each of the observation positions R0 to R3 near the boundary portion D of the well <NUM>. f0 to f3 shown in <FIG> are times during which the auto-focus control is performed for the respective observation positions R0 to R3, Tx is a scanning time between adjacent observation positions (a time during which the imaging optical system <NUM> is relatively moved with respect to the stage <NUM>), and Td is a scanning time from the observation position R2 to the observation position R3.

As shown in <FIG>, for the observation position R0, the auto-focus control starts at a time t0, and the auto-focus control ends at a time t1. Then, after scanning from the observation position R0 to the observation position R1, the auto-focus control of the observation position R1 is started at a time t2, and the auto-focus control ends at a time t3. Then, the auto-focus control of the observation position R2 is started from the time t3 when the auto-focus control of the observation position R1 ends, and at a time t4 before a time t5 when the imaging optical system <NUM> reaches the observation position R2, the auto-focus control of the observation position R2 ends. That is, during a scanning time Td from the observation position R1 to the observation position R2, the auto-focus control of the observation position R2 ends.

Then, at the time t5 when the imaging optical system <NUM> reaches the observation position R2, since the auto-focus control of the observation position R2 has already ended, a phase difference image at the observation position R2 is immediately captured, and the scanning is performed toward the next observation position R3. Then, after scanning from the observation position R2 to the observation position R3, the auto-focus control of the observation position R3 starts at a time t6, and the auto-focus control ends at a time t7.

In the case of the auto-focus control in the related art, since the auto-focus control of the observation position R2 is started from a time point when the imaging optical system <NUM> reaches the observation position R2 (the time t5 in <FIG>), its imaging time becomes longer, and particularly, in a case where the thickness of the bottom portion of the well <NUM> varies, the time loss becomes larger.

In the present embodiment, as described above, it is possible to reduce the imaging time by making the start timing of the auto-focus control of the observation position R2 immediately after the boundary portion D earlier.

As for the observation positions other than the observation position R2 immediately after the boundary portion D, as described above, the auto-focus control is started from the time point when each observation position is reached. Further, in the present embodiment, the auto-focus control of the observation position R2 immediately after the boundary portion D is started from the time point t3 when the auto-focus control of the observation position R1 immediately before the boundary portion D ends, but the present disclosure is not limited to thereto, and the auto-focus control of the observation position R2 immediately after the boundary portion D may be started at any other time point as long as it is before the time t5 when the imaging optical system <NUM> reaches the observation position R2 after the time point t3 when the auto-focus control of the observation position R ends. That is, any other time point during Td shown in <FIG> may be used.

Further, in the present embodiment, the time for the auto-focus control of the observation position R2 immediately after the boundary portion D (for example, the time Td shown in <FIG>, which is the time from the time point when the imaging optical system <NUM> reaches the observation position R1 to the time point when the imaging optical system <NUM> reaches the observation position R2) is set to be longer than the time for the auto-focus control of the observation position other than the observation position R2 immediately after the boundary portion D (for example, the time from the time t0 to the time t2 shown in <FIG>, which is the time from the time point when the imaging optical system <NUM> reaches the observation position R0 to the time point when the imaging optical system <NUM> reaches the observation position R1).

In addition, in a case where the auto-focus control of the observation position R2 immediately after the boundary portion D is performed as described above, it is necessary to specify coordinates of the observation position R1 immediately before the boundary portion D and the observation position R2 immediately after the boundary portion D in the X-Y plane. That is, it is necessary to specify the boundary portion D. Therefore, in the present embodiment, the boundary portion D between the adjacent wells <NUM> is detected by the above-described first or second auto-focus displacement sensors 18a or 18b. Specifically, since the bottom surface of the well <NUM> does not exist at the boundary portion D, a detection signal detected by the first or second auto-focus displacement sensor 18a or 18b obviously varies. Accordingly, for example, it is possible to detect the boundary portion D by determining whether the detection signal detected by the first or second auto-focus displacement sensors 18a or 18b is within a range of a preset threshold value.

Then, a configuration of the microscope control device <NUM> that controls the microscope device <NUM> will be described. <FIG> is a block diagram showing a configuration of the microscope observation system according to this embodiment. With respect to the microscope device <NUM>, a block diagram of a partial configuration controlled by respective sections of the microscope control device <NUM> is shown.

