WO2019202979A1 - Observation device, observation device operation method, and observation control program - Google Patents

Observation device, observation device operation method, and observation control program Download PDF

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Publication number
WO2019202979A1
WO2019202979A1 PCT/JP2019/014725 JP2019014725W WO2019202979A1 WO 2019202979 A1 WO2019202979 A1 WO 2019202979A1 JP 2019014725 W JP2019014725 W JP 2019014725W WO 2019202979 A1 WO2019202979 A1 WO 2019202979A1
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WIPO (PCT)
Prior art keywords
unit
imaging
observation
optical system
control
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PCT/JP2019/014725
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French (fr)
Japanese (ja)
Inventor
一英 長谷川
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富士フイルム株式会社
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Priority to JP2020514060A priority Critical patent/JPWO2019202979A1/en
Publication of WO2019202979A1 publication Critical patent/WO2019202979A1/en

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    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B21/00—Microscopes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/21—Polarisation-affecting properties
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
    • G01N21/41—Refractivity; Phase-affecting properties, e.g. optical path length
    • G01N21/45—Refractivity; Phase-affecting properties, e.g. optical path length using interferometric methods; using Schlieren methods
    • G—PHYSICS
    • G02—OPTICS
    • G02B—OPTICAL ELEMENTS, SYSTEMS OR APPARATUS
    • G02B7/00—Mountings, adjusting means, or light-tight connections, for optical elements
    • G02B7/28—Systems for automatic generation of focusing signals