The microscope control device <NUM> generally controls the microscope device <NUM>, and particularly, includes the auto-focus controller <NUM>, the scanning controller <NUM>, and the display controller <NUM>.

The microscope control device <NUM> is configured of a computer including a central processing unit, a semiconductor memory, a hard disk, and the like, and an embodiment of an observation device control program of the present disclosure is installed in the hard disk. Further, as the observation device control program is executed by the central processing unit, the auto-focus controller <NUM>, the scanning controller <NUM>, and the display controller <NUM> shown in <FIG> execute their functions.

The auto-focus controller <NUM> performs the auto-focus control by operating the operation section <NUM> on the basis of the information on the Z-directional position of the culture container <NUM> detected by the detection section <NUM> as described above. Further, as described above, the auto-focus controller <NUM> of the present embodiment switches a starting timing of the auto-focus control between the observation position immediately after the boundary portion between the adjacent wells and the observation position other than the observation position immediately after the boundary portion.

Here, the auto-focus controller <NUM> stores relationships between the information on the Z-directional position of the culture container <NUM>, a voltage applied to the imaging lens 14d for changing the focal length of the imaging lens 14d, the amount of movement of the imaging lens 14d in the optical axis direction, the amount of movement of the imaging element <NUM> in the optical axis direction, the amount of movement of the stage <NUM> in the optical axis direction, the amount of movement of the objective lens 14b in the optical axis direction, a voltage applied to the objective lens 14b for changing the focal length of the objective lens 14b, and the amount of movement of the focal length changing optical system <NUM> in advance as a table. This table is referred to as a first table.

The auto-focus controller <NUM> respectively obtains the voltage applied to the imaging lens 14d for changing the focal length of the imaging lens 14d, the amount of movement of the imaging lens 14d in the optical axis direction, the amount of movement of the imaging element <NUM> in the optical axis direction, the amount of movement of the stage <NUM> in the optical axis direction, the amount of movement of the objective lens 14b in the optical axis direction, the voltage applied to the objective lens 14b for changing the focal length, and the amount of movement of the focal length changing optical system <NUM>, with reference to the first table, on the basis of the input information on the Z-directional position of the culture container <NUM>. In the following description, the voltage applied to the imaging lens 14d for changing the focal length of the imaging lens 14d, the amount of movement of the imaging lens 14d in the optical axis direction, the amount of movement of the imaging element <NUM> in the optical axis direction, the amount of movement of the stage <NUM> in the optical axis direction, the amount of movement of the objective lens 14b in the optical axis direction, the voltage applied to the objective lens 14b for changing the focal length of the objective lens 14b, and the amount of movement of the focal length changing optical system <NUM> are referred to as focus control amounts.

The auto-focus controller <NUM> outputs control signals corresponding to the focus control amounts to the first operation section 15A to the seventh operation section <NUM> in order to control the operation section <NUM>. Specifically, the focus control amount is acquired with reference to the first table on the basis of the position information of the stage <NUM> acquired as described later. Thus, the focal length of the imaging lens 14d is changed by the first operation section 15A, and thus, the focal length of the imaging optical system <NUM> is changed. Further, the imaging lens 14d is moved in the optical axis direction by the second operation section 15B. The imaging element <NUM> is moved in the optical axis direction by the third operation section 15C. Further, the stage <NUM> is moved in the optical axis direction by the fourth operation section 15D. In addition, the objective lens 14b is moved in the optical axis direction by the fifth operation section 15E. The focal length of the objective lens 14b is changed by the sixth operation section 15F, and thus, the focal length of the imaging optical system <NUM> is changed. Further, the focal position of the imaging optical system <NUM> is changed by the seventh operation section <NUM>, and thus, the focal length of the imaging optical system <NUM> is changed. Through these seven operations, the auto-focus control is performed.

The change of the focal length of the imaging lens 14d by the first operation section 15A, the change of the focal length of the objective lens 14b by the sixth operation section 15F, and the change of the focal length changing optical system <NUM> by the seventh operation section <NUM> correspond to a first operation. The movement of the imaging lens 14d in the optical axis direction by the second operation section 15B corresponds to a second operation. The movement of the imaging element <NUM> in the optical axis direction by the third operation section 15C corresponds to a third operation. The movement of the stage <NUM> in the optical axis direction by the fourth operation section 15D corresponds to a fourth operation. The movement of the objective lens 14b in the optical axis direction by the fifth operation section 15E corresponds to a fifth operation.