Definitions

  • the present invention relates to an observation apparatus for observing an observation object accommodated in a container, an operation method of the observation apparatus, and an observation control program.
  • 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.
  • 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 cells that can be applied in regenerative medicine, drug development, disease elucidation, and the like.
  • an imaging optical system is used to scan the range of a culture vessel such as a well plate to obtain an image for each observation position. After that, it has been proposed to perform so-called tiling photography, in which images for each observation position are combined.
  • NA numerical aperture
  • culture containers used in microscopic observation are mass-produced by, for example, injection molding of polystyrene resin, and are often disposable types, so that the manufacturing accuracy is not very good.
  • the observation surface on which the cells settle that is, the bottom surface of the culture container may be curved and / or inclined.
  • the curvature and / or inclination of the bottom surface of the culture vessel varies depending on the type of the culture vessel, such as a difference in manufacturer, and the range of production error may be different.
  • the focal position of the imaging optical system is adjusted to the bottom surface in the culture vessel.
  • the observation region is set large in order to increase the imaging time. In this case, or when the curve and / or inclination is large, as shown in FIG. 18, if the observation surface is out of the range of depth of field, the captured image becomes out of focus and the image is blurred. .
  • FIG. 17 a microscope including two imaging units 16A and 16B capable of forming optical images of different focal planes F1 and F2 on one imaging optical system 14 has been proposed. .
  • the range of the depth of field is expanded by overlapping the depth of field.
  • imaging is performed by using a plurality of imaging elements for one imaging optical system and performing focus adjustment for each imaging element (for each angle of view) using a variable apex angle prism provided for each imaging element.
  • an image acquisition apparatus capable of focusing on the entire region.
  • images are captured at different focal positions, that is, suitable focal positions for each observation region by photographing different observation regions in the imaging region at the focal positions adjusted in the respective observation regions. Since the acquired images are acquired, the images acquired by photographing at different focal positions are images representing different observation regions, and a plurality of images are not acquired in one observation region.
  • Patent Document 2 proposes an image acquisition device in which an optical path is branched into two, one is an optical path for image acquisition, and the other is an optical path for focus control, and an imaging unit is provided on each optical path. .
  • an imaging unit is used for focus control, a plurality of images for observation in one observation region are not acquired.
  • the defocus amount between the two imaging units 16A and 16B is fixed.
  • the defocus amount may be small, but when the fixed defocus amount is larger than the required defocus amount, it is acquired by photographing. In some cases, the quality of the captured image may be degraded.
  • the present invention has been made in view of the above circumstances, and an object of the present invention is to provide an observation apparatus, an operation method of the observation apparatus, and an observation control program that can suppress deterioration in image quality of a captured image.
  • the observation device of the present invention includes an acquisition unit that acquires position information indicating the position of the bottom surface of a container that stores an observation target, a plurality of imaging units that can form optical images with different focal planes, and a container And at least one operation unit capable of changing at least one of the position and orientation of at least one of the plurality of imaging units with respect to the imaging optical system that forms an optical image indicating the observed object. Based on the position information acquired by the unit, the operation unit is driven to change at least one of the position and orientation of at least one of the plurality of imaging units, and each of the plurality of imaging units has a focal plane. And a control unit that performs control to form different optical images.
  • the “focal plane” means a plane including the focal point of the optical image and a surface on the object side.
  • position of the imaging unit means the position of the imaging unit in the optical axis direction of the imaging optical system, and “posture of the imaging unit” means the position of the imaging unit with respect to the optical axis of the imaging optical system. Means tilt.
  • the “bottom surface of the container” is the bottom surface in the container that accommodates the observation target, and means the installation surface of the observation target.
  • the “container” may have any form as long as it can accommodate the observation target.
  • a container having a form having a bottom part and a wall part continuous to the bottom part such as a petri dish, a dish, a flask, or a well plate, can be used as a container.
  • a micro-channel device or the like in which a fine channel is formed on a plate-like member can be used as a container.
  • what has a plate-like form like a slide glass can also be used as a container.
  • the observation apparatus of the present invention includes an optical path dividing unit that divides an optical path of light transmitted through the observation target into a plurality of optical paths, Each of the plurality of imaging units may be disposed on each of the plurality of optical paths divided by the optical path dividing unit.
  • At least one imaging unit can change the position on the optical axis of the imaging optical system with respect to the imaging optical system
  • the control unit may perform control to change the position on the optical axis of at least one imaging unit based on the position information acquired by the acquisition unit.
  • At least one imaging unit can change the inclination of the light receiving surface with respect to the imaging optical system on the optical axis
  • the control unit may perform control to change the tilt on the optical axis of the light receiving surface of at least one imaging unit in accordance with the tilt of the bottom surface based on the position information acquired by the acquiring unit.
  • At least one imaging unit can change the position on the optical axis of the imaging optical system with respect to the imaging optical system and the inclination of the light receiving surface with respect to the imaging optical system on the optical axis.
  • the control unit changes the position on the optical axis of at least one imaging unit, and in accordance with the inclination of the bottom surface based on the position information acquired by the acquisition unit, You may perform control which changes the inclination on the optical axis of the light-receiving surface of at least 1 imaging part.
  • control unit when the control unit has a difference in height of the bottom surface based on the position information acquired by the acquisition unit, which is greater than a predetermined threshold, at least one of the position and orientation of at least one imaging unit. You may perform control which changes one side.
  • the observation apparatus of the present invention further includes an imaging optical system and an imaging unit, and a drive unit that relatively moves at least one of the container on a specific intersection plane that intersects the optical axis of the imaging optical system.
  • the acquisition unit corresponds to a position preceding the imaging optical system along the direction in which the observation region of the imaging optical system moves according to the relative movement of the imaging optical system and the imaging unit and the container. You may acquire the positional information which shows the position of the bottom face of the container to perform.
  • the “specific intersection plane” means an intersection plane that intersects the optical axis.
  • the driving unit moves at least one of the imaging optical system, the imaging unit, and the container in the main scanning direction and the sub-scanning direction orthogonal to the main scanning direction at the intersection plane
  • the acquisition unit may acquire the shape information of the bottom surface based on the previously acquired position information in the main scanning direction and the position information in the main scanning direction after being moved in the sub scanning direction.
  • the “position of the bottom surface of the container” is the position of the bottom surface of the container in the direction perpendicular to the intersecting surface
  • the “shape information of the bottom surface” is at least information on the height difference of the bottom surface of the container, or It is information including either one of the information on the inclination of the bottom surface of the container.
  • the drive unit moves at least one of the imaging optical system, the imaging unit, and the container in the main scanning direction and the sub-scanning direction orthogonal to the main scanning direction at the intersecting plane
  • the acquisition unit may acquire position information indicating the bottom surface of the container corresponding to a position preceding the imaging optical system in the main scanning direction and two or more pieces of position information that are different in the sub-scanning direction.
  • “acquiring two or more pieces of position information different in the sub-scanning direction” is not limited to two or more pieces of position information arranged in a straight line along the sub-scanning direction. If the above position information can be acquired, two or more pieces of position information that are not arranged on a straight line may be used.
  • the observation apparatus of the present invention may include an image processing unit that generates a single image by combining a plurality of images acquired by a plurality of imaging units.
  • observation apparatus of the present invention may include a selection unit that selects an appropriate image from a plurality of images acquired by a plurality of imaging units.
  • the operation method of the observation apparatus of the present invention is to acquire position information indicating the position of the bottom surface of the container that accommodates the observation target by the acquisition unit, and based on the position information acquired by the acquisition unit, by driving the operation unit, A plurality of imaging units that change at least one of the position and orientation of at least one imaging unit among a plurality of imaging units with respect to an imaging optical system that forms an optical image indicating an observation object accommodated in a container.
  • Each of these includes a method of operating an observation apparatus that forms optical images with different focal planes.
  • the observation control program of the present invention includes a computer, It functions as an acquisition unit and a control unit included in the observation apparatus.
  • Another observation apparatus includes a memory for storing instructions to be executed by a computer, A processor configured to execute stored instructions, the processor comprising: Obtain position information indicating the position of the bottom surface of the container containing the observation target, Based on the acquired position information, at least one of the position and orientation of the imaging unit capable of changing at least one of the position and orientation is changed, and an optical image having a different focal plane is formed on each of the plurality of imaging units. The process which performs control to perform is performed.
  • the acquisition unit that acquires position information indicating the position of the bottom surface of the container that accommodates the observation target, and the position and orientation with respect to the imaging optical system that forms an optical image that indicates the observation target accommodated in the container
  • a plurality of imaging units each including at least one imaging unit capable of changing at least one of them and capable of forming optical images with different focal planes, and position and orientation based on position information acquired by the acquisition unit
  • a control unit that controls at least one of the position and orientation of the imaging unit that can change at least one to form an optical image with a different focal plane on each of the plurality of imaging units.
  • the defocus amount can be changed according to the shape of the bottom surface.
  • the observation surface can be positioned within the depth of field, it is possible to prevent the captured image from being out of focus and becoming a blurred image due to the observation surface being out of the range of the depth of field. be able to.
  • the difference in height of the bottom surface of the container is relatively small, the image quality of the captured image can be maintained by reducing the defocus amount.
  • the height difference of the bottom surface of the container is relatively large, it is possible to suppress a decrease in the image quality of the captured image by increasing the defocus amount.
  • FIG. 1 is a schematic diagram illustrating an example of the configuration of a microscope apparatus to which the observation apparatus of the present embodiment is applied.
  • FIG. 2 is a schematic diagram illustrating an example of the configuration of an imaging optical system included in the microscope apparatus main body according to the first embodiment.
  • the microscope apparatus 1 includes a microscope apparatus body 10 and a microscope control apparatus 20 (see FIG. 4).
  • the microscope apparatus 1 is an example of an observation apparatus according to the present invention.
  • the microscope apparatus body 10 captures cultured cells that are observation targets and acquires a phase difference image.
  • the microscope apparatus main body 10 includes a white light source 11 that emits white light, a condenser lens 12, a slit plate 13, an imaging optical system 14, an operation unit 15, and an imaging unit 16A.
  • the imaging unit 16B and the detection unit 18 are provided.
  • the operation unit 15 includes a first operation unit 15A, a second operation unit 15B, a third operation unit 15C, a fourth operation unit 15D, a fifth operation unit 15E, and a sixth operation unit 15F.
  • the operations of the first to sixth operation units 15A to 15F will be described later.
  • the slit plate 13 is provided with a ring-shaped slit that transmits white light to the light-shielding plate that blocks white light emitted from the white light source 11, and the ring shape is obtained when white light passes through the slit. Illumination light L is formed.
  • the imaging optical system 14 forms an image of the phase difference image obtained by observing the culture vessel 50 on the imaging unit 16A and the imaging unit 16B.
  • FIG. 2 is a diagram showing a detailed configuration of the imaging optical system 14.
  • the imaging optical system 14 includes a phase difference lens 14a and an imaging lens 14d.
  • the phase difference lens 14a includes an objective lens 14b and a phase plate 14c.
  • the phase plate 14 c is formed by forming a phase ring on a substrate that is transparent to the wavelength of the illumination light L.
  • the slit size of the slit plate 13 described above is in a conjugate relationship with the phase ring of the phase plate 14c.
  • phase film that shifts the phase of incident light by 1 ⁇ 4 wavelength and a neutral density filter that attenuates incident light are formed in a ring shape.
  • the phase is shifted by 1 ⁇ 4 wavelength and its brightness is weakened.
  • most of the diffracted light diffracted by the observation object passes through the transparent plate of the phase plate 14c, and its phase and brightness do not change.
  • 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 unit 15E included in the operation unit 15 shown in FIG.
  • the optical axis direction of the objective lens 14b and the Z direction are the same direction.
  • Autofocus control is performed by the movement of the objective lens 14b in the Z direction, and the contrast of the phase difference image acquired by the imaging unit 16A and the imaging unit 16B is adjusted.
  • the fifth operation unit 15 moves the phase difference lens 14a in the Z direction.
  • the present invention is not limited to this, and only the objective lens 14b may be moved in the Z direction. .
  • phase difference lens 14a or the imaging optical system 14 having different magnifications may be configured to be exchangeable.
  • the replacement of the phase difference lens 14a or the imaging optical system 14 may be performed automatically or manually by a user.
  • the objective lens 14b of this embodiment consists of a liquid lens which can change a focal distance as an example.
  • the lens is not limited to the liquid lens, and any lens such as a liquid crystal lens and a shape deforming lens can be used.
  • the applied voltage is changed by the sixth operating unit 15F included in the operating unit 15 shown in FIG.
  • the focal length of the imaging optical system 14 is changed.
  • the autofocus control is also performed by changing the focal length of the objective lens 14b, and the contrast of the phase difference image acquired by the imaging unit 16A and the imaging unit 16B is adjusted.
  • the imaging lens 14 d receives light indicating a phase difference image that has passed through the phase difference lens 14 a, and this light forms an image on the imaging surface 16 ⁇ / b> A of the imaging unit 16.
  • the imaging lens 14d is a liquid lens whose focal length can be changed. Note that as long as the focal length can be changed, the lens is not limited to the liquid lens, and any lens such as a liquid crystal lens and a shape deforming lens can be used.
  • the applied voltage is changed by the first operating unit 15A included in the operating unit 15 shown in FIG. As a result, the focal length of the imaging optical system 14 is changed. Autofocus control is performed by changing the focal length of the imaging lens 14d, and the contrast of the phase difference images acquired by the imaging unit 16A and the imaging unit 16B is adjusted.
  • the imaging lens 14d is moved in the optical axis direction of the imaging lens 14d by the second operating unit 15B included in the operating unit 15 shown in FIG.
  • the optical axis direction of the imaging lens 14d and the Z direction are the same direction.
  • Autofocus control is performed by the movement of the imaging lens 14d in the Z direction, and the contrast of the phase difference images acquired by the imaging unit 16A and the imaging unit 16B is adjusted.
  • the microscope apparatus body 10 includes an optical path dividing unit 19 that divides the optical path of the light emitted from the imaging optical system 14 into a plurality of optical paths.
  • the optical path splitting unit 19 is configured by a beam splitter, transmits light emitted from the imaging optical system 14, and transmits light emitted from the imaging optical system 14 to the imaging optical system 14. Reflects in a direction different from the optical axis.
  • the optical path splitting unit 19 of this embodiment deflects the light emitted from the imaging optical system 14 at a right angle to the optical axis of the imaging optical system 14.
  • the optical path splitting unit 19 of the present embodiment deflects the light emitted from the imaging optical system 14 at right angles to the optical axis of the imaging optical system 14, but the present invention is not limited to this. It is not limited to a right angle as long as the deflected light can be guided to the imaging unit 16B.
  • the optical path splitting unit 19 of the present embodiment is configured by a beam splitter.
  • the present invention is not limited to this, as long as it reflects a part of incident light and transmits a part thereof. Any beam splitter such as a mirror type or a prism type may be used. However, it is more preferable to use a beam splitter that separates incident light into 50% and 50% from the viewpoint of equalizing the image quality of the imaging unit 16A and the imaging unit 16B.
  • the microscope apparatus 1 of the present embodiment has a configuration including two imaging units, that is, the imaging unit 16A and the imaging unit 16B. However, in a microscope apparatus including three imaging units, for example, the light is first incident by a beam splitter. The light may be separated into 33% and 66%, and the incident light separated at 66% may be separated into 50% and 50% by the next beam splitter.
  • the imaging unit 16A receives the light reflected by the optical path splitting unit 19 that is an image of the observation target imaged by the imaging lens 14d, and uses a phase difference image representing the observation target as an observation image. Output.
  • the imaging unit 16B receives light that is an image of the observation target imaged by the imaging lens 14d and has passed through the optical path dividing unit 19, and outputs a phase difference image that represents the observation target as an observation image. To do.
  • the imaging unit 16A and the imaging unit 16B each include an imaging element such as a charge-coupled device (CCD) image sensor or a complementary metal-oxide semiconductor (CMOS) image sensor.
  • an imaging device provided with a color filter of RGB (Red Green Blue) may be used, or a monochrome imaging device may be used.
  • the imaging unit 16B can change the position of the imaging optical system 14 on the optical axis. Specifically, the imaging unit 16B is moved in the Z direction by the third operating unit 15C included in the operating unit 15 illustrated in FIG. In the present embodiment, the optical axis of the imaging optical system 14 and the Z direction are the same direction. The defocus amount between the imaging unit 16A and the imaging unit 16B is changed by the movement of the imaging unit 16B in the Z direction. A method for changing the defocus amount will be described later in detail.
  • the detection unit 18 detects the position in the Z direction (vertical direction) of the bottom surface of the culture vessel 50 installed on the stage 51.
  • the detection unit 18 includes a first displacement sensor 18a and a second displacement sensor 18b.
  • the first displacement sensor 18a and the second displacement sensor 18b are provided side by side in the X direction shown in FIG. 1 with the phase difference lens 14a interposed therebetween.
  • the first displacement sensor 18a and the second displacement sensor 18b in this embodiment are laser displacement meters, which irradiate the culture vessel 50 with laser light and detect the reflected light, thereby detecting Z on the bottom surface of the culture vessel 50. Detect the position of the direction.
  • the bottom surface of the culture vessel 50 means the boundary surface between the bottom of the culture vessel 50 and the cell that is the observation target, that is, the installation surface of the observation target. That is, the bottom surface in the culture vessel 50.
  • the bottom of the culture vessel 50 means a bottom wall that forms the bottom of the culture vessel 50.
  • the position of the bottom surface of the culture vessel 50 in the Z direction is, for example, the detection unit 18 as a reference plane, and the value of the reflected light signal detected by the detection unit 18 is the position of the bottom surface of the culture vessel 50 in the Z direction. Is a value representing.
  • the value of the position in the Z direction on the bottom surface of the culture vessel 50 is the value of the reflected light signal detected by the detection unit 18, but is not limited thereto, and the reflected light signal is not limited thereto.
  • a value obtained by converting the above value into a distance, that is, the distance from the detection unit 18 to the bottom surface of the culture vessel 50 may be set as the position of the bottom surface of the culture vessel 50 in the Z direction.
  • the detection unit 18 further detects the reflected light reflected from the bottom surface of the bottom of the culture vessel 50, whereby the reflected light reflected from the bottom surface of the bottom of the culture vessel 50 from the value of the reflected light signal reflected from the bottom surface of the culture vessel 50.
  • a value representing the thickness of the bottom of the culture vessel 50 may be calculated by subtracting the light value, and this value may be used as a value representing the position of the bottom surface of the culture vessel 50 in the Z direction. Further, a value representing the position in the Z direction may be converted into an actual distance value and used.
  • the laser displacement sensor can use a specular reflection optical system measuring instrument.
  • the detection part 18 is not restricted to a laser displacement sensor, For example, a confocal type sensor can also be used.
  • the position information in the Z direction of the bottom surface of the culture vessel 50 detected by the detection unit 18 is output to the acquisition unit 23A described later.
  • the acquiring unit 23A outputs the input position information to the focus adjusting unit 24 described later.
  • the focus adjustment unit 24 adjusts the focus position of the imaging optical system 14 based on the input position information to acquire a focus control amount, and the control unit 22 described later performs focus control acquired by the focus adjustment unit 24. Based on the amount, the operation unit 15 is controlled to perform autofocus control.
  • the detection of the position in the Z direction of the culture vessel 50 by the first displacement sensor 18a and the second displacement sensor 18b and the adjustment of the focus position by the focus adjustment unit 24 will be described in detail later.
  • a stage 51 is provided between the slit plate 13, the phase difference lens 14 a and the detection unit 18.
  • a culture vessel 50 in which cells to be observed are accommodated is installed on the stage 51.
  • the culture container 50 of this embodiment is an example of the container used in the observation apparatus according to the present invention.
  • the container is not limited to the culture container 50 and may have any form as long as it can accommodate an observation target.
  • a container having a form having a bottom part and a wall part continuous to the bottom part such as a petri dish, a dish, a flask, or a well plate, can be used as a container.
  • the observation target is not limited to the cultured one, and may be any of blood, various particles, fibers, and the like.
  • the cells contained in the culture vessel 50 include pluripotent stem cells such as iPS cells and ES cells, nerves, skin, myocardium and liver cells induced to differentiate from the stem cells, and skin, retina extracted from the human body, There are myocardium, blood cells, nerve and organ cells.
  • the stage 51 is moved in the X and Y directions orthogonal to each other by a horizontal driving unit 17 (see FIG. 4) described later.
  • the X direction and the Y direction are directions in a specific intersection plane that intersects the optical axis of the imaging optical system 14, and are directions orthogonal to each other in the specific intersection plane.
  • the specific intersection plane is a horizontal plane as an example. Therefore, the X direction and the Y direction are directions orthogonal to the Z direction, and are directions orthogonal to each other in the horizontal plane.
  • the X direction is the main scanning direction
  • the Y direction is the sub-scanning direction.
  • the stage 51 has a rectangular opening 51 a at the center.
  • the culture vessel 50 is installed on the member forming the opening 51a, and the phase difference image of the cells in the culture vessel 50 is configured to pass through the opening 51a.
  • the stage 51 is moved in the Z direction by the fourth operating unit 15D, whereby the culture vessel 50 is moved in the Z direction.
  • the direction perpendicular to the surface of the stage 51 where the culture vessel 50 is installed and the Z direction are the same direction.
  • the autofocus control is also performed by the movement of the stage 51 in the Z direction, and the contrast of the phase difference image acquired by the imaging unit 16A and the imaging unit 16B is adjusted.
  • the first operating unit 15A and the sixth operating unit 15F include, for example, a voltage variable circuit.
  • the first operation unit 15A changes the voltage applied to the imaging lens 14d based on a control signal output from the control unit 22 described later.
  • the sixth operation unit 15F changes the voltage applied to the objective lens 14b based on a control signal output from the control unit 22 described later.
  • the second operation unit 15B, the third operation unit 15C, the fourth operation unit 15D, and the fifth operation unit 15E include, as an example, a piezoelectric element and a drive source that applies a high voltage. Drive based on the output control signal.
  • the operation unit 15 is configured to pass the phase difference image that has passed through the phase difference lens 14a and the imaging lens 14d as it is.
  • the configuration of the second operating unit 15B, the third operating unit 15C, the fourth operating unit 15D, and the fifth operating unit 15E is not limited to the piezoelectric element, and the imaging lens 14d, the imaging unit 16B, and the stage 51.
  • the objective lens 14b phase difference lens 14a
  • the number of piezoelectric elements (also referred to as piezo elements) constituting the second operating unit 15B, the third operating unit 15C, the fourth operating unit 15D, and the fifth operating unit 15E is not limited to one, but is a plurality. There may be.
  • FIG. 4 is a block diagram showing a configuration of the microscope apparatus 1 of the present embodiment.
  • the block diagram of the one part structure controlled by each part of the microscope control apparatus 20 is shown.
  • the microscope control device 20 includes a CPU (Central Processing Unit) 21, a primary storage unit 25A, a secondary storage unit 25B, an external I / F (Interface) 28A, and the like.
  • the CPU 21 includes a control unit 22, an acquisition unit 23 ⁇ / b> A, a processing unit 23 ⁇ / b> B, and a focus adjustment unit 24, and controls the entire microscope apparatus 1.
  • the primary storage unit 25A is a volatile memory used as a work area or the like when executing various programs.
  • An example of the primary storage unit 25A is a RAM (Random Access Memory).
  • the secondary storage unit 25B is a nonvolatile memory in which various programs, various parameters, and the like are stored in advance, and an example of the observation control program 26 according to the technique of the present disclosure is installed.
  • the CPU 21 reads the observation control program 26 from the secondary storage unit 25B and develops the read observation control program 26 in the primary storage unit 25A.
  • the CPU 21 operates as the control unit 22, the acquisition unit 23A, the processing unit 23B, and the focus adjustment unit 24 by executing the observation control program 26 developed in the primary storage unit 25A.
  • the secondary storage unit 25B stores position information 27 described later.
  • An example of the secondary storage unit 25B is an EEPROM (Electrically Erasable Programmable Read-Only Memory) or a flash memory.
  • the external I / F 28 ⁇ / b> A controls transmission / reception of various information between the microscope apparatus main body 10 and the microscope control apparatus 20.
  • the CPU 21, the primary storage unit 25 ⁇ / b> A, and the secondary storage unit 25 ⁇ / b> B are connected to the bus line 28.
  • the external I / F 28A is also connected to the bus line 28.
  • the observation control program 26 is recorded and distributed on a recording medium such as a DVD (Digital Versatile Disc) and a CD-ROM (Compact Disc Read Only Memory), and is installed in the computer from the recording medium.
  • a recording medium such as a DVD (Digital Versatile Disc) and a CD-ROM (Compact Disc Read Only Memory)
  • the observation control program 26 is stored in a storage device or network storage of a server computer connected to the network in a state where it can be accessed from the outside, and is installed after being downloaded to the computer in response to an external request. You may be made to do.
  • the position information 27 is position information in the Z direction of the bottom surface of the culture vessel 50 acquired by the acquisition unit 23A.
  • the general-purpose computer may be implemented by a dedicated computer.
  • the dedicated computer may be firmware that executes a program recorded in a nonvolatile memory such as a built-in ROM (Read-Only Memory) and a flash memory.
  • a dedicated ASIC Application Specific Integrated Circuit
  • FPGA Field-Programmable Gate Array
  • the program instructions stored in the dedicated circuit may be combined with the program instructions executed by a general-purpose CPU programmed to use the program of the dedicated circuit.
  • program instructions may be executed by any combination of hardware configurations of computers.
  • the acquisition unit 23A acquires position information indicating the position of the bottom surface of the culture vessel 50. Specifically, position information in the Z direction of the bottom surface of the culture vessel 50 detected by the detection unit 18 is acquired.
  • the processing unit 23B performs various processes such as gamma correction, luminance / color difference conversion, and compression processing on the image signal acquired by the imaging unit 16A and / or the imaging unit 16B. Further, the processing unit 23B outputs an image signal obtained by performing various processes to the control unit 22 described later for each frame at a specific frame rate. Further, the processing unit 23B combines a phase difference image of each observation region imaged by the microscope apparatus body 10, that is, a combined image obtained by the imaging unit 16A and / or the imaging unit 16B, thereby combining one composite position. A phase difference image is generated. Note that the processing unit 23B of this embodiment is an example of an image processing unit of the present invention.
  • the focus adjustment unit 24 adjusts the focus position of the imaging optical system 14 based on the position information in the Z direction of the bottom surface of the culture vessel 50 acquired by the acquisition unit 23A. Based on the position information on the bottom surface, the focus adjustment unit 24 has the first operation unit 15A, the second operation unit 15B, the fourth operation unit 15D, the fifth operation unit 15E, and the sixth operation unit 15F.
  • the movement amount, that is, the focus control amount is acquired for each of them, and each focus control amount is output to the control unit 22.