The scanning controller <NUM> drives and controls the horizontal driving section <NUM>, to thereby move the stage <NUM> in the X direction and the Y direction, and move the culture container <NUM> in the X direction and the Y direction. The horizontal driving section <NUM> is configured by an actuator such as a piezoelectric element.

In this embodiment, as described above, the stage <NUM> is moved in the X direction and the Y direction under the control of the scanning controller <NUM>, the observation position in the culture container <NUM> is scanned in a two-dimensional manner, and a phase difference image at each observation position is captured. <FIG> is a diagram showing a scanning position of an observation position in the culture container <NUM> using a solid line M. In this embodiment, a well plate having six wells <NUM> is used as the culture container <NUM>.

As shown in <FIG>, the observation region in the culture container <NUM> is scanned from a scanning start point S to a scanning end point E along the solid line M, by the movement of the stage <NUM> in the X direction and the Y direction. That is, the observation region is scanned in a positive direction (a rightward direction in <FIG>) of the X direction, is scanned in the Y direction (a downward direction in <FIG>), and then, is scanned in a reverse negative direction (in a leftward direction in <FIG>). Then, the observation region is scanned in the Y direction again, and then, is scanned in the positive direction of the X direction again. In this way, by repeating the reciprocating movement of the stage <NUM> in the X direction and the movement of the stage <NUM> in the Y direction, the observation region is scanned in the culture container <NUM> in a two-dimensional manner.

Next, returning to <FIG>, the display controller <NUM> combines phase difference images at the respective observation positions imaged by the microscope device <NUM> to generate one composite phase difference image, and displays the composite phase difference image on the display device <NUM>.

The display device <NUM> displays the composite phase difference image generated by the display controller <NUM> as described above. For example, the display device <NUM> includes a liquid crystal display, or the like. Further, the display device <NUM> may be formed by a touch panel, and may also be used as the input device <NUM>.

The input device <NUM> includes a mouse, a keyboard, and the like, and receives various setting inputs by the user. The input device <NUM> according to this embodiment receives a setting input such as a change command of the magnification of the phase difference lens 14a or a change command of the moving velocity of the stage <NUM>, for example.

Next, an operation of the microscope observation system according to this embodiment will be described with reference to a flowchart shown in <FIG>. First, the culture container <NUM> in which cells that are observation targets are contained is provided on the stage <NUM> (step ST10). Then, the stage <NUM> is moved so that the observation position of the imaging optical system <NUM> is set to the position of the scanning start point S shown in <FIG>, and the movement of the stage <NUM> is started (step ST12).

Here, in this embodiment, as described above, the Z-directional position of the culture container <NUM> is precedently detected with respect to each observation position, and at a time point when the imaging optical system <NUM> is moved up to the observation position, imaging for a phase difference image is performed. Further, the detection of the Z-directional position of the culture container <NUM> and the capturing of the phase difference image are performed while scanning the observation position, and capturing of a phase difference image at a certain observation position and detection of the Z-directional position of the culture container <NUM> at a forward position in the scanning direction with reference to the observation position are performed in parallel.

Specifically, in a case where the stage <NUM> is moved forward in an arrow direction shown in <FIG>, the Z-directional position of the culture container <NUM> is detected by the first auto-focus displacement sensor 18a (step ST14), and information on the detected position is acquired by the auto-focus controller <NUM>. The auto-focus controller <NUM> stores the acquired information on the Z-directional position of the culture container <NUM> together with X-Y coordinates of the observation position of the culture container <NUM>.

Further, the first auto-focus displacement sensor 18a performs a process of detecting a boundary portion of a well together with the detection of the Z-directional position of the culture container <NUM> (step ST16). In a case where the boundary portion of the well is detected, X-Y coordinates thereof are stored.

Then, in step ST14, the imaging optical system <NUM> moves toward the observation position where the position of the culture container <NUM> is detected by the first auto-focus displacement sensor 18a and the auto-focus control of the observation position is performed, but as described above, the start timing of the auto-focus control is switched depending on the observation position.

Specifically, in a case where the observation position has not reached the boundary portion between the adjacent wells (step ST18; NO), the auto-focus control is started from a time point when each observation position has been reached (step ST20). Specifically, the focus control amount is acquired on the basis of the information on the Z-directional position of the culture container <NUM> at each observation position, and the auto-focus control is performed on the basis of the focus control amount.

On the other hand, in a case where the observation position immediately before the boundary portion between the adjacent wells has been reached, the auto-focus control of an observation position immediately after a boundary portion of the next well (the first observation position of the next well) starts from a time point when the auto-focus control of the observation position immediately before the boundary portion ends (step ST22). That is, the focus control amount is acquired on the basis of the information on the Z-directional position of the culture container <NUM> at the observation position immediately after the boundary portion, and the auto-focus control is performed on the basis of the focus control amount.

Then, after the auto-focus control ends for each observation position (step ST24), imaging for a phase difference image is performed (step ST26). The phase difference image at each observation position is output from the imaging element <NUM> to the display controller <NUM> for storage.

Further, in a case where the forward movement is terminated, and then, the movement is switched to a returning movement as shown in <FIG>, a displacement sensor to be used is switched from the first auto-focus displacement sensor 18a to the second auto-focus displacement sensor 18b.

In addition, in a case where the entire scanning is not terminated at this time point (step ST28; NO), the stage <NUM> is reversely moved again, and then, the processes of steps ST14 to ST28 are performed.

The displacement sensor to be used is switched whenever the movement direction of the stage <NUM> is switched, and the processes of steps ST14 to ST26 are repeatedly performed until the entire scanning is terminated. Further, at a time point when the observation position reaches the position of the scanning end point E shown in <FIG>, the entire scanning is terminated (step ST28; YES).

After the entire scanning is terminated, the display controller <NUM> combines phase difference images in the respective observation regions R to generate a composite phase difference image (step ST30), and displays the generated composite phase difference image on the display device <NUM> (step ST32).

According to the microscope observation system of the above-described embodiment, in a case where the auto-focus control for each observation position in the culture container <NUM> is performed, since the start timing of the auto-focus control for each observation position is switched on the basis of the boundary portion D between the adjacent wells <NUM> in the scanning direction of the observation position, it is possible to efficiently perform the auto-focus control, to thereby reduce the imaging time.

In addition, since the auto-focus control is performed by the first to seventh operation sections 15A to <NUM>, it is possible to perform the auto-focus control with high speed compared with a case where the auto-focus control is performed by only one operation.

In the above-described embodiment, a boundary portion between wells is detected by the first or second auto-focus displacement sensor 18a or 18b, but the present disclosure is not limited to thereto, and the boundary portion between the wells may be acquired and stored in advance. Specifically, as shown in <FIG>, a storage unit <NUM> that stores information on the position of the boundary portion between the wells may be provided, and the auto-focus controller <NUM> may switch the start timing of the auto-focus control on the basis of the information on the position of the boundary portion stored in the storage unit <NUM>.

In a case where the information on the position of the boundary portion between the wells is stored in this manner, identification information may be assigned to each culture container <NUM>, and a table (hereinafter, referred to as a second table) in which the identification information is associated with the information on the position of the boundary portion between the wells may be set in advance. By providing such a second table, for example, even in a case where the culture container <NUM> having a different number of wells is installed, it is possible to appropriately switch the start timing of the auto-focus control as described above. The identification information of the culture container <NUM> may be set and input by a user using the input device <NUM>, or a barcode or an RFID (radio frequency identification) tag storing the identification information may be provided for the culture container <NUM>, and the identification information may be read therefrom.

In the microscope observation system of this embodiment, the boundary portion between the wells is detected by the first and second auto-focus displacement sensors 18a and 18b, but the present disclosure is not limited thereto, and a well boundary detecting sensor other than the first and second auto-focus displacement sensors 18a and 18b may be provided.

In the microscope observation system of the above embodiment, a well plate having a plurality of wells <NUM> is used as the culture container <NUM>, but a petri dish may be used as the container of the present disclosure, and a plurality of petri dishes may be installed on the stage <NUM>. In the above-described embodiment, the start timing of the auto-focus control for each observation position is switched on the basis of the boundary portion between the adjacent wells <NUM> in the scanning direction of the observation position, but in a case where the plurality of petri dishes are installed on the stage <NUM>, the start timing of the auto-focus control for each observation position may be set to be switched on the basis of a boundary portion between adjacent petri dishes in the scanning direction of the observation position.