  • the position information in the Z direction of the bottom surface of the culture vessel 50, the voltage applied to the imaging lens 14d for changing the focal length of the imaging lens 14d, and the amount of movement of the imaging lens 14d in the optical axis direction The table is an example showing the relationship between the amount of movement of the stage 51 in the optical axis direction, the amount of movement of the objective lens 14b in the optical axis direction, and the voltage applied to the objective lens 14b for changing the focal length of the objective lens 14b. Is previously stored in the secondary storage unit 25B.
  • the focus adjustment unit 24 refers to the above table based on the position information in the Z direction of the culture vessel 50 acquired by the acquisition unit 23A, and applies the voltage applied to the imaging lens 14d and the optical axis direction of the imaging lens 14d. , The amount of movement of the stage 51 in the optical axis direction, the amount of movement of the objective lens 14b in the optical axis direction, and the applied voltage to the objective lens 14b.
  • the voltage applied to the imaging lens 14d, the movement amount of the imaging lens 14d in the optical axis direction, the movement amount of the stage 51 in the optical axis direction, the movement amount of the objective lens 14b in the optical axis direction, The voltage applied to the objective lens 14b is referred to as a focus control amount.
  • the position information in the Z direction of the bottom surface of the culture vessel 50, the voltage applied to the imaging lens 14d, the amount of movement of the imaging lens 14d in the optical axis direction, the amount of movement of the stage 51 in the optical axis direction, and the objective lens 14b What shows the relationship between the movement amount in the optical axis direction and the voltage applied to the objective lens 14b is not limited to the table, and may be an equation, for example. What shows the above relationship is the voltage applied from the position information to the imaging lens 14d, the amount of movement of the imaging lens 14d in the optical axis direction, the amount of movement of the stage 51 in the optical axis direction, and the movement of the objective lens 14b in the optical axis direction. Any method may be used as long as the amount and the voltage applied to the objective lens 14b can be derived.
  • the control unit 22 focuses each of the first operation unit 15A, the second operation unit 15B, the fourth operation unit 15D, the fifth operation unit 15E, and the sixth operation unit 15F acquired by the focus adjustment unit 24.
  • a control signal based on the control amount is output to each of the first operation unit 15A, the second operation unit 15B, the fourth operation unit 15D, the fifth operation unit 15E, and the sixth operation unit 15F.
  • the focal length of the imaging lens 14d is changed by the first operating unit 15A, and the focal length of the imaging optical system 14 is changed.
  • the imaging lens 14d is moved in the optical axis direction by the second operation unit 15B.
  • the stage 51 is moved in the optical axis direction by the fourth operation unit 15D.
  • the objective lens 14b is moved in the optical axis direction by the fifth operating unit 15E.
  • the focal length of the objective lens 14b is changed by the sixth operation unit 15F, and the focal length of the imaging optical system 14 is changed.
  • the autofocus control is performed by these five operations.
  • control unit 22 drives and controls the horizontal direction drive unit 17, thereby moving the stage 51 in the X direction and the Y direction, and moving the culture vessel 50 in the X direction and the Y direction.
  • the horizontal drive unit 17 includes a known moving mechanism for moving in the horizontal direction and a drive source such as a motor.
  • the horizontal driving unit 17 is an example of the driving unit of the present invention.
  • the stage 51 is moved in the X direction and the Y direction by the control of the control unit 22, the imaging optical system 14 is scanned two-dimensionally in the culture vessel 50, and the imaging unit 16A and the imaging unit 16B are A phase difference image at each observation position by the imaging optical system 14 is acquired. That is, the imaging unit 16A and the imaging unit 16B each acquire a phase difference image for each of a plurality of imaging regions (fields of view) divided within one well.
  • control unit 22 causes the display device 30 to display one composite phase difference image generated by the processing unit 23B combining the phase difference images of the respective observation regions photographed by the microscope apparatus body 10. It also functions as a part.
  • control unit 22 moves the imaging unit 16B in the optical axis direction by the third operation unit 15C based on the position information indicating the position of the bottom surface of the culture vessel 50 acquired by the acquisition unit 23A, and the imaging unit Control is performed to form optical images with different focal planes on each of 16A and imaging unit 16B.
  • FIG. 5 is a diagram for explaining the adjustment of the defocus amount in the microscope apparatus 1.
  • the focal plane in the imaging unit 16A is a focal plane F1
  • the focal plane in the imaging unit 16B is a focal plane F2.
  • the focal plane means a plane in which the optical image is in focus, that is, in focus.
  • the imaging unit 16A and the imaging unit 16B are arranged with the focal plane F1 of the imaging unit 16A different from the focal plane F2 of the imaging unit 16B.
  • the observation target when the observation target is observed or photographed in the microscope apparatus 1, it can be considered that the observation target is in focus even at a distance away from the focus position, that is, the focal plane, in the optical axis direction.
  • the range is called depth of field (DOF).
  • DOF depth of field
  • the focal plane F1 of the imaging unit 16A and the focal plane F2 of the imaging unit 16B there are depths of field DOF1 and DOF2, respectively.
  • the depth of field DOF1 and the depth of field DOF2 are different values depending on performance such as the numerical aperture NA of the imaging units 16A and 16B, the magnification of the objective lens 14b, and the imaging lens 14d.
  • the user sets the input device 40 in advance. Used and stored in the secondary storage unit 25B.
  • the method for calculating the depth of field is not particularly limited, and can be calculated using a known technique.
  • the depth of field DOF1 and the depth of field DOF2 are set to overlap at least partially. can do.
  • the depth of field DOF in the microscope apparatus 1 can be expanded as compared with the case where there is one imaging unit.
  • the depth of field DOF1 and the depth of field DOF2 can be expanded up to twice as much as when there is one imaging unit.
  • control unit 22 performs control to change the position in the Z direction of the imaging unit 16B based on the position information in the Z direction of the culture vessel 50 acquired by the acquisition unit 23A. That is.
  • the position of the focal plane F2 of the imaging unit 16B is moved in the Z direction, and the depth of field DOF2 is moved in the Z direction. That is, the control unit 22 moves the depth of field DOF2 in the Z direction to change the overlapping area between the depth of field DOF1 and the depth of field DOF2, and sets the depth of field DOF in the microscope apparatus 1 to Control to change.
  • the depth of field DOF1 of the imaging unit 16A and the depth of field DOF2 of the imaging unit 16B are in the range of ⁇ 6 ⁇ m, that is, 12 ⁇ m, respectively.
  • the amount of shift in the Z direction between the focal plane F1 of the imaging unit 16A and the focal plane F2 of the imaging unit 16B is referred to as a defocus amount.
  • the defocus amount is 0 because there is no shift in the Z direction.
  • the amount is 12 ⁇ m.
  • the distance d is 10 2 times, so the distance d is defocus amount ⁇ 10 2 . Control for changing the defocus amount in the microscope apparatus 1 will be described in detail later.
  • an input device 40 and a display device 30 are connected to the microscope control device 20 by a bus line 28.
  • the display device 30 displays the composite phase difference image generated by the control unit 22 as described above, and includes, for example, a liquid crystal display. 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 and a keyboard as an example, and accepts various setting inputs by the user.
  • the input device 40 receives setting inputs such as an instruction to change the magnification of the phase difference lens 14a and an instruction to change the moving speed of the stage.
  • FIG. 6 is a flowchart showing an example of the operation of the microscope apparatus according to the first embodiment
  • FIG. 7 is a diagram showing an example of the scanning position of the observation region in the culture container of the microscope apparatus according to the first embodiment
  • FIG. FIG. 9 is a flowchart for explaining an example of the arrangement of displacement sensors in the microscope apparatus according to the embodiment
  • FIG. 9 is a flowchart illustrating an example of defocus amount adjustment processing of the microscope apparatus according to the first embodiment
  • FIG. It is a figure for demonstrating an example of acquisition of the height difference of the bottom face in the microscope apparatus which concerns on a form.
  • step S ⁇ b> 1 the control unit 22 drives the horizontal driving unit 17 to place the stage 51 on which the culture vessel 50 containing the cells to be observed is placed.
  • the observation region of the imaging optical system 14 is positioned at the scanning start point S shown in FIG. 7 as an example, and scanning of the culture vessel 50 by the observation region is started.
  • the stage 51 is moved in the X direction and the Y direction by the control of the control unit 22, and the observation region of the imaging optical system 14 is moved two-dimensionally in the culture vessel 50 to scan the culture vessel 50. Then, a phase difference image of each observation area is acquired.
  • the solid line M indicates the scanning position of the observation region in the culture vessel 50.
  • the observation region of the imaging optical system 14 moves along the solid line M from the scanning start point S to the scanning end point E by the above movement of the stage 51. That is, the observation area is moved in the positive direction of the X direction (right direction in FIG. 7), then moved in the Y direction (downward direction in FIG. 7), and moved in the opposite negative direction (left direction in FIG. 7). Is done. Next, the observation area moves again in the Y direction and is moved again in the positive direction. In this way, the culture vessel 50 is scanned two-dimensionally by repeatedly performing the reciprocating movement in the X direction and the movement in the Y direction of the observation region.
  • the control unit 22 causes the detection unit 18 to detect the position information of the bottom surface of the culture vessel 50, and the acquisition unit 23A acquires the position information detected by the detection unit 18.
  • a first displacement sensor 18a and a second displacement sensor 18b are provided side by side in the X direction with the imaging optical system 14 interposed therebetween.
  • the observation region R of the imaging optical system 14 is moved two-dimensionally within the culture vessel 50 as described above.
  • the control unit 22 determines whether the culture vessel 50 and the imaging optical system 14 are connected. Control that causes the detection unit 18 to acquire position information indicating the position of the bottom surface of the culture vessel 50 corresponding to the position preceding the imaging optical system 14 along the direction in which the observation region R moves in accordance with the relative movement. I do.
  • the position of the culture container 50 in the Z direction is detected at a position in front of the movement direction of the observation region R relative to the position of the observation region R of the imaging optical system 14 with respect to the culture container 50.
  • the observation region R moves in the direction of the arrow shown in FIG. 8 (the right direction in FIG. 8)
  • the observation region of the first displacement sensor 18a and the second displacement sensor 18b are detected by the first displacement sensor 18a on the front side in the R movement direction.
  • the observation region R when the observation region R is moving in the direction opposite to the arrow direction in FIG. 8 (the left direction in FIG. 8), the observation region R out of the first displacement sensor 18a and the second displacement sensor 18b.
  • the position of the culture vessel 50 in the Z direction is detected by the second displacement sensor 18b on the front side in the R movement direction.
  • control unit 22 switches between detection using the first displacement sensor 18a and detection using the second displacement sensor 18b according to the moving direction of the observation region R.
  • step S3 the control unit 22 performs control to adjust the defocus amount between the imaging unit 16A and the imaging unit 16B based on the position information acquired by the acquisition unit 23A.
  • FIG. 9 is a flowchart illustrating an example of a defocus amount adjustment process of the microscope apparatus according to the first embodiment
  • FIG. 10 is a diagram for explaining an example of acquisition of the height difference of the bottom surface in the microscope apparatus according to the first embodiment. is there.
  • the control unit 22 acquires the height difference of the bottom surface based on the position information of the bottom surface acquired by the acquisition unit 23 ⁇ / b> A.
  • the position of the bottom surface of the culture vessel 50 in the Z direction is acquired in time series by the detection unit 18 in the X direction and / or the Y direction.
  • Position information of the bottom surface is detected along each line such as a line along which the observation area moves, that is, a movement line M1 of the current observation area and a movement line M2 of the previous observation area.
  • the first displacement shown in FIG. A height difference is acquired from the position in the Z direction of the culture vessel 50 detected in advance at the position of the sensor 18a and the position in the Z direction of the culture vessel 50 detected in advance at the position of the observation region R shown in FIG. Thereby, the height difference of the bottom face in the X direction of the culture vessel 50 can be acquired.
  • FIG. 11 is a diagram for explaining another example of the arrangement of the displacement sensor in the microscope apparatus according to the first embodiment
  • FIG. 12 shows still another arrangement of the displacement sensor in the microscope apparatus according to the first embodiment
  • FIG. 13 is a diagram for explaining an example
  • FIG. 13 is a diagram for explaining another example for obtaining the height difference of the bottom surface in the microscope apparatus according to the first embodiment.
  • the first displacement sensor 18a and the second displacement sensor 18b are provided at positions shifted in the Y direction from the imaging optical system 14 as compared with the embodiment of FIG. ing.
  • the bottom surface along each line such as a line L along which the observation region moves, that is, a movement line L1 of the current observation region and a movement line L2 of the previous observation region. Position information is detected.
  • the first region shown in FIG. A difference in height in the X direction from the position in the Z direction of the culture vessel 50 detected in advance at the position of the displacement sensor 18a and the position in the Z direction of the culture vessel 50 detected in advance in the position of the observation region R shown in FIG. get. Thereby, the height difference of the bottom face in the X direction of the culture vessel 50 can be acquired.
  • the observation region R in the X direction moves from the position shown in FIG. 11 to the position where the position of the culture vessel 50 in the Z direction is detected by the first displacement sensor 18a, the first region shown in FIG.
  • the height difference in the Y direction is acquired from the detected position of the culture vessel 50 in the Z direction. Thereby, the height difference of the bottom face in the Y direction of the culture vessel 50 can be acquired.
  • the first displacement sensor 18a and the second displacement sensor 18b are connected to the imaging optical system as shown in FIG. 11 rather than the embodiment shown in FIG.
  • the position in the Z direction acquired at a position closer to the observation area can be used in one observation area as shown in FIG.
  • the accuracy of the height difference of the bottom surface of the culture vessel 50 in the direction can be improved.
  • a third displacement sensor 18c and a fourth displacement sensor 18d may be provided on the opposite side with respect to each other.
  • the position information of the bottom surface at different positions in the Y direction is detected simultaneously along the movement line of the observation region.
  • step S ⁇ b> 22 the control unit 22 stores the obtained height difference of the bottom surface of the culture vessel 50 in the secondary storage unit 25 ⁇ / b> B.
  • step S23 the control unit 22 performs control to move the position of the imaging unit 16B in the Z direction based on the height difference of the bottom surface of the culture vessel 50 stored in the secondary storage unit 25B.
  • the position in the Z direction of the imaging unit 16B where the defocus amount is 0 is set as a default.
  • the control unit 22 does not move the imaging unit 16B.
  • the control unit 22 moves the imaging unit 16B in the Z direction by the third operating unit 15C.
  • the depth of field DOF of the microscope apparatus 1 may be a value larger than 14 ⁇ m. Accordingly, in order to set the defocus amount to a value larger than 2 ⁇ m, the control unit 22 moves the imaging unit 16B in the Z direction by a distance longer than 2 ⁇ m.
  • the depth of field DOF of the microscope apparatus 1 only needs to be a value larger than 24 ⁇ m, so that the defocus amount is controlled to 12 ⁇ m.
  • the unit 22 moves the 12 ⁇ m imaging unit 16B in the Z direction. That is, the control unit 22 causes the depth of field DOF1 of the imaging unit 16A and the depth of field DOF2 of the imaging unit 16B to continue without overlapping.
  • the control unit 22 moves the imaging unit 16B in the Z direction based on the height difference of the bottom surface of the culture vessel 50, the observation surface can be positioned within the depth of field. Since the surface is out of the range of the depth of field, it is possible to prevent the captured image from being out of focus and becoming a blurred image.
  • the height difference of the bottom surface of the culture vessel 50 is relatively small, the image quality of the captured image can be maintained by reducing the defocus amount.
  • the difference in height of the bottom surface of the culture vessel 50 is relatively large, it is possible to suppress a decrease in the image quality of the captured image by increasing the defocus amount.
  • the adjustment method of the defocus amount based on the height difference of the bottom surface has been described, the defocus amount may be adjusted in consideration of other length measuring machines or mechanical error factors.
  • step S23 When the control unit 22 moves the imaging unit 16B in step S23, the process returns to FIG. 6 and the control unit 22 continues to perform the processing from step S4.
  • step S4 the focus adjustment unit 24 acquires the focus control amount based on the position information acquired by the acquisition unit 23A in step S2.
  • the focus adjusting unit 24 refers to the table stored in the secondary storage unit 25B, applies the voltage to the imaging lens 14d, the amount of movement of the imaging lens 14d in the optical axis direction, and the stage 51.
  • the amount of movement in the optical axis direction, the amount of movement of the objective lens 14b in the optical axis direction, and the voltage applied to the objective lens 14b are respectively acquired as focus control amounts.
  • the acquisition unit 23A interpolates the position information detected at the position of the displacement sensor 18a by a known technique, thereby acquiring the position information at the same position as the position of the imaging optical system 14 in the Y direction.
  • the acquisition unit 23A interpolates the position information simultaneously detected by the first displacement sensor 18a and the third displacement sensor 18c by a known technique, so that the imaging optical system 14 The position information at the same position as the position in the Y direction is acquired.
  • step S5 the control unit 22 associates the focus control amount acquired by the focus adjustment unit 24 in step S3 with the position on the XY coordinate of the detection position of the position information on the bottom surface of the culture vessel 50, and performs secondary processing. It memorize
  • the focus control amount acquisition and storage processing in steps S4 and S5 can be performed in parallel with the control processing for adjusting the defocus amount in step S3 by the control unit 22.
  • step S ⁇ b> 6 the control unit 22 drives the horizontal driving unit 17 so that the observation region R moves toward the position where the position of the culture vessel 50 is detected by the first displacement sensor 18 a. Moving.
  • step S7 the control unit 22 acquires the focus control amount stored in the secondary storage unit 25B immediately before the observation region R reaches the position where the position of the culture vessel 50 is detected.
  • step S8 the control unit 22 performs autofocus control based on the acquired focus control amount. That is, the control unit 22 controls the first operation unit 15A to the sixth operation unit 15F based on the acquired focus control amount, thereby changing the focal lengths of the imaging lens 14d and the objective lens 14b.
  • the imaging lens 14d, the imaging unit 16, the stage 51, and the objective lens 14b are moved in the Z direction.
  • step S9 when the observation region R reaches the position where the position of the culture vessel 50 is detected, the imaging unit 16A and the imaging unit 16B capture a phase difference image.
  • the phase difference image of the observation region R is output from the imaging unit 16A and the imaging unit 16B to the control unit 22 and stored.
  • the position of the culture vessel 50 is detected in advance for each observation region R, and when the observation region R reaches the detection position, the imaging unit 16A and the imaging unit Imaging by 16B is performed.
  • the position detection of the culture vessel 50 and the imaging by the imaging unit 16A and the imaging unit 16B are performed while moving the observation region R, and the imaging of the observation region R at a certain position by the imaging unit 16A and the imaging unit 16B, The position detection of the culture vessel 50 at the front position with respect to the movement direction with respect to the position is performed in parallel.
  • the culture container by the detection unit 18 at a position ahead of the observation region R in the movement direction is acquired in parallel.
  • the movement of the imaging unit 16B according to the height difference of the bottom surface of the culture vessel 50 is performed after the imaging by the imaging unit 16B in step S9.
  • step S10 if the control unit 22 has not moved the observation region R to the acceleration / deceleration region range R2 shown in FIG. 7 by driving the horizontal direction driving unit 17, the determination is negative, Control proceeds to step S2 shown in FIG.
  • step S10 the control unit 22 drives the horizontal direction driving unit 17 to move the observation region R to the acceleration / deceleration region range R2 shown in FIG.
  • the determination is affirmed and FIG.
  • the process proceeds to step S11 shown in FIG.
  • step S11 the control unit 22 switches the displacement sensor to be used from the first displacement sensor 18a to the second displacement sensor 18b.
  • the detection timing of the position of the culture vessel 50 in each observation region R and the phase difference image The imaging timing is shifted in time. Accordingly, the autofocus control is performed after the position of the culture vessel 50 is detected by the first displacement sensor 18a or the second displacement sensor 18b and before the observation region R reaches the detection position. .
  • step S12 the processing unit 23B determines whether or not all scanning has been completed. If all the scans are not completed in step S12, the determination is negative and the process proceeds to step S2 shown in FIG. Each time the control unit 22 moves the observation region R to the acceleration / deceleration range R1, R2, the control unit 22 switches the displacement sensor to be used, and the processing from step S2 to step S11 is performed until all scanning is completed. Is repeated.
  • step S12 when all the scans are completed, that is, when the control unit 22 causes the observation region R to reach the position of the scan end point E shown in FIG. 7, the determination is affirmed and the control unit 22 terminates all scanning.
  • the processing unit 23B combines the phase difference images of the observation regions R to generate a combined phase difference image in step S13.
  • phase difference images of the same observation region R are acquired by the imaging unit 16A and the imaging unit 16B.
  • the processing unit 23B divides the phase difference image into 64 ⁇ 64 pixels, and selects the better image quality for each divided region. Select and synthesize. At this time, for example, the higher contrast in the image can be selected as an image with good image quality.
  • the relative positions of the imaging unit 16A and the imaging unit 16B may be corrected using a known camera calibration technique.
  • step S14 the control unit 22 causes the display device 30 to display the combined phase difference image generated by the processing unit 23B, and the series of processes by the microscope device 1 is completed.
  • the control unit 22 changes the position of the imaging unit 16B based on the position information acquired by the acquisition unit 23A, and causes each of the imaging unit 16A and the imaging unit 16B to have a focal plane. Since the phase difference image is acquired by forming different optical images, the defocus amount can be changed according to the shape of the bottom surface of the culture vessel 50. As a result, since the observation surface can be positioned within the depth of field, it is possible to prevent the captured image from being out of focus and becoming a blurred image due to the observation surface being out of the range of the depth of field. be able to. In addition, when the height difference of the bottom surface of the culture vessel 50 is relatively small, the image quality of the captured image can be maintained by reducing the defocus amount. Moreover, when the difference in height of the bottom surface of the culture vessel 50 is relatively large, it is possible to suppress a decrease in the image quality of the captured image by increasing the defocus amount.
  • FIG. 14 is a block diagram showing an example of the configuration of the microscope apparatus according to the second embodiment. Note that FIG. 14 is an apparatus that further includes the selection unit 23C in the microscope apparatus 1 of the above-described embodiment of FIG. 4, and other configurations are the same as in the above-described embodiment. Only different parts will be described in detail.
  • the microscope apparatus of the present embodiment includes a selection unit 23C.
  • the selection unit 23C selects an appropriate image from the two images acquired by the imaging unit 16A and the imaging unit 16B. That is, the more focused image is selected. Specifically, an image with a higher contrast in the image may be selected, or by obtaining and comparing the in-focus positions when the images of the imaging unit 16A and the imaging unit 16B are acquired, the focus is increased. You may select the image that has.
  • step S13 when the processing unit 23B generates a composite phase difference image in step S13, an image selected by the selection unit 23C for each observation region is employed.
  • the third operation unit 15C moves the imaging unit 16B in the Z direction.
  • the present invention is not limited to this, and the inclination of the bottom surface of the culture vessel 50 is determined.
  • the posture of the imaging unit 16B may be changed.
  • FIG. 15 is a diagram illustrating adjustment of the defocus amount in the microscope apparatus according to the third embodiment.
  • the third operation unit 15 ⁇ / b> C includes, as an example, two piezoelectric elements and a drive source that applies a high voltage, and is driven based on a control signal output from the control unit 22.
  • the two piezoelectric elements are disposed at a distance from the bottom surface of the imaging unit 16B.
  • At least one piezoelectric element is driven by the control unit 22 in accordance with the inclination of the bottom surface of the culture vessel 50, whereby the posture of the imaging unit 16B is changed.
  • the two piezoelectric elements may be driven relatively.
  • the number of piezoelectric elements constituting the third operating unit 15C is not limited to two and may be two or more.
  • a table indicating the relationship between the height difference and the bottom surface inclination is stored in advance in the secondary storage unit 25B, and the control unit 22 acquires the bottom surface inclination of the culture vessel 50 by referring to this table.
  • what shows the relationship between a height difference and the inclination of a bottom face is not limited to a table, For example, a formula may be sufficient. Any method showing the above relationship may be used as long as the inclination of the bottom surface can be derived from the height difference.
  • the inclination of the bottom surface is acquired and stored instead of the height difference of the bottom surface of the culture vessel 50.
  • the control unit 22 tilts the imaging unit 16B according to the inclination of the bottom surface of the culture vessel 50, not the height difference of the bottom surface of the culture vessel 50.
  • control unit 22 controls the tilt of the bottom surface of the culture vessel 50 and the tilt of the image capturing unit 16B by tilting the image capturing unit 16B in accordance with the tilt of the bottom surface of the culture vessel 50.
  • the imaging part 16B May be provided with a function of moving the Z in the Z direction. That is, the posture of the imaging unit 16B and the position in the Z direction may be changed according to the shape such as the inclination of the bottom surface of the culture vessel 50.
  • the inclination of the light receiving surface of the imaging unit is changed by changing the attitude of the imaging unit, but only the inclination of the light receiving surface of the imaging unit is changed without changing the attitude of the imaging unit.
  • the configuration may be changed.
  • control unit 22 can move the imaging unit 16B in the Z direction by driving the two piezoelectric elements with the same driving amount.
  • one or more piezoelectric elements for moving the imaging unit 16B in the Z direction may be further provided.
  • control unit 22 changes the position and orientation of the imaging unit 16B in the Z direction, thereby performing finer control that matches the shape of the bottom surface of the culture vessel 50 as compared to the above-described embodiment. Therefore, it is possible to further suppress deterioration in image quality of images acquired by the imaging unit 16A and the imaging unit 16B.
  • control unit 22 determines the position of the imaging unit 16B in the Z direction and Control to change at least one of the postures of the imaging unit 16B is performed.
  • the observation surface is within the depth of field in one shooting by the imaging unit 16A and the imaging unit 16B.
  • the control unit 22 divides one observation region R into a plurality of observation regions in which the observation surface is within the depth of field, and the imaging interval is shorter than that in the above embodiment. Control for causing the unit 16A and the imaging unit 16B to perform imaging a plurality of times is performed.
  • the observation surface can be positioned within the depth of field, and thus the captured image is prevented from being out of focus and being out of focus due to the observation surface being out of the range of the depth of field. be able to.
  • the control unit 22 performs control to change the position and / or orientation in the Z direction of only the imaging unit 16B.
  • the present invention is not limited to this, and only the imaging unit 16A or The movement of both the imaging unit 16A and the imaging unit 16B may be controlled.
  • an operation unit that moves the imaging unit 16A in the X-axis direction that is, the optical axis direction of the light reflected by the optical path dividing unit 19 is newly provided.
  • two piezoelectric elements are newly provided on the left side of the imaging unit 16A, for example, in FIG. 15, so that the imaging unit 16A can move in the X direction.
  • this operation unit can have the same configuration as the third operation unit 16C.
  • the microscope apparatus of the embodiment described above includes two imaging units
  • the present invention is not limited to this, and may include, for example, three or more imaging units.
  • two beam splitters may be used as the optical path dividing unit 19.
  • the present invention is applied to a phase contrast microscope.
  • the present invention is not limited to the phase contrast microscope, and is applied to observation of other microscopes such as a differential interference microscope and a bright field microscope. Can do.
  • the case where the observation control program 26 is read from the secondary storage unit 25B is exemplified, but it is not always necessary to store the observation control program 26 in the secondary storage unit 25B from the beginning.
  • an arbitrary portable storage medium 250 such as an SSD (Solid State Drive), a USB (Universal Serial Bus) memory, or a DVD-ROM (Digital versatile disc-Read Only Memory) is used.
  • the observation control program 26 may be stored.
  • the observation control program 26 of the storage medium 250 is installed in the microscope control apparatus 20, and the installed observation control program 26 is executed by the CPU 21.
  • the observation control program 26 is stored in a storage unit such as another computer or server device connected to the microscope apparatus 1 via a communication network (not shown), and the observation control program 26 requests the microscope apparatus body 10. It may be downloaded according to. In this case, the downloaded observation control program 26 is executed by the CPU 21.
  • observation control process described in the above embodiments is merely an example. Therefore, it goes without saying that unnecessary steps may be deleted, new steps may be added, and the processing order may be changed within a range not departing from the spirit.
  • Microscope device 10 Microscope unit 11
  • Slit plate 14 Imaging optical system 14a phase difference lens 14b
  • Microscope control device 22 Control unit 23A acquisition unit 23B processing section 23C selector 24 Focus adjustment unit 25A primary storage 25B Secondary storage unit 250 storage media 26 Observation control program 27

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Abstract

The present invention suppresses photographed image quality degradation in an observation device, observation device operation method, and observation control program. An observation device according to the present invention comprises: an acquisition unit for acquiring position information indicating the position of the bottom surface of a container accommodating an object of observation; a plurality of imaging units capable of forming optical images having different focal planes; at least one operation unit capable of changing at least one from among the position and orientation of at least one of the imaging units from among the plurality of imaging units in relation to an image formation optical system for forming an optical image of the object of observation accommodated in the container; and a control unit for carrying out control in which at least one from among the position and orientation of at least one of the imaging units from among the plurality of imaging units is changed through the driving of the operation unit on the basis of the position information acquired by the acquisition unit and the plurality of imaging units are made to form optical images having different focal planes.

Description

観察装置、観察装置の作動方法、及び観察制御プログラムObservation apparatus, operation method of observation apparatus, and observation control program

　本発明は、容器内に収容された観察対象を観察する観察装置、観察装置の作動方法、及び観察制御プログラムに関するものである。
The present invention relates to an observation apparatus for observing an observation object accommodated in a container, an operation method of the observation apparatus, and an observation control program.

　従来、ＥＳ（Embryonic Stem）細胞及びｉＰＳ（Induced Pluripotent Stem）細胞等の多能性幹細胞や分化誘導された細胞等を顕微鏡等で撮像し、その画像の特徴を捉えることで細胞の分化状態などを判定する方法が提案されている。ＥＳ細胞及びｉＰＳ細胞等の多能性幹細胞は、種々の組織の細胞に分化する能力を備えており、再生医療、薬の開発、病気の解明などにおいて応用が可能な細胞として注目されている。このような細胞を顕微鏡で撮像する際には、高倍率な広視野画像を取得するため、例えばウェルプレート等の培養容器の範囲内を結像光学系によって走査し、観察位置毎の画像を取得した後、その観察位置毎の画像を結合する、いわゆるタイリング撮影を行うことが提案されている。
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 cells that can be applied in regenerative medicine, drug development, disease elucidation, and the like. When such cells are imaged with a microscope, in order to obtain a wide-field image with a high magnification, for example, an imaging optical system is used to scan the range of a culture vessel such as a well plate to obtain an image for each observation position. After that, it has been proposed to perform so-called tiling photography, in which images for each observation position are combined.

　上述したように、細胞を撮像する際に使用される顕微鏡においては、細胞の細部を正しく観察するためにより高い分解能が要求される。分解能を高くするためには、対物光学系の開口数（ＮＡ：Numerical Aperture）が必要となる。ＮＡの大きな高倍率の対物光学系を使用した顕微鏡においては、分解能は向上するが、被写界深度が浅くなってしまう。
As described above, a microscope used for imaging a cell requires higher resolution in order to correctly observe the details of the cell. In order to increase the resolution, the numerical aperture (NA) of the objective optical system is required. In a microscope using a high-magnification objective optical system with a large NA, the resolution is improved, but the depth of field becomes shallow.

　一方、一般的に、顕微鏡観察において用いられる培養容器は、例えばポリスチレン樹脂の射出成型によって大量生産されており、使い捨てタイプのものが多く、製造精度があまり良くない。培養容器において、細胞が定着する観察面すなわち培養容器の底面には、湾曲及び／又は傾斜が生じている場合もある。培養容器の底面の湾曲及び／又は傾斜については、例えば製造メーカの違い等、培養容器の種類によって異なり、製造誤差の範囲も異なる場合がある。
On the other hand, in general, culture containers used in microscopic observation are mass-produced by, for example, injection molding of polystyrene resin, and are often disposable types, so that the manufacturing accuracy is not very good. In the culture container, the observation surface on which the cells settle, that is, the bottom surface of the culture container may be curved and / or inclined. The curvature and / or inclination of the bottom surface of the culture vessel varies depending on the type of the culture vessel, such as a difference in manufacturer, and the range of production error may be different.

　ここで、上述したような観察領域毎の画像を取得する際、培養容器内の底面に結像光学系の焦点位置を合わせるが、撮像時間の高速化のために観察領域が大きく設定されている場合、あるいは上記湾曲及び／又は傾斜が大きい場合等において、図１８に示すように、観察面が被写界深度の範囲から外れると、焦点が合わずに撮像画像がボケた画像になってしまう。
Here, when acquiring an image for each observation region as described above, the focal position of the imaging optical system is adjusted to the bottom surface in the culture vessel. However, the observation region is set large in order to increase the imaging time. In this case, or when the curve and / or inclination is large, as shown in FIG. 18, if the observation surface is out of the range of depth of field, the captured image becomes out of focus and the image is blurred. .

　そこで、図１７に示すように、１つの結像光学系１４に対して、異なる焦点面Ｆ１，Ｆ２の光学像を結像可能な２つの撮像部１６Ａ，１６Ｂを備えた顕微鏡が提案されている。このような顕微鏡においては、被写界深度を重ねることにより、被写界深度の範囲を拡張している。
Therefore, as shown in FIG. 17, a microscope including two imaging units 16A and 16B capable of forming optical images of different focal planes F1 and F2 on one imaging optical system 14 has been proposed. . In such a microscope, the range of the depth of field is expanded by overlapping the depth of field.

　特許文献１には、１つの結像光学系に対して複数の撮像素子を用い、撮像素子ごとに設けた可変頂角プリズムにより撮像素子ごと（画角ごと）のフォーカス調整を行うことで、撮像領域全域でフォーカスを合わせることができるようにした画像取得装置が提案されている。特許文献１に記載の画像取得装置においては、撮像領域内において異なる観察領域を各々の観察領域で調整された焦点位置で撮影することにより、観察領域毎に異なる焦点位置すなわち好適な焦点位置で撮影された画像を取得しているため、異なる焦点位置で撮影して取得した画像は異なる観察領域を表す画像であり、１つの観察領域において複数の画像は取得されていない。
In Patent Document 1, imaging is performed by using a plurality of imaging elements for one imaging optical system and performing focus adjustment for each imaging element (for each angle of view) using a variable apex angle prism provided for each imaging element. There has been proposed an image acquisition apparatus capable of focusing on the entire region. In the image acquisition device described in Patent Document 1, images are captured at different focal positions, that is, suitable focal positions for each observation region by photographing different observation regions in the imaging region at the focal positions adjusted in the respective observation regions. Since the acquired images are acquired, the images acquired by photographing at different focal positions are images representing different observation regions, and a plurality of images are not acquired in one observation region.

　また、特許文献２には、光路を２つに分岐し、一方を画像取得用の光路、他方を焦点制御用の光路として各光路上にそれぞれ撮像手段を設けた画像取得装置が提案されている。特許文献２に記載の画像取得装置においては、一方の撮像手段は、焦点制御用に使用されているため、１つの観察領域において観察するための複数の画像は取得されていない。
Patent Document 2 proposes an image acquisition device in which an optical path is branched into two, one is an optical path for image acquisition, and the other is an optical path for focus control, and an imaging unit is provided on each optical path. . In the image acquisition device described in Patent Document 2, since one imaging unit is used for focus control, a plurality of images for observation in one observation region are not acquired.

特開２０１５－１３５４４１号公報JP2015-135441A 特開２０１３－２１０６７２号公報JP 2013-210672A

　一般的に、上記のような２つの撮像部１６Ａ，１６Ｂを備えた顕微鏡において、２つの撮像部１６Ａ，１６Ｂ間のデフォーカス量は固定されている。しかしながら、培養容器の底面における湾曲及び／又は傾斜が小さい場合には、デフォーカス量は少なくて良いが、固定されたデフォーカス量が必要とするデフォーカス量よりも多い場合には、撮影により取得した画像の画質が低下してしまう場合がある。
In general, in a microscope including the two imaging units 16A and 16B as described above, the defocus amount between the two imaging units 16A and 16B is fixed. However, when the curvature and / or inclination at the bottom of the culture vessel is small, the defocus amount may be small, but when the fixed defocus amount is larger than the required defocus amount, it is acquired by photographing. In some cases, the quality of the captured image may be degraded.

　本発明は上記事情に鑑みなされたものであり、撮像画像の画質の低下を抑制できる観察装置及び観察装置の作動方法並びに観察制御プログラムを提供することを目的とする。
The present invention has been made in view of the above circumstances, and an object of the present invention is to provide an observation apparatus, an operation method of the observation apparatus, and an observation control program that can suppress deterioration in image quality of a captured image.

　本発明の観察装置は、観察対象を収容する容器の底面の位置を示す位置情報を取得する取得部と、各々に焦点面が異なる光学像を結像可能な複数の撮像部と、容器に収容された観察対象を示す光学像を結像させる結像光学系に対し、複数の撮像部の中の少なくとも１つの撮像部の位置及び姿勢の少なくとも一方を変更可能な少なくとも１つの動作部と、取得部により取得された位置情報に基づいて、動作部を駆動して複数の撮像部の中の少なくとも１つの撮像部の位置及び姿勢の少なくとも一方を変更し、複数の撮像部の各々に、焦点面が異なる光学像を結像させる制御を行う制御部と、を含む。
The observation device of the present invention includes an acquisition unit that acquires position information indicating the position of the bottom surface of a container that stores an observation target, a plurality of imaging units that can form optical images with different focal planes, and a container And at least one operation unit capable of changing at least one of the position and orientation of at least one of the plurality of imaging units with respect to the imaging optical system that forms an optical image indicating the observed object. Based on the position information acquired by the unit, the operation unit is driven to change at least one of the position and orientation of at least one of the plurality of imaging units, and each of the plurality of imaging units has a focal plane. And a control unit that performs control to form different optical images.

　なお、本発明において、「焦点面」は、光学像の焦点を含む面であって、対象物側の面を意味する。また、本発明において、「撮像部の位置」は、結像光学系の光軸方向における撮像部の位置を意味し、「撮像部の姿勢」は、結像光学系の光軸に対する撮像部の傾きを意味する。
In the present invention, the “focal plane” means a plane including the focal point of the optical image and a surface on the object side. In the present invention, “position of the imaging unit” means the position of the imaging unit in the optical axis direction of the imaging optical system, and “posture of the imaging unit” means the position of the imaging unit with respect to the optical axis of the imaging optical system. Means tilt.

　また、本発明において、「容器の底面」は、観察対象を収容する容器内の底面であり、観察対象の設置面を意味する。
In the present invention, the “bottom surface of the container” is the bottom surface in the container that accommodates the observation target, and means the installation surface of the observation target.