Specifically, the above-described well <NUM> may be replaced with a petri dish, and the auto-focus control of an observation position immediately after a boundary portion of the petri dish may be started, for example, from a time point when the auto-focus control of an observation position immediately before the boundary portion is terminated. Then, for an observation position other than the observation position immediately after the boundary portion, the auto-focus control may be started from a time point when the imaging optical system <NUM> reaches the observation position.

In the above embodiment, the Z-directional position of the bottom surface of the culture container <NUM> is detected, but for example, in a case where the bottom surface of the culture container <NUM> has a flaw or adhesion of dirt and a detection signal detected by the detection section <NUM> is abnormal, it is not possible to appropriately perform the auto-focus control. <FIG> is a diagram showing s Z-directional position based on a detection signal detected by the detection section <NUM> in a case where the bottom surface of the culture container <NUM> has a flaw or adhesion of dirt. A range of S1 shown in <FIG> corresponds to a range of the bottom surface of each well <NUM>, and a range of S3 corresponds to a range of the boundary portion D of the wells <NUM>. Then, a range of S2 shown in <FIG> corresponds to a range of the flaw or adhesion of dirt on the bottom surface of the culture container <NUM>.

Accordingly, in a case where the detection signal detected by the detection section <NUM> is abnormal, the auto-focus controller <NUM> may perform, for an observation position where the abnormal detection signal is detected, the auto-focus control based on a detection signal of the detection section <NUM> at an observation position immediately after the observation position in the scanning direction of the observation position. In the case of the detection signal as shown in <FIG>, the detection signal detected by the detection section <NUM> is not used for the range of S2, and the auto-focus control is performed using detection signal of the observation position immediately before and/or immediately after the range of S2.

Specifically, for example, the auto-focus control of the observation position in the range of S2 may be performed using an average value of the detection signal of the observation position immediately before the range of S2 and the detection signal of the observation position immediately after the range of S2. Further, the detection signal of the observation position immediately before the range of S2 or the detection signal of the observation position immediately after the range of S2, instead of the average value may be used, and linear interpolation may be performed using a detection signal of an observation position immediately before the range of S2 and a detection signal of an observation position immediately after the range of S2 to acquire the detection signal of the observation position in the range of S2. Further, linear interpolation may be performed using detection signals of two or more observation positions before the range of S2 and detection signals of two or more observation positions after the range of S2, instead of the observation positions immediately before and immediately after the range of S2, to acquire the detection signal in the range of S2.

In the above-described embodiment, the observation position in the culture container <NUM> is scanned by moving the stage <NUM>, but the present disclosure is not limited thereto, and an imaging system that includes the imaging optical system <NUM>, the detection section <NUM>, and the imaging element <NUM> may be configured to be moved. Further, both the stage <NUM> and the imaging system may be configured to be moved.

In the above-described embodiments, the operation section <NUM> performs the auto-focus control by the first to seventh operation sections 15A to <NUM>, but instead, only the first to fourth operation sections 15A to 15D, and the sixth to seventh operation sections 15F and <NUM> may be provided. Further, the auto-focus control may be performed using only one of the first to fourth operation sections 15A to 15D, and the sixth to seventh operation sections 15F and <NUM>. In this case, the auto-focus control may be further performed using the fifth operation section 15E. Further, only one of the first to fourth operation sections 15A to 15D, and the sixth to seventh operation sections 15F and <NUM> may be provided. In this case, similarly, the fifth operation section 15E may be further provided, and the auto-focus control may be configured to be performed using the fifth operation section 15E. In addition, the auto-focus control may be performed using a plurality of operation sections among the first to fourth operation sections 15A to 15D, and the sixth to seventh operation sections 15F and <NUM>. In this case, similarly, the auto-focus control may be performed further using the fifth operation section 15E.

Further, in the above-described embodiments, the focal length changing optical system <NUM> is disposed between the imaging optical system <NUM> and the imaging element <NUM>, but may be disposed between the imaging optical system <NUM> and the stage <NUM>.

In the above-described embodiments, the focal length of the imaging optical system <NUM> is changed by the first operation section 15A, the sixth operation section 15F, and the seventh operation section <NUM>, but the focal length of the imaging optical system <NUM> may be changed by only any one or two of the first operation section 15A, the sixth operation section 15F, and the seventh operation section <NUM>.