　また、本発明において、「容器」は、観察対象を収容することができればどのような形態を有するものであってもよい。例えば、シャーレ、ディッシュ、フラスコ又はウェルプレート等のように、底部及び底部に連続する壁部を有する形態を有するものを容器として用いることができる。また、板状の部材に微細な流路が形成されたマイクロ流路デバイス等を容器として用いることもできる。さらに、スライドガラスのように、板状の形態を有するものも容器として用いることができる。
In the present invention, the “container” may have any form as long as it can accommodate the observation target. For example, a container having a form having a bottom part and a wall part continuous to the bottom part, such as a petri dish, a dish, a flask, or a well plate, can be used as a container. Further, a micro-channel device or the like in which a fine channel is formed on a plate-like member can be used as a container. Furthermore, what has a plate-like form like a slide glass can also be used as a container.

　また、本発明の観察装置は、観察対象を透過した光の光路を複数の光路に分割する光路分割部を含み、

　複数の撮像部の各々は、光路分割部により分割された複数の光路の各々に配置されていてもよい。
Moreover, the observation apparatus of the present invention includes an optical path dividing unit that divides an optical path of light transmitted through the observation target into a plurality of optical paths,

Each of the plurality of imaging units may be disposed on each of the plurality of optical paths divided by the optical path dividing unit.

　また、本発明の観察装置は、少なくとも１つの撮像部が、結像光学系に対する結像光学系の光軸上の位置が変更可能であり、

　制御部は、取得部により取得された位置情報に基づいて、少なくとも１つの撮像部の光軸上の位置を変更する制御を行ってもよい。
In the observation apparatus of the present invention, at least one imaging unit can change the position on the optical axis of the imaging optical system with respect to the imaging optical system,

The control unit may perform control to change the position on the optical axis of at least one imaging unit based on the position information acquired by the acquisition unit.

　また、本発明の観察装置は、少なくとも１つの撮像部が、結像光学系に対する受光面の光軸上における傾きが変更可能であり、

　制御部は、取得部により取得された位置情報に基づく底面の傾きに合わせて、少なくとも１つの撮像部の受光面の光軸上における傾きを変更する制御を行ってもよい。
In the observation apparatus of the present invention, at least one imaging unit can change the inclination of the light receiving surface with respect to the imaging optical system on the optical axis,

The control unit may perform control to change the tilt on the optical axis of the light receiving surface of at least one imaging unit in accordance with the tilt of the bottom surface based on the position information acquired by the acquiring unit.

　また、本発明の観察装置は、少なくとも１つの撮像部が、結像光学系に対する結像光学系の光軸上の位置、及び結像光学系に対する受光面の光軸上における傾きが変更可能であり、

　制御部は、取得部により取得された位置情報に基づいて、少なくとも１つの撮像部の光軸上の位置を変更する制御、及び取得部により取得された位置情報に基づく底面の傾きに合わせて、少なくとも１つの撮像部の受光面の光軸上における傾きを変更する制御を行ってもよい。
In the observation apparatus of the present invention, at least one imaging unit can change the position on the optical axis of the imaging optical system with respect to the imaging optical system and the inclination of the light receiving surface with respect to the imaging optical system on the optical axis. Yes,

Based on the position information acquired by the acquisition unit, the control unit changes the position on the optical axis of at least one imaging unit, and in accordance with the inclination of the bottom surface based on the position information acquired by the acquisition unit, You may perform control which changes the inclination on the optical axis of the light-receiving surface of at least 1 imaging part.

　なお、本発明において、「合わせて」は、完全に一致するものだけではなく、許容される範囲内の誤差を含めた意味合いでの「合わせて」を意図する。
In the present invention, “together” is intended not only to completely match, but also to “together” in a meaning including an error within an allowable range.

　また、本発明の観察装置は、制御部が、取得部により取得された位置情報に基づく底面の高低差が予め定められた閾値よりも大きい場合に、少なくとも１つの撮像部の位置及び姿勢の少なくとも一方を変更する制御を行ってもよい。
In the observation apparatus of the present invention, when the control unit has a difference in height of the bottom surface based on the position information acquired by the acquisition unit, which is greater than a predetermined threshold, at least one of the position and orientation of at least one imaging unit. You may perform control which changes one side.

　また、本発明の観察装置は、結像光学系及び撮像部と、容器との少なくとも一方を結像光学系の光軸に交差する特定の交差面において相対的に移動させる駆動部とをさらに含み、　取得部は、結像光学系及び撮像部と、容器との相対的移動に応じて結像光学系の観察領域が移動する方向に沿って、かつ結像光学系よりも先行する位置に対応する容器の底面の位置を示す位置情報を取得してもよい。
In addition, the observation apparatus of the present invention further includes an imaging optical system and an imaging unit, and a drive unit that relatively moves at least one of the container on a specific intersection plane that intersects the optical axis of the imaging optical system. The acquisition unit corresponds to a position preceding the imaging optical system along the direction in which the observation region of the imaging optical system moves according to the relative movement of the imaging optical system and the imaging unit and the container. You may acquire the positional information which shows the position of the bottom face of the container to perform.

　なお、本発明において、「特定の交差面」は、光軸に交差する交差面を意味する。
In the present invention, the “specific intersection plane” means an intersection plane that intersects the optical axis.

　また、本発明の観察装置は、駆動部が、結像光学系及び撮像部と、容器との少なくとも一方を、交差面において主走査方向及び主走査方向に直交する副走査方向に移動させ、

　取得部は、先に取得した主走査方向における位置情報と、副走査方向に移動させた後の主走査方向における位置情報とに基づいて底面の形状情報を取得してもよい。
Further, in the observation apparatus of the present invention, the driving unit moves at least one of the imaging optical system, the imaging unit, and the container in the main scanning direction and the sub-scanning direction orthogonal to the main scanning direction at the intersection plane,

The acquisition unit may acquire the shape information of the bottom surface based on the previously acquired position information in the main scanning direction and the position information in the main scanning direction after being moved in the sub scanning direction.

　なお、本発明において、「容器の底面の位置」は、交差面に直交する方向における容器の底面の位置であり、「底面の形状情報」は、少なくとも容器の底面の高低差の情報か、又は容器の底面の傾きの情報のどちらか一方を含む情報である。
In the present invention, the “position of the bottom surface of the container” is the position of the bottom surface of the container in the direction perpendicular to the intersecting surface, and the “shape information of the bottom surface” is at least information on the height difference of the bottom surface of the container, or It is information including either one of the information on the inclination of the bottom surface of the container.

　また、本発明の観察装置は、駆動部が、結像光学系及び撮像部と、容器との少なくとも一方を、交差面において主走査方向及び主走査方向に直交する副走査方向に移動し、

　取得部が、主走査方向において結像光学系よりも先行する位置に対応する容器の底面を示す位置情報であって、かつ、副走査方向において異なる２つ以上の位置情報を取得してもよい。
In the observation apparatus of the present invention, the drive unit moves at least one of the imaging optical system, the imaging unit, and the container in the main scanning direction and the sub-scanning direction orthogonal to the main scanning direction at the intersecting plane,

The acquisition unit may acquire position information indicating the bottom surface of the container corresponding to a position preceding the imaging optical system in the main scanning direction and two or more pieces of position information that are different in the sub-scanning direction. .

　なお、本発明において「副走査方向において異なる２つ以上の位置情報を取得する」は、副走査方向に沿って直線上に並んだ２つ以上の位置情報に限られず、副走査方向における２つ以上の位置情報が取得できれば直線上に並んでいない２つ以上の位置情報であってもよい。
In the present invention, “acquiring two or more pieces of position information different in the sub-scanning direction” is not limited to two or more pieces of position information arranged in a straight line along the sub-scanning direction. If the above position information can be acquired, two or more pieces of position information that are not arranged on a straight line may be used.

　また、本発明の観察装置は、複数の撮像部によって取得した複数の画像をそれぞれ合成して１枚の画像を生成する画像処理部を備えてもよい。
The observation apparatus of the present invention may include an image processing unit that generates a single image by combining a plurality of images acquired by a plurality of imaging units.

　また、本発明の観察装置は、複数の撮像部によって取得した複数の画像から、適切な画像を選択する選択部を備えてもよい。
In addition, the observation apparatus of the present invention may include a selection unit that selects an appropriate image from a plurality of images acquired by a plurality of imaging units.

　本発明の観察装置の作動方法は、観察対象を収容する容器の底面の位置を示す位置情報を取得部で取得し、取得部により取得された位置情報に基づき、動作部を駆動することにより、容器に収容された観察対象を示す光学像を結像させる結像光学系に対して、複数の撮像部の中の少なくとも１つの撮像部の位置及び姿勢の少なくとも一方を変更し、複数の撮像部の各々に、焦点面が異なる光学像を結像させる、観察装置の作動方法を含む。
The operation method of the observation apparatus of the present invention is to acquire position information indicating the position of the bottom surface of the container that accommodates the observation target by the acquisition unit, and based on the position information acquired by the acquisition unit, by driving the operation unit, A plurality of imaging units that change at least one of the position and orientation of at least one imaging unit among a plurality of imaging units with respect to an imaging optical system that forms an optical image indicating an observation object accommodated in a container. Each of these includes a method of operating an observation apparatus that forms optical images with different focal planes.

　本発明の観察制御プログラムは、コンピュータを、

　上記観察装置に含まれる取得部及び制御部として機能させる。
The observation control program of the present invention includes a computer,

It functions as an acquisition unit and a control unit included in the observation apparatus.

　なお、本発明による観察装置の作動方法をコンピュータに実行させるプログラムとして提供してもよい。
In addition, you may provide the operating method of the observation apparatus by this invention as a program which makes a computer perform.

　本発明による他の観察装置は、コンピュータに実行させるための命令を記憶するメモリと、

　記憶された命令を実行するよう構成されたプロセッサとを備え、プロセッサは、

　観察対象を収容する容器の底面の位置を示す位置情報を取得し、

　取得された位置情報に基づいて、位置及び姿勢の少なくとも一方を変更可能な撮像部の位置及び姿勢の少なくとも一方を変更して、複数の撮像部の各々に、焦点面が異なる光学像を結像させる制御を行う処理を実行する。
Another observation apparatus according to the present invention includes a memory for storing instructions to be executed by a computer,

A processor configured to execute stored instructions, the processor comprising:

Obtain position information indicating the position of the bottom surface of the container containing the observation target,

Based on the acquired position information, at least one of the position and orientation of the imaging unit capable of changing at least one of the position and orientation is changed, and an optical image having a different focal plane is formed on each of the plurality of imaging units. The process which performs control to perform is performed.

　本発明によれば、観察対象を収容する容器の底面の位置を示す位置情報を取得する取得部と、容器に収容された観察対象を示す光学像を結像させる結像光学系に対する位置及び姿勢の少なくとも一方を変更可能な少なくとも１つの撮像部を含み、各々に焦点面が異なる光学像を結像可能な複数の撮像部と、取得部により取得された位置情報に基づいて、位置及び姿勢の少なくとも一方を変更可能な撮像部の位置及び姿勢の少なくとも一方を変更して、複数の撮像部の各々に、焦点面が異なる光学像を結像させる制御を行う制御部と、を含むので、容器の底面の形状に応じてデフォーカス量を変更することができる。これにより、観察面を被写界深度内に位置させることができるので、観察面が被写界深度の範囲から外れることによって、焦点が合わずに撮像画像がボケた画像になるのを防止することができる。また、容器の底面の高低差が比較的小さい場合には、デフォーカス量を少なくすることによって撮像された画像の画質を維持することができる。また、容器の底面の高低差が比較的大きい場合には、デフォーカス量を多くすることによって撮像された画像の画質の低下を抑制することができる。
According to the present invention, the acquisition unit that acquires position information indicating the position of the bottom surface of the container that accommodates the observation target, and the position and orientation with respect to the imaging optical system that forms an optical image that indicates the observation target accommodated in the container A plurality of imaging units each including at least one imaging unit capable of changing at least one of them and capable of forming optical images with different focal planes, and position and orientation based on position information acquired by the acquisition unit A control unit that controls at least one of the position and orientation of the imaging unit that can change at least one to form an optical image with a different focal plane on each of the plurality of imaging units. The defocus amount can be changed according to the shape of the bottom surface. As a result, since the observation surface can be positioned within the depth of field, it is possible to prevent the captured image from being out of focus and becoming a blurred image due to the observation surface being out of the range of the depth of field. be able to. In addition, when the difference in height of the bottom surface of the container is relatively small, the image quality of the captured image can be maintained by reducing the defocus amount. Further, when the height difference of the bottom surface of the container is relatively large, it is possible to suppress a decrease in the image quality of the captured image by increasing the defocus amount.

第１実施形態に係る顕微鏡装置の構成の一例を示す模式図である。It is a schematic diagram which shows an example of a structure of the microscope apparatus which concerns on 1st Embodiment. 第１実施形態に係る顕微鏡装置本体に含まれる撮像光学系の構成の一例を示す模式図である。It is a schematic diagram which shows an example of a structure of the imaging optical system contained in the microscope apparatus main body which concerns on 1st Embodiment. 第１実施形態に係る顕微鏡装置本体に含まれるステージの構成の一例を示す斜視図である。It is a perspective view which shows an example of the structure of the stage contained in the microscope apparatus main body which concerns on 1st Embodiment. 第１実施形態に係る顕微鏡装置の構成の一例を示すブロック図である。It is a block diagram which shows an example of a structure of the microscope apparatus which concerns on 1st Embodiment. 第１実施形態に係る顕微鏡装置におけるデフォーカス量の調整を説明する図である。It is a figure explaining adjustment of the defocus amount in the microscope apparatus concerning a 1st embodiment. 第１実施形態に係る顕微鏡装置の作用の一例を示すフローチャートである。It is a flowchart which shows an example of an effect | action of the microscope apparatus which concerns on 1st Embodiment. 第１実施形態に係る顕微鏡装置の培養容器内における観察領域の走査位置の一例を示す図である。It is a figure which shows an example of the scanning position of the observation area | region in the culture container of the microscope apparatus which concerns on 1st Embodiment. 第１実施形態に係る顕微鏡装置における変位センサの配置の一例を説明するための図である。It is a figure for demonstrating an example of arrangement | positioning of the displacement sensor in the microscope apparatus which concerns on 1st Embodiment. 第１実施形態に係る顕微鏡装置のデフォーカス量調整処理の一例を示すフローチャートである。It is a flowchart which shows an example of the defocusing amount adjustment process of the microscope apparatus which concerns on 1st Embodiment. 第１実施形態に係る顕微鏡装置における底面の高低差の取得の一例を説明するための図である。It is a figure for demonstrating an example of acquisition of the height difference of the bottom face in the microscope apparatus which concerns on 1st Embodiment. 第１実施形態に係る顕微鏡装置における変位センサの配置の他の一例を説明するための図である。It is a figure for demonstrating another example of arrangement | positioning of the displacement sensor in the microscope apparatus which concerns on 1st Embodiment. 第１実施形態に係る顕微鏡装置における変位センサの配置のさらに他の一例を説明するための図である。It is a figure for demonstrating another example of arrangement | positioning of the displacement sensor in the microscope apparatus which concerns on 1st Embodiment. 第１実施形態に係る顕微鏡装置における底面の高低差の取得に他の一例を説明するための図である。It is a figure for demonstrating another example to the acquisition of the height difference of the bottom face in the microscope apparatus which concerns on 1st Embodiment. 第２実施形態に係る顕微鏡装置の構成の一例を示すブロック図である。It is a block diagram which shows an example of a structure of the microscope apparatus which concerns on 2nd Embodiment. 第３実施形態に係る顕微鏡装置におけるデフォーカス量の調整を説明する図である。It is a figure explaining adjustment of the amount of defocuss in the microscope apparatus concerning a 3rd embodiment. 第１～第３実施形態に係る観察制御プログラムが記憶された記憶媒体から観察制御プログラムが顕微鏡制御装置にインストールされる態様の一例を示す概念図The conceptual diagram which shows an example of the aspect by which an observation control program is installed in the microscope control apparatus from the storage medium in which the observation control program which concerns on 1st-3rd embodiment was memorize | stored 従来の顕微鏡装置におけるデフォーカス量を説明する図である。It is a figure explaining the defocus amount in the conventional microscope apparatus. 従来の顕微鏡装置における問題点を説明するための図である。It is a figure for demonstrating the problem in the conventional microscope apparatus.

　以下、本発明の実施形態による観察装置、観察装置の作動方法、及び観察制御プログラムの一実施形態を適用した顕微鏡装置について、図面を参照しながら詳細に説明する。図１は、本実施形態の観察装置を適用した顕微鏡装置の構成の一例を示す模式図、図２は、第１実施形態に係る顕微鏡装置本体に含まれる撮像光学系の構成の一例を示す模式図である。
Hereinafter, an observation apparatus according to an embodiment of the present invention, a method for operating the observation apparatus, and a microscope apparatus to which an embodiment of an observation control program is applied will be described in detail with reference to the drawings. FIG. 1 is a schematic diagram illustrating an example of the configuration of a microscope apparatus to which the observation apparatus of the present embodiment is applied. FIG. 2 is a schematic diagram illustrating an example of the configuration of an imaging optical system included in the microscope apparatus main body according to the first embodiment. FIG.

　顕微鏡装置１は、顕微鏡装置本体１０と顕微鏡制御装置２０とを備える（図４参照）。

なお、顕微鏡装置１は本発明に係る観察装置の一例である。顕微鏡装置本体１０は、観察対象である培養された細胞を撮像して位相差画像を取得する。具体的には、顕微鏡装置本体１０は、一例として図２に示すように、白色光を出射する白色光源１１、コンデンサレンズ１２、スリット板１３、結像光学系１４、動作部１５、撮像部１６Ａ、撮像部１６Ｂ及び検出部１８を備える。
The microscope apparatus 1 includes a microscope apparatus body 10 and a microscope control apparatus 20 (see FIG. 4).

The microscope apparatus 1 is an example of an observation apparatus according to the present invention. The microscope apparatus body 10 captures cultured cells that are observation targets and acquires a phase difference image. Specifically, as shown in FIG. 2 as an example, the microscope apparatus main body 10 includes a white light source 11 that emits white light, a condenser lens 12, a slit plate 13, an imaging optical system 14, an operation unit 15, and an imaging unit 16A. The imaging unit 16B and the detection unit 18 are provided.

　動作部１５は、第１の動作部１５Ａ、第２の動作部１５Ｂ、第３の動作部１５Ｃ、第４の動作部１５Ｄ、第５の動作部１５Ｅ、及び第６の動作部１５Ｆを備える。第１～第６の動作部１５Ａ～１５Ｆの動作は後述する。
The operation unit 15 includes a first operation unit 15A, a second operation unit 15B, a third operation unit 15C, a fourth operation unit 15D, a fifth operation unit 15E, and a sixth operation unit 15F. The operations of the first to sixth operation units 15A to 15F will be described later.

　スリット板１３は、白色光源１１から出射された白色光を遮光する遮光板に対して白色光を透過するリング形状のスリットが設けられたものであり、白色光がスリットを通過することによってリング状の照明光Ｌが形成される。
The slit plate 13 is provided with a ring-shaped slit that transmits white light to the light-shielding plate that blocks white light emitted from the white light source 11, and the ring shape is obtained when white light passes through the slit. Illumination light L is formed.

　結像光学系１４は、培養容器５０を観察した位相差像を撮像部１６Ａ及び撮像部１６Ｂに結像する。図２は、結像光学系１４の詳細な構成を示す図である。図２に示すように、結像光学系１４は、位相差レンズ１４ａ及び結像レンズ１４ｄを備える。また、位相差レンズ１４ａは、対物レンズ１４ｂ及び位相板１４ｃを備える。位相板１４ｃは、照明光Ｌの波長に対して透明な基板に対して位相リングを形成したものである。なお、上述したスリット板１３のスリットの大きさは、位相板１４ｃの位相リングと共役な関係にある。
The imaging optical system 14 forms an image of the phase difference image obtained by observing the culture vessel 50 on the imaging unit 16A and the imaging unit 16B. FIG. 2 is a diagram showing a detailed configuration of the imaging optical system 14. As shown in FIG. 2, the imaging optical system 14 includes a phase difference lens 14a and an imaging lens 14d. The phase difference lens 14a includes an objective lens 14b and a phase plate 14c. The phase plate 14 c is formed by forming a phase ring on a substrate that is transparent to the wavelength of the illumination light L. The slit size of the slit plate 13 described above is in a conjugate relationship with the phase ring of the phase plate 14c.

　位相リングは、入射された光の位相を１／４波長ずらす位相膜と、入射された光を減光する減光フィルタとがリング状に形成されたものである。位相リングに入射された直接光は、位相リングを通過することによって位相が１／４波長ずれ、かつその明るさが弱められる。一方、観察対象によって回折された回折光は大部分が位相板１４ｃの透明板を通過し、その位相及び明るさは変化しない。
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 ¼ wavelength and its brightness is weakened. On the other hand, most of the diffracted light diffracted by the observation object passes through the transparent plate of the phase plate 14c, and its phase and brightness do not change.

　対物レンズ１４ｂを有する位相差レンズ１４ａは、図１に示す動作部１５に含まれる第５の動作部１５Ｅによって、対物レンズ１４ｂの光軸方向に移動される。なお、本実施形態においては、対物レンズ１４ｂの光軸方向とＺ方向（鉛直方向）とは同じ方向である。

対物レンズ１４ｂのＺ方向への移動によってオートフォーカス制御が行われ、撮像部１６Ａ及び撮像部１６Ｂによって取得される位相差画像のコントラストが調整される。
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 unit 15E included in the operation unit 15 shown in FIG. In the present embodiment, the optical axis direction of the objective lens 14b and the Z direction (vertical direction) are the same direction.

Autofocus control is performed by the movement of the objective lens 14b in the Z direction, and the contrast of the phase difference image acquired by the imaging unit 16A and the imaging unit 16B is adjusted.

　また、第５の動作部１５は、位相差レンズ１４ａをＺ方向に移動させるものとしたが、本発明はこれに限られるものではなく、対物レンズ１４ｂのみをＺ方向に移動させる構成としてもよい。
The fifth operation unit 15 moves the phase difference lens 14a in the Z direction. However, the present invention is not limited to this, and only the objective lens 14b may be moved in the Z direction. .

　また、位相差レンズ１４ａの倍率を変更可能な構成としてもよい。具体的には、異なる倍率を有する位相差レンズ１４ａ又は結像光学系１４を交換可能に構成するようにしてもよい。位相差レンズ１４ａ又は結像光学系１４の交換は、自動的に行うようにしてもよいし、ユーザが手動で行うようにしてもよい。
Moreover, it is good also as a structure which can change the magnification of the phase difference lens 14a. Specifically, the phase difference lens 14a or the imaging optical system 14 having different magnifications may be configured to be exchangeable. The replacement of the phase difference lens 14a or the imaging optical system 14 may be performed automatically or manually by a user.

　また、本実施形態の対物レンズ１４ｂは、一例として焦点距離を変更可能な液体レンズからなる。なお、焦点距離を変更可能であれば、液体レンズに限定されるものではなく、液晶レンズ及び形状変形レンズ等、任意のレンズを用いることができる。対物レンズ１４ｂは、図１に示す動作部１５に含まれる第６の動作部１５Ｆによって、印加される電圧が変更されて、焦点距離が変更される。これにより、結像光学系１４の焦点距離が変更される。対物レンズ１４ｂの焦点距離の変更によってもオートフォーカス制御が行われ、撮像部１６Ａ及び撮像部１６Ｂによって取得される位相差画像のコントラストが調整される。
Moreover, the objective lens 14b of this embodiment consists of a liquid lens which can change a focal distance as an example. Note that as long as the focal length can be changed, the lens is not limited to the liquid lens, and any lens such as a liquid crystal lens and a shape deforming lens can be used. In the objective lens 14b, the applied voltage is changed by the sixth operating unit 15F included in the operating unit 15 shown in FIG. As a result, the focal length of the imaging optical system 14 is changed. The autofocus control is also performed by changing the focal length of the objective lens 14b, and the contrast of the phase difference image acquired by the imaging unit 16A and the imaging unit 16B is adjusted.

　結像レンズ１４ｄは、位相差レンズ１４ａを通過した位相差画像を示す光が入射され、この光が撮像部１６の撮像面１６Ａに結像する。本実施形態において、結像レンズ１４ｄは、焦点距離を変更可能な液体レンズからなる。なお、焦点距離を変更可能であれば、液体レンズに限定されるものではなく、液晶レンズ及び形状変形レンズ等、任意のレンズを用いることができる。結像レンズ１４ｄは、図１に示す動作部１５に含まれる第１の動作部１５Ａによって、印加する電圧が変更されて、焦点距離が変更される。これにより、結像光学系１４の焦点距離が変更される。結像レンズ１４ｄの焦点距離の変更によってオートフォーカス制御が行われ、撮像部１６Ａ及び撮像部１６Ｂによって取得される位相差画像のコントラストが調整される。
The imaging lens 14 d receives light indicating a phase difference image that has passed through the phase difference lens 14 a, and this light forms an image on the imaging surface 16 </ b> A of the imaging unit 16. In the present embodiment, the imaging lens 14d is a liquid lens whose focal length can be changed. Note that as long as the focal length can be changed, the lens is not limited to the liquid lens, and any lens such as a liquid crystal lens and a shape deforming lens can be used. In the imaging lens 14d, the applied voltage is changed by the first operating unit 15A included in the operating unit 15 shown in FIG. As a result, the focal length of the imaging optical system 14 is changed. Autofocus control is performed by changing the focal length of the imaging lens 14d, and the contrast of the phase difference images acquired by the imaging unit 16A and the imaging unit 16B is adjusted.

　また、結像レンズ１４ｄは、図１に示す動作部１５に含まれる第２の動作部１５Ｂによって結像レンズ１４ｄの光軸方向に移動される。なお、本実施形態においては、結像レンズ１４ｄの光軸方向とＺ方向（鉛直方向）とは同じ方向である。結像レンズ１４ｄのＺ方向への移動によってオートフォーカス制御が行われ、撮像部１６Ａ及び撮像部１６Ｂによって取得される位相差画像のコントラストが調整される。
The imaging lens 14d is moved in the optical axis direction of the imaging lens 14d by the second operating unit 15B included in the operating unit 15 shown in FIG. In the present embodiment, the optical axis direction of the imaging lens 14d and the Z direction (vertical direction) are the same direction. Autofocus control is performed by the movement of the imaging lens 14d in the Z direction, and the contrast of the phase difference images acquired by the imaging unit 16A and the imaging unit 16B is adjusted.

　図１に示すように、顕微鏡装置本体１０は、結像光学系１４から出射された光の光路を複数の光路に分割する光路分割部１９を備える。本実施形態において、光路分割部１９はビームスプリッタで構成されており、結像光学系１４から出射された光を透過し、かつ結像光学系１４から出射された光を結像光学系１４の光軸とは異なる方向に反射する。本実施形態の光路分割部１９は、結像光学系１４から出射された光を、結像光学系１４の光軸に対して直角に偏向する。なお、本実施形態の光路分割部１９は、結像光学系１４から出射された光を、結像光学系１４の光軸に対して直角に偏向するものとしたが、本発明はこれに限られるものではなく、偏向した後の光を撮像部１６Ｂに導くことができれば特に直角に限定されない。
As shown in FIG. 1, the microscope apparatus body 10 includes an optical path dividing unit 19 that divides the optical path of the light emitted from the imaging optical system 14 into a plurality of optical paths. In the present embodiment, the optical path splitting unit 19 is configured by a beam splitter, transmits light emitted from the imaging optical system 14, and transmits light emitted from the imaging optical system 14 to the imaging optical system 14. Reflects in a direction different from the optical axis. The optical path splitting unit 19 of this embodiment deflects the light emitted from the imaging optical system 14 at a right angle to the optical axis of the imaging optical system 14. The optical path splitting unit 19 of the present embodiment deflects the light emitted from the imaging optical system 14 at right angles to the optical axis of the imaging optical system 14, but the present invention is not limited to this. It is not limited to a right angle as long as the deflected light can be guided to the imaging unit 16B.

　また、本実施形態の光路分割部１９はビームスプリッタで構成されているが、本発明はこれに限られるものではなく、入射した光の一部を反射し、一部を透過するものであれば、ミラー型やプリズム型等いずれのビームスプリッタを使用してもよい。ただし、撮像部１６Ａと撮像部１６Ｂとの画質を同等にする観点から、入射光を５０％と５０％に分離するビームスプリッタを使用することがより好ましい。なお、本実施形態の顕微鏡装置１は、撮像部１６Ａ及び撮像部１６Ｂの２つの撮像部を備えた構成であるが、撮像部を３つ備えた顕微鏡装置においては、例えば、先ずビームスプリッタによって入射光を３３％と６６％に分離し、６６％で分離された入射光を次のビームスプリッタによって５０％と５０％に分離する構成にしてもよい。
The optical path splitting unit 19 of the present embodiment is configured by a beam splitter. However, the present invention is not limited to this, as long as it reflects a part of incident light and transmits a part thereof. Any beam splitter such as a mirror type or a prism type may be used. However, it is more preferable to use a beam splitter that separates incident light into 50% and 50% from the viewpoint of equalizing the image quality of the imaging unit 16A and the imaging unit 16B. Note that the microscope apparatus 1 of the present embodiment has a configuration including two imaging units, that is, the imaging unit 16A and the imaging unit 16B. However, in a microscope apparatus including three imaging units, for example, the light is first incident by a beam splitter. The light may be separated into 33% and 66%, and the incident light separated at 66% may be separated into 50% and 50% by the next beam splitter.

　撮像部１６Ａは、結像レンズ１４ｄによって結像された観察対象の像を表す光であって、光路分割部１９で反射された光を受光して、観察対象を表す位相差画像を観察画像として出力する。
The imaging unit 16A receives the light reflected by the optical path splitting unit 19 that is an image of the observation target imaged by the imaging lens 14d, and uses a phase difference image representing the observation target as an observation image. Output.

　撮像部１６Ｂは、結像レンズ１４ｄによって結像された観察対象の像を表す光であって、光路分割部１９を透過した光を受光して、観察対象を表す位相差画像を観察画像として出力する。
The imaging unit 16B receives light that is an image of the observation target imaged by the imaging lens 14d and has passed through the optical path dividing unit 19, and outputs a phase difference image that represents the observation target as an observation image. To do.

　撮像部１６Ａ及び撮像部１６Ｂは、それぞれＣＣＤ（Charge-Coupled Device）イメージセンサまたはＣＭＯＳ（Complementary Metal-Oxide Semiconductor）イメージセンサ等の撮像素子を備える。撮像素子は、ＲＧＢ（Red Green Blue）のカラーフィルタが設けられた撮像素子を用いてもよいし、モノクロの撮像素子を用いるようにしてもよい。
The imaging unit 16A and the imaging unit 16B each include an imaging element such as a charge-coupled device (CCD) image sensor or a complementary metal-oxide semiconductor (CMOS) image sensor. As the imaging device, an imaging device provided with a color filter of RGB (Red Green Blue) may be used, or a monochrome imaging device may be used.

　また、撮像部１６Ｂは、結像光学系１４の光軸上の位置が変更可能である。具体的には、撮像部１６Ｂは、図１に示す動作部１５に含まれる第３の動作部１５ＣによってＺ方向に移動される。なお、本実施形態においては、結像光学系１４の光軸とＺ方向とは同じ方向である。撮像部１６ＢのＺ方向への移動によって撮像部１６Ａと撮像部１６Ｂとの間のデフォーカス量を変更する。なおデフォーカス量の変更方法については後で詳細に説明する。
Further, the imaging unit 16B can change the position of the imaging optical system 14 on the optical axis. Specifically, the imaging unit 16B is moved in the Z direction by the third operating unit 15C included in the operating unit 15 illustrated in FIG. In the present embodiment, the optical axis of the imaging optical system 14 and the Z direction are the same direction. The defocus amount between the imaging unit 16A and the imaging unit 16B is changed by the movement of the imaging unit 16B in the Z direction. A method for changing the defocus amount will be described later in detail.

　検出部１８は、ステージ５１に設置された培養容器５０の底面のＺ方向（鉛直方向）の位置を検出する。検出部１８は、具体的には、第１の変位センサ１８ａ及び第２の変位センサ１８ｂを含む。第１の変位センサ１８ａ及び第２の変位センサ１８ｂは、位相差レンズ１４ａを挟んで、図１に示すＸ方向に並べて設けられている。本実施形態における第１の変位センサ１８ａ及び第２の変位センサ１８ｂはレーザ変位計であり、培養容器５０にレーザ光を照射し、その反射光を検出することによって、培養容器５０の底面のＺ方向の位置を検出する。なお、培養容器５０の底面とは、培養容器５０の底部と観察対象である細胞との境界面、すなわち観察対象の設置面を意味する。つまり培養容器５０内の底面である。ここで、培養容器５０の底部とは、培養容器５０の底を形成する底壁を意味する。
The detection unit 18 detects the position in the Z direction (vertical direction) of the bottom surface of the culture vessel 50 installed on the stage 51. Specifically, the detection unit 18 includes a first displacement sensor 18a and a second displacement sensor 18b. The first displacement sensor 18a and the second displacement sensor 18b are provided side by side in the X direction shown in FIG. 1 with the phase difference lens 14a interposed therebetween. The first displacement sensor 18a and the second displacement sensor 18b in this embodiment are laser displacement meters, which irradiate the culture vessel 50 with laser light and detect the reflected light, thereby detecting Z on the bottom surface of the culture vessel 50. Detect the position of the direction. Note that the bottom surface of the culture vessel 50 means the boundary surface between the bottom of the culture vessel 50 and the cell that is the observation target, that is, the installation surface of the observation target. That is, the bottom surface in the culture vessel 50. Here, the bottom of the culture vessel 50 means a bottom wall that forms the bottom of the culture vessel 50.