Further, in the above-described embodiments, the culture container <NUM> is moved in the optical axis direction by moving the stage <NUM> in the optical axis direction using the fourth operation section 15D. However, instead of moving the stage <NUM> in the optical axis direction, a mechanism for moving the culture container <NUM> in the optical axis direction may be provided, and only the culture container <NUM> may be moved in the optical axis direction.

Further, in the above-described embodiments, the optical system that moves the first and second wedge prisms <NUM> and <NUM> is used as the focal length changing optical system <NUM> for changing the focal length of the imaging optical system <NUM>. However, an optical element capable of changing a focal length, such as a liquid lens, a liquid crystal lens, a shape deformable lens, or the like, may be used as the focal length changing optical system. For example, instead of the focal length changing optical system <NUM> for moving the first and second wedge prisms <NUM> and <NUM>, as shown in <FIG>, a focal length changing optical system <NUM> including an optical element capable of changing a focal length may be provided between the imaging optical system <NUM> and the imaging element <NUM>. In this case, the focal length changing optical system <NUM> is configured such that an applied voltage is changed by an eighth operation section <NUM> to change the focal length. The focal length changing optical system <NUM> may be disposed between the imaging optical system <NUM> and the stage <NUM>. The focal length changing optical system <NUM> may be disposed in addition to the focal length changing optical system <NUM>.

Further, in the above embodiment, the first auto-focus displacement sensor 18a and the second auto-focus displacement sensor 18b are provided side by side in the X direction with the phase difference lens 14a being interposed therebetween, but as shown in <FIG>, a third auto-focus displacement sensor 18c and a fourth auto-focus displacement sensor 18d may be provided side by side in the Y direction with the phase difference lens 14a being interposed therebetween.

Thus, it is possible to reciprocating the observation region R, and also, to move the observation region R as shown in <FIG>. That is, in <FIG>, the observation region R is moved from the scanning start point S in a positive direction (a rightward direction in <FIG>) in the X direction, is moved in a positive direction (a downward direction in <FIG>) in the Y direction, is moved in a negative direction (in a leftward direction in <FIG>) in the X direction, and then, is moved in a negative direction (an upward direction in <FIG>) in the Y direction. Thus, it is possible to scan the inside of the culture container <NUM> in a two-dimensional manner by repeatedly moving the observation region R in the X direction and the Y direction. In this case, it is possible to detect a boundary portion between wells by the third and fourth auto-focus displacement sensors 18c and 18d, in addition to the first and second auto-focus displacement sensors 18a and 18b.

Further, in the above-described embodiment, the present disclosure is applied to the phase difference microscope, but the present disclosure is not limited to the phase difference microscope, and may be applied to a different microscope such as a differential interference microscope or a bright field microscope.

Claim 1:
An observation device comprising:
an imaging optical system (<NUM>) having an imaging lens (14d) configured to form an image of an observation target in a plurality of containers (<NUM>) in which the observation target is contained, wherein each container (<NUM>) is a well of a well plate;
an operation section (<NUM>) configured to perform at least one of a first operation of changing a focal length of the imaging optical system (<NUM>), a second operation of moving the imaging lens (14d) in an optical axis direction, or a fourth operation of moving the container (<NUM>) in the optical axis direction;
a horizontal driving section (<NUM>) configured to move at least one of the container (<NUM>) or the imaging optical system (<NUM>) in a horizontal plane;
a scanning controller (<NUM>) configured to control the horizontal driving section (<NUM>) and moves at least one of the container (<NUM>) or the imaging optical system (<NUM>) to scan an observation position (R0-R3) in the container (<NUM>); and
an auto-focus controller (<NUM>) configured to control the operation section (<NUM>) and performs an auto-focus control for each observation position (R0-R3),
wherein the auto-focus controller (<NUM>) is configured to, for each observation position (R0-R3),
detect, during the scanning movement, whether the observation position (R0-R3) has reached the boundary portion (D) between the adjacent containers (<NUM>) in a scanning direction of the observation position (R0-R3), and
switch a start timing of the auto-focus control depending upon whether the observation position (R0-R3) has reached the boundary portion (D) between the adjacent containers (<NUM>).