　本実施形態において培養容器５０の底面のＺ方向の位置は、一例として、検出部１８を基準面とし、検出部１８が検出した反射光の信号の値を培養容器５０の底面のＺ方向の位置を表す値とする。
In this embodiment, the position of the bottom surface of the culture vessel 50 in the Z direction is, for example, the detection unit 18 as a reference plane, and the value of the reflected light signal detected by the detection unit 18 is the position of the bottom surface of the culture vessel 50 in the Z direction. Is a value representing.

　なお、本実施形態において培養容器５０の底面のＺ方向の位置の値は、検出部１８が検出した反射光の信号の値としたが、これに限定されるものではなく、上記反射光の信号の値を距離に換算した値すなわち検出部１８から培養容器５０の底面までの距離を培養容器５０の底面のＺ方向の位置としてもよい。また検出部１８が培養容器５０の底部の下面で反射した反射光をさらに検出することによって、培養容器５０の底面で反射した反射光の信号の値から培養容器５０の底部の下面で反射した反射光の値を減算して培養容器５０の底部の厚さを表す値を算出し、この値を培養容器５０の底面のＺ方向の位置を表す値としてもよい。またこのＺ方向の位置を表す値を実際の距離の値に換算して使用してもよい。
In the present embodiment, the value of the position in the Z direction on the bottom surface of the culture vessel 50 is the value of the reflected light signal detected by the detection unit 18, but is not limited thereto, and the reflected light signal is not limited thereto. A value obtained by converting the above value into a distance, that is, the distance from the detection unit 18 to the bottom surface of the culture vessel 50 may be set as the position of the bottom surface of the culture vessel 50 in the Z direction. Further, the detection unit 18 further detects the reflected light reflected from the bottom surface of the bottom of the culture vessel 50, whereby the reflected light reflected from the bottom surface of the bottom of the culture vessel 50 from the value of the reflected light signal reflected from the bottom surface of the culture vessel 50. A value representing the thickness of the bottom of the culture vessel 50 may be calculated by subtracting the light value, and this value may be used as a value representing the position of the bottom surface of the culture vessel 50 in the Z direction. Further, a value representing the position in the Z direction may be converted into an actual distance value and used.

　なお、レーザ変位センサは、正反射光学系計測器を使用することができる。また検出部１８は、レーザ変位センサに限られず、例えば共焦点式センサを使用することもできる。
The laser displacement sensor can use a specular reflection optical system measuring instrument. Moreover, the detection part 18 is not restricted to a laser displacement sensor, For example, a confocal type sensor can also be used.

　検出部１８によって検出された培養容器５０の底面のＺ方向の位置情報は、後述する取得部２３Ａに出力される。取得部２３Ａは、入力された位置情報を後述する焦点調節部２４に出力する。焦点調節部２４は、入力された位置情報に基づいて結像光学系１４の焦点位置を調節してフォーカス制御量を取得し、後述する制御部２２が、焦点調節部２４によって取得されたフォーカス制御量に基づいて動作部１５を制御し、オートフォーカス制御を行う。なお、第１の変位センサ１８ａ及び第２の変位センサ１８ｂによる培養容器５０のＺ方向の位置の検出及び焦点調節部２４による焦点位置の調節については、後で詳述する。
The position information in the Z direction of the bottom surface of the culture vessel 50 detected by the detection unit 18 is output to the acquisition unit 23A described later. The acquiring unit 23A outputs the input position information to the focus adjusting unit 24 described later. The focus adjustment unit 24 adjusts the focus position of the imaging optical system 14 based on the input position information to acquire a focus control amount, and the control unit 22 described later performs focus control acquired by the focus adjustment unit 24. Based on the amount, the operation unit 15 is controlled to perform autofocus control. The detection of the position in the Z direction of the culture vessel 50 by the first displacement sensor 18a and the second displacement sensor 18b and the adjustment of the focus position by the focus adjustment unit 24 will be described in detail later.

　スリット板１３と位相差レンズ１４ａ及び検出部１８との間には、ステージ５１が設けられている。ステージ５１上には、観察対象である細胞が収容された培養容器５０が設置される。なお本実施形態の培養容器５０は本発明に係る観察装置において使用される容器の一例である。容器としては、培養容器５０に限られず、観察対象を収容することができればどのような形態を有するものであってもよい。例えば、シャーレ、ディッシュ、フラスコ又はウェルプレート等のように、底部及び底部に連続する壁部を有する形態を有するものを容器として用いることができる。
A stage 51 is provided between the slit plate 13, the phase difference lens 14 a and the detection unit 18. On the stage 51, a culture vessel 50 in which cells to be observed are accommodated is installed. In addition, the culture container 50 of this embodiment is an example of the container used in the observation apparatus according to the present invention. The container is not limited to the culture container 50 and may have any form as long as it can accommodate an observation target. For example, a container having a form having a bottom part and a wall part continuous to the bottom part, such as a petri dish, a dish, a flask, or a well plate, can be used as a container.

　また、板状の部材に微細な流路が形成されたマイクロ流路デバイス等を容器として用いることもできる。さらに、スライドガラスのように、板状の形態を有するものも容器として用いることができる。また、観察対象は、培養されたものに限られるものではなく、例えば、血液、各種の粒子、又は繊維等のいずれであってもよい。また、培養容器５０に収容される細胞としては、ｉＰＳ細胞及びＥＳ細胞といった多能性幹細胞、幹細胞から分化誘導された神経、皮膚、心筋及び肝臓の細胞、並びに人体から取り出された皮膚、網膜、心筋、血球、神経及び臓器の細胞等がある。
Further, a micro-channel device or the like in which a fine channel is formed on a plate-like member can be used as a container. Furthermore, what has a plate-like form like a slide glass can also be used as a container. Further, the observation target is not limited to the cultured one, and may be any of blood, various particles, fibers, and the like. The cells contained in the culture vessel 50 include pluripotent stem cells such as iPS cells and ES cells, nerves, skin, myocardium and liver cells induced to differentiate from the stem cells, and skin, retina extracted from the human body, There are myocardium, blood cells, nerve and organ cells.

　ステージ５１は、後述する水平方向駆動部１７（図４参照）によって互いに直交するＸ方向及びＹ方向に移動する。Ｘ方向及びＹ方向は、結像光学系１４の光軸に交差する特定の交差面内における方向であり、特定の交差面内において互いに直交する方向である。なお、本実施形態において、特定の交差面は、一例として水平面とする。よって、Ｘ方向及びＹ方向は、Ｚ方向に直交する方向であり、水平面内において互いに直交する方向である。本実施形態においては、Ｘ方向を主走査方向とし、Ｙ方向を副走査方向とする。
The stage 51 is moved in the X and Y directions orthogonal to each other by a horizontal driving unit 17 (see FIG. 4) described later. The X direction and the Y direction are directions in a specific intersection plane that intersects the optical axis of the imaging optical system 14, and are directions orthogonal to each other in the specific intersection plane. In the present embodiment, the specific intersection plane is a horizontal plane as an example. Therefore, the X direction and the Y direction are directions orthogonal to the Z direction, and are directions orthogonal to each other in the horizontal plane. In the present embodiment, the X direction is the main scanning direction, and the Y direction is the sub-scanning direction.

　一例として図３に示すように、ステージ５１は、中央に矩形の開口５１ａが形成されている。この開口５１ａを形成する部材の上に培養容器５０が設置され、培養容器５０内の細胞の位相差画像が開口５１ａを通過するように構成されている。
As an example, as shown in FIG. 3, the stage 51 has a rectangular opening 51 a at the center. The culture vessel 50 is installed on the member forming the opening 51a, and the phase difference image of the cells in the culture vessel 50 is configured to pass through the opening 51a.

　また、ステージ５１は、第４の動作部１５ＤによってＺ方向に移動され、これにより、培養容器５０がＺ方向に移動される。本実施形態においては、ステージ５１における培養容器５０が設置される面に垂直な方向とＺ方向とは同じ方向である。ステージ５１のＺ方向への移動によってもオートフォーカス制御が行われ、撮像部１６Ａ及び撮像部１６Ｂによって取得される位相差画像のコントラストが調整される。
Further, the stage 51 is moved in the Z direction by the fourth operating unit 15D, whereby the culture vessel 50 is moved in the Z direction. In the present embodiment, the direction perpendicular to the surface of the stage 51 where the culture vessel 50 is installed and the Z direction are the same direction. The autofocus control is also performed by the movement of the stage 51 in the Z direction, and the contrast of the phase difference image acquired by the imaging unit 16A and the imaging unit 16B is adjusted.

　第１の動作部１５Ａ及び第６の動作部１５Ｆは、例えば電圧可変回路を含む。第１の動作部１５Ａは、後述する制御部２２から出力された制御信号に基づいて、結像レンズ１４ｄに印加する電圧を変更する。第６の動作部１５Ｆは、後述する制御部２２から出力された制御信号に基づいて、対物レンズ１４ｂに印加する電圧を変更する。
The first operating unit 15A and the sixth operating unit 15F include, for example, a voltage variable circuit. The first operation unit 15A changes the voltage applied to the imaging lens 14d based on a control signal output from the control unit 22 described later. The sixth operation unit 15F changes the voltage applied to the objective lens 14b based on a control signal output from the control unit 22 described later.

　第２の動作部１５Ｂ、第３の動作部１５Ｃ、第４の動作部１５Ｄ及び第５の動作部１５Ｅは、一例として圧電素子及び高電圧を印加させる駆動源を含み、後述する制御部２２から出力された制御信号に基づいて駆動する。なお、動作部１５は、位相差レンズ１４ａ及び結像レンズ１４ｄを通過した位相差画像をそのまま通過させる構成となっている。また、第２の動作部１５Ｂ、第３の動作部１５Ｃ、第４の動作部１５Ｄ及び第５の動作部１５Ｅの構成は圧電素子に限定されず、結像レンズ１４ｄ、撮像部１６Ｂ、ステージ５１及び対物レンズ１４ｂ（位相差レンズ１４ａ）をＺ方向に移動可能なものであればよく、例えば各種モータやソレノイド等を含むものであってもよいし、その他の公知な構成を用いることができる。また、第２の動作部１５Ｂ、第３の動作部１５Ｃ、第４の動作部１５Ｄ及び第５の動作部１５Ｅを構成する圧電素子（ピエゾ素子ともいう）は、１つに限られず複数個であってもよい。
The second operation unit 15B, the third operation unit 15C, the fourth operation unit 15D, and the fifth operation unit 15E include, as an example, a piezoelectric element and a drive source that applies a high voltage. Drive based on the output control signal. The operation unit 15 is configured to pass the phase difference image that has passed through the phase difference lens 14a and the imaging lens 14d as it is. Further, the configuration of the second operating unit 15B, the third operating unit 15C, the fourth operating unit 15D, and the fifth operating unit 15E is not limited to the piezoelectric element, and the imaging lens 14d, the imaging unit 16B, and the stage 51. In addition, the objective lens 14b (phase difference lens 14a) may be moved as long as it can move in the Z direction. For example, it may include various motors, solenoids, and the like, and other known configurations may be used. In addition, the number of piezoelectric elements (also referred to as piezo elements) constituting the second operating unit 15B, the third operating unit 15C, the fourth operating unit 15D, and the fifth operating unit 15E is not limited to one, but is a plurality. There may be.

　次に、顕微鏡装置本体１０を制御する顕微鏡制御装置２０の構成について説明する。一例として図４は、本実施形態の顕微鏡装置１の構成を示すブロック図である。なお、顕微鏡装置本体１０については、顕微鏡制御装置２０の各部により制御される一部の構成のブロック図を示している。
Next, the configuration of the microscope control apparatus 20 that controls the microscope apparatus body 10 will be described. As an example, FIG. 4 is a block diagram showing a configuration of the microscope apparatus 1 of the present embodiment. In addition, about the microscope apparatus main body 10, the block diagram of the one part structure controlled by each part of the microscope control apparatus 20 is shown.

　顕微鏡制御装置２０は、ＣＰＵ（Central Processing Unit）２１、一次記憶部２５Ａ、二次記憶部２５Ｂ及び外部Ｉ／Ｆ（Interface）２８Ａ等を備えている。ＣＰＵ２１は、制御部２２、取得部２３Ａ、処理部２３Ｂ及び焦点調節部２４を備え、顕微鏡装置１の全体を制御する。一次記憶部２５Ａは、各種プログラムの実行時のワークエリア等として用いられる揮発性のメモリである。一次記憶部２５Ａの一例としては、ＲＡＭ（Random Access Memory）が挙げられる。二次記憶部２５Ｂは、各種プログラム及び各種パラメータ等を予め記憶した不揮発性のメモリであり、本開示の技術に係る観察制御プログラム２６の一例がインストールされている。ＣＰＵ２１は、二次記憶部２５Ｂから観察制御プログラム２６を読み出し、読み出した観察制御プログラム２６を一次記憶部２５Ａに展開する。ＣＰＵ２１は、一次記憶部２５Ａに展開した観察制御プログラム２６を実行することで制御部２２、取得部２３Ａ、処理部２３Ｂ、及び焦点調節部２４として動作する。
The microscope control device 20 includes a CPU (Central Processing Unit) 21, a primary storage unit 25A, a secondary storage unit 25B, an external I / F (Interface) 28A, and the like. The CPU 21 includes a control unit 22, an acquisition unit 23 </ b> A, a processing unit 23 </ b> B, and a focus adjustment unit 24, and controls the entire microscope apparatus 1. The primary storage unit 25A is a volatile memory used as a work area or the like when executing various programs. An example of the primary storage unit 25A is a RAM (Random Access Memory). The secondary storage unit 25B is a nonvolatile memory in which various programs, various parameters, and the like are stored in advance, and an example of the observation control program 26 according to the technique of the present disclosure is installed. The CPU 21 reads the observation control program 26 from the secondary storage unit 25B and develops the read observation control program 26 in the primary storage unit 25A. The CPU 21 operates as the control unit 22, the acquisition unit 23A, the processing unit 23B, and the focus adjustment unit 24 by executing the observation control program 26 developed in the primary storage unit 25A.

　また、二次記憶部２５Ｂは、後述する位置情報２７を記憶している。二次記憶部２５Ｂの一例としては、ＥＥＰＲＯＭ（Electrically Erasable Programmable Read-Only Memory）又はフラッシュメモリ等が挙げられる。外部Ｉ／Ｆ２８Ａは顕微鏡装置本体１０と顕微鏡制御装置２０との間の各種情報の送受信を司る。ＣＰＵ２１、一次記憶部２５Ａ、及び二次記憶部２５Ｂは、バスライン２８に接続されている。また、外部Ｉ／Ｆ２８Ａも、バスライン２８に接続されている。
Further, the secondary storage unit 25B stores position information 27 described later. An example of the secondary storage unit 25B is an EEPROM (Electrically Erasable Programmable Read-Only Memory) or a flash memory. The external I / F 28 </ b> A controls transmission / reception of various information between the microscope apparatus main body 10 and the microscope control apparatus 20. The CPU 21, the primary storage unit 25 </ b> A, and the secondary storage unit 25 </ b> B are connected to the bus line 28. The external I / F 28A is also connected to the bus line 28.

　観察制御プログラム２６は、ＤＶＤ（Digital Versatile Disc）及びＣＤ－ＲＯＭ（Compact Disc Read Only Memory）などの記録媒体に記録されて配布され、その記録媒体からコンピュータにインストールされる。又は、観察制御プログラム２６は、ネットワークに接続されたサーバコンピュータの記憶装置もしくはネットワークストレージに対して、外部からアクセス可能な状態で記憶され、外部からの要求に応じてコンピュータにダウンロードされた後に、インストールされるようにしてもよい。
The observation control program 26 is recorded and distributed on a recording medium such as a DVD (Digital Versatile Disc) and a CD-ROM (Compact Disc Read Only Memory), and is installed in the computer from the recording medium. Alternatively, the observation control program 26 is stored in a storage device or network storage of a server computer connected to the network in a state where it can be accessed from the outside, and is installed after being downloaded to the computer in response to an external request. You may be made to do.

　位置情報２７は、取得部２３Ａによって取得された培養容器５０の底面のＺ方向の位置情報である。
The position information 27 is position information in the Z direction of the bottom surface of the culture vessel 50 acquired by the acquisition unit 23A.

　また、上記では、汎用コンピュータが顕微鏡制御装置２０として機能する場合について説明したが、専用コンピュータによって実施されてもよい。専用コンピュータは、内蔵されたＲＯＭ（Read-Only Memory）及びフラッシュメモリなど、不揮発メモリに記録されたプログラムを実行するファームウェアであってもよい。さらに、この顕微鏡制御装置２０の少なくとも一部の機能を実行するためのプログラムを永久的に記憶するＡＳＩＣ（Application Specific Integrated Circuit：特定用途向け集積回路）やＦＰＧＡ(Field-Programmable Gate Array)などの専用回路を設けるようにしてもよい。あるいは、専用回路に記憶されたプログラム命令と、専用回路のプログラムを利用するようにプログラムされた汎用のＣＰＵによって実行されるプログラム命令と組み合わせるようにしてもよい。以上のように、コンピュータのハードウェア構成をどのように組み合わせてプログラム命令を実行してもよい。
In the above description, the case where the general-purpose computer functions as the microscope control device 20 has been described. However, the general-purpose computer may be implemented by a dedicated computer. The dedicated computer may be firmware that executes a program recorded in a nonvolatile memory such as a built-in ROM (Read-Only Memory) and a flash memory. Furthermore, a dedicated ASIC (Application Specific Integrated Circuit) or FPGA (Field-Programmable Gate Array) that permanently stores a program for executing at least a part of the functions of the microscope control device 20 A circuit may be provided. Alternatively, the program instructions stored in the dedicated circuit may be combined with the program instructions executed by a general-purpose CPU programmed to use the program of the dedicated circuit. As described above, program instructions may be executed by any combination of hardware configurations of computers.

　取得部２３Ａは、培養容器５０の底面の位置を示す位置情報を取得する。具体的には、検出部１８によって検出された培養容器５０の底面のＺ方向の位置情報を取得する。
The acquisition unit 23A acquires position information indicating the position of the bottom surface of the culture vessel 50. Specifically, position information in the Z direction of the bottom surface of the culture vessel 50 detected by the detection unit 18 is acquired.

　処理部２３Ｂは、撮像部１６Ａ及び／又は撮像部１６Ｂによって取得された画像信号に対して、ガンマ補正、輝度・色差変換、及び圧縮処理等の各種処理を行う。また、処理部２３Ｂは、各種処理を行って得た画像信号を特定のフレームレートで１フレーム毎に後述する制御部２２に出力する。また、処理部２３Ｂは、顕微鏡装置本体１０によって撮影された各観察領域の位相差画像、すなわち撮像部１６Ａ及び／又は撮像部１６Ｂによって取得された撮影画像を結合することによって、１枚の合成位相差画像を生成する。なお、本実施形態の処理部２３Ｂは、本発明の画像処理部の一例である。
The processing unit 23B performs various processes such as gamma correction, luminance / color difference conversion, and compression processing on the image signal acquired by the imaging unit 16A and / or the imaging unit 16B. Further, the processing unit 23B outputs an image signal obtained by performing various processes to the control unit 22 described later for each frame at a specific frame rate. Further, the processing unit 23B combines a phase difference image of each observation region imaged by the microscope apparatus body 10, that is, a combined image obtained by the imaging unit 16A and / or the imaging unit 16B, thereby combining one composite position. A phase difference image is generated. Note that the processing unit 23B of this embodiment is an example of an image processing unit of the present invention.

　焦点調節部２４は、取得部２３Ａによって取得された培養容器５０の底面のＺ方向の位置情報に基づいて、結像光学系１４の焦点位置を調節する。焦点調節部２４は、底面の位置情報に基づいて、第１の動作部１５Ａ、第２の動作部１５Ｂ、第４の動作部１５Ｄ、第５の動作部１５Ｅ、及び第６の動作部１５Ｆのそれぞれに対して移動量すなわちフォーカス制御量を取得し、制御部２２に各々のフォーカス制御量を出力する。具体的には、培養容器５０の底面のＺ方向の位置情報と、結像レンズ１４ｄの焦点距離を変更するための結像レンズ１４ｄへの印加電圧、結像レンズ１４ｄの光軸方向の移動量、ステージ５１の光軸方向の移動量、対物レンズ１４ｂの光軸方向の移動量、及び対物レンズ１４ｂの焦点距離を変更するための対物レンズ１４ｂへの印加電圧との関係を示す一例であるテーブルを、予め二次記憶部２５Ｂに記憶しておく。
The focus adjustment unit 24 adjusts the focus position of the imaging optical system 14 based on the position information in the Z direction of the bottom surface of the culture vessel 50 acquired by the acquisition unit 23A. Based on the position information on the bottom surface, the focus adjustment unit 24 has the first operation unit 15A, the second operation unit 15B, the fourth operation unit 15D, the fifth operation unit 15E, and the sixth operation unit 15F. The movement amount, that is, the focus control amount is acquired for each of them, and each focus control amount is output to the control unit 22. Specifically, the position information in the Z direction of the bottom surface of the culture vessel 50, the voltage applied to the imaging lens 14d for changing the focal length of the imaging lens 14d, and the amount of movement of the imaging lens 14d in the optical axis direction The table is an example showing the relationship between the amount of movement of the stage 51 in the optical axis direction, the amount of movement of the objective lens 14b in the optical axis direction, and the voltage applied to the objective lens 14b for changing the focal length of the objective lens 14b. Is previously stored in the secondary storage unit 25B.

　焦点調節部２４は、取得部２３Ａによって取得された培養容器５０のＺ方向の位置情報に基づいて、上記テーブルを参照して、結像レンズ１４ｄへの印加電圧、結像レンズ１４ｄの光軸方向の移動量、ステージ５１の光軸方向の移動量、対物レンズ１４ｂの光軸方向の移動量、及び対物レンズ１４ｂへの印加電圧をそれぞれ取得する。なお、以降の説明においては、結像レンズ１４ｄへの印加電圧、結像レンズ１４ｄの光軸方向の移動量、ステージ５１の光軸方向の移動量、対物レンズ１４ｂの光軸方向の移動量、及び対物レンズ１４ｂへの印加電圧をフォーカス制御量と称する。
The focus adjustment unit 24 refers to the above table based on the position information in the Z direction of the culture vessel 50 acquired by the acquisition unit 23A, and applies the voltage applied to the imaging lens 14d and the optical axis direction of the imaging lens 14d. , The amount of movement of the stage 51 in the optical axis direction, the amount of movement of the objective lens 14b in the optical axis direction, and the applied voltage to the objective lens 14b. In the following description, the voltage applied to the imaging lens 14d, the movement amount of the imaging lens 14d in the optical axis direction, the movement amount of the stage 51 in the optical axis direction, the movement amount of the objective lens 14b in the optical axis direction, The voltage applied to the objective lens 14b is referred to as a focus control amount.

　なお、培養容器５０の底面のＺ方向の位置情報と、結像レンズ１４ｄへの印加電圧、結像レンズ１４ｄの光軸方向の移動量、ステージ５１の光軸方向の移動量、対物レンズ１４ｂの光軸方向の移動量、及び対物レンズ１４ｂへの印加電圧の関係を示すものは、テーブルに限定されるものではなく、例えば式であってもよい。上記関係を示すものは、位置情報から結像レンズ１４ｄへの印加電圧、結像レンズ１４ｄの光軸方向の移動量、ステージ５１の光軸方向の移動量、対物レンズ１４ｂの光軸方向の移動量、及び対物レンズ１４ｂへの印加電圧を導出できれば何れの方法を使用してもよい。
Note that the position information in the Z direction of the bottom surface of the culture vessel 50, the voltage applied to the imaging lens 14d, the amount of movement of the imaging lens 14d in the optical axis direction, the amount of movement of the stage 51 in the optical axis direction, and the objective lens 14b What shows the relationship between the movement amount in the optical axis direction and the voltage applied to the objective lens 14b is not limited to the table, and may be an equation, for example. What shows the above relationship is the voltage applied from the position information to the imaging lens 14d, the amount of movement of the imaging lens 14d in the optical axis direction, the amount of movement of the stage 51 in the optical axis direction, and the movement of the objective lens 14b in the optical axis direction. Any method may be used as long as the amount and the voltage applied to the objective lens 14b can be derived.

　制御部２２は、焦点調節部２４が取得した第１の動作部１５Ａ、第２の動作部１５Ｂ、第４の動作部１５Ｄ、第５の動作部１５Ｅ、及び第６の動作部１５Ｆのそれぞれフォーカス制御量に基づく制御信号を、第１の動作部１５Ａ、第２の動作部１５Ｂ、第４の動作部１５Ｄ、第５の動作部１５Ｅ、及び第６の動作部１５Ｆのそれぞれに対して出力する。これにより、第１の動作部１５Ａにより結像レンズ１４ｄの焦点距離が変更されて結像光学系１４の焦点距離が変更される。また、第２の動作部１５Ｂにより結像レンズ１４ｄが光軸方向に移動する。また、第４の動作部１５Ｄによりステージ５１が光軸方向に移動する。また、第５の動作部１５Ｅにより対物レンズ１４ｂが光軸方向に移動する。第６の動作部１５Ｆにより対物レンズ１４ｂの焦点距離が変更されて結像光学系１４の焦点距離が変更される。これらの５つの動作により、オートフォーカス制御が行われる。
The control unit 22 focuses each of the first operation unit 15A, the second operation unit 15B, the fourth operation unit 15D, the fifth operation unit 15E, and the sixth operation unit 15F acquired by the focus adjustment unit 24. A control signal based on the control amount is output to each of the first operation unit 15A, the second operation unit 15B, the fourth operation unit 15D, the fifth operation unit 15E, and the sixth operation unit 15F. . Thereby, the focal length of the imaging lens 14d is changed by the first operating unit 15A, and the focal length of the imaging optical system 14 is changed. In addition, the imaging lens 14d is moved in the optical axis direction by the second operation unit 15B. In addition, the stage 51 is moved in the optical axis direction by the fourth operation unit 15D. Further, the objective lens 14b is moved in the optical axis direction by the fifth operating unit 15E. The focal length of the objective lens 14b is changed by the sixth operation unit 15F, and the focal length of the imaging optical system 14 is changed. The autofocus control is performed by these five operations.

　また、制御部２２は、水平方向駆動部１７を駆動制御し、これによりステージ５１をＸ方向及びＹ方向に移動させて、培養容器５０をＸ方向及びＹ方向に移動させる。なお水平方向駆動部１７は、水平方向に移動させるための公知の移動機構と、例えばモータ等の駆動源とを含む。なお、水平方向駆動部１７は本発明の駆動部の一例である。本実施形態においては、制御部２２による制御によってステージ５１をＸ方向及びＹ方向に移動させ、結像光学系１４を培養容器５０内において２次元状に走査し、撮像部１６Ａ及び撮像部１６Ｂが結像光学系１４による各観察位置の位相差画像を取得する。すなわち、撮像部１６Ａ及び撮像部１６Ｂは、それぞれ１つのウェル内で分割された複数の撮像領域（視野）毎の位相差画像を取得する。
Further, the control unit 22 drives and controls the horizontal direction drive unit 17, thereby moving the stage 51 in the X direction and the Y direction, and moving the culture vessel 50 in the X direction and the Y direction. The horizontal drive unit 17 includes a known moving mechanism for moving in the horizontal direction and a drive source such as a motor. The horizontal driving unit 17 is an example of the driving unit of the present invention. In the present embodiment, the stage 51 is moved in the X direction and the Y direction by the control of the control unit 22, the imaging optical system 14 is scanned two-dimensionally in the culture vessel 50, and the imaging unit 16A and the imaging unit 16B are A phase difference image at each observation position by the imaging optical system 14 is acquired. That is, the imaging unit 16A and the imaging unit 16B each acquire a phase difference image for each of a plurality of imaging regions (fields of view) divided within one well.

　また、制御部２２は、顕微鏡装置本体１０によって撮影された各観察領域の位相差画像を処理部２３Ｂが結合することによって生成された１枚の合成位相差画像を表示装置３０に表示させる表示制御部としても機能する。
Further, the control unit 22 causes the display device 30 to display one composite phase difference image generated by the processing unit 23B combining the phase difference images of the respective observation regions photographed by the microscope apparatus body 10. It also functions as a part.

　また、制御部２２は、取得部２３Ａにより取得された培養容器５０の底面の位置を示す位置情報に基づいて、第３の動作部１５Ｃにより撮像部１６Ｂを光軸方向に移動させて、撮像部１６Ａ及び撮像部１６Ｂの各々に、焦点面が異なる光学像を結像させる制御を行なう。
Further, the control unit 22 moves the imaging unit 16B in the optical axis direction by the third operation unit 15C based on the position information indicating the position of the bottom surface of the culture vessel 50 acquired by the acquisition unit 23A, and the imaging unit Control is performed to form optical images with different focal planes on each of 16A and imaging unit 16B.

　ここで、図５に顕微鏡装置１におけるデフォーカス量の調整を説明する図を示す。一例として図５に示すように、撮像部１６Ａにおける焦点面を焦点面Ｆ１、撮像部１６Ｂにおける焦点面を焦点面Ｆ２とする。本実施形態において、焦点面は光学像の焦点があっている、すなわちピントが合っている面を意味する。本実施形態において、撮像部１６Ａと撮像部１６Ｂは、撮像部１６Ａの焦点面Ｆ１と撮像部１６Ｂの焦点面Ｆ２とを異ならせて配置される。
Here, FIG. 5 is a diagram for explaining the adjustment of the defocus amount in the microscope apparatus 1. As an example, as shown in FIG. 5, the focal plane in the imaging unit 16A is a focal plane F1, and the focal plane in the imaging unit 16B is a focal plane F2. In the present embodiment, the focal plane means a plane in which the optical image is in focus, that is, in focus. In the present embodiment, the imaging unit 16A and the imaging unit 16B are arranged with the focal plane F1 of the imaging unit 16A different from the focal plane F2 of the imaging unit 16B.

　一般的に、顕微鏡装置１において、観察対象を観察又は撮影する場合、最も焦点が合っている位置つまり焦点面から、観察対象が光軸方向に離れた距離においても、焦点が合っていると見なせる範囲を被写界深度（D.O.F:Depth Of Field）という。撮像部１６Ａの焦点面Ｆ１及び撮像部１６Ｂの焦点面Ｆ２においても、それぞれ被写界深度ＤＯＦ１，ＤＯＦ２が存在する。被写界深度ＤＯＦ１及び被写界深度ＤＯＦ２は、撮像部１６Ａ，１６Ｂの開口数ＮＡ、対物レンズ１４ｂ及び結像レンズ１４ｄの倍率等の性能により異なる値であり、例えば予めユーザが入力装置４０を使用して二次記憶部２５Ｂに記憶しておく。なお、被写界深度の算出方法については特に限定されず、公知の技術を使用して算出することができる。
In general, when the observation target is observed or photographed in the microscope apparatus 1, it can be considered that the observation target is in focus even at a distance away from the focus position, that is, the focal plane, in the optical axis direction. The range is called depth of field (DOF). Also in the focal plane F1 of the imaging unit 16A and the focal plane F2 of the imaging unit 16B, there are depths of field DOF1 and DOF2, respectively. The depth of field DOF1 and the depth of field DOF2 are different values depending on performance such as the numerical aperture NA of the imaging units 16A and 16B, the magnification of the objective lens 14b, and the imaging lens 14d. For example, the user sets the input device 40 in advance. Used and stored in the secondary storage unit 25B. The method for calculating the depth of field is not particularly limited, and can be calculated using a known technique.

　本実施形態においては、撮像部１６Ａの焦点面Ｆ１と撮像部１６Ｂの焦点面Ｆ２とを異ならせて配置しているので、被写界深度ＤＯＦ１と被写界深度ＤＯＦ２を少なくとも一部重ねて設定することができる。これにより、撮像部が１つの場合と比較して顕微鏡装置１における被写界深度ＤＯＦを拡張することができる。なお、被写界深度ＤＯＦ１と被写界深度ＤＯＦ２を重ねることなく連続して設定することにより、撮像部が１つの場合よりも最大で２倍、被写界深度ＤＯＦを拡張することができる。
In the present embodiment, since the focal plane F1 of the imaging unit 16A and the focal plane F2 of the imaging unit 16B are arranged differently, the depth of field DOF1 and the depth of field DOF2 are set to overlap at least partially. can do. Thereby, the depth of field DOF in the microscope apparatus 1 can be expanded as compared with the case where there is one imaging unit. In addition, by setting the depth of field DOF1 and the depth of field DOF2 continuously without overlapping each other, the depth of field DOF can be expanded up to twice as much as when there is one imaging unit.

　ここで、本実施形態において特徴的なのは、制御部２２が、取得部２３Ａによって取得された培養容器５０のＺ方向の位置情報に基づいて、撮像部１６ＢのＺ方向の位置を変更する制御を行うことである。撮像部１６ＢのＺ方向の位置を変更することにより、撮像部１６Ｂの焦点面Ｆ２の位置をＺ方向に移動させて、被写界深度ＤＯＦ２をＺ方向に移動させる。つまり、制御部２２は、被写界深度ＤＯＦ２をＺ方向に移動させることにより、被写界深度ＤＯＦ１と被写界深度ＤＯＦ２との重複領域を変更し、顕微鏡装置１における被写界深度ＤＯＦを変更する制御を行う。
Here, what is characteristic in the present embodiment is that the control unit 22 performs control to change the position in the Z direction of the imaging unit 16B based on the position information in the Z direction of the culture vessel 50 acquired by the acquisition unit 23A. That is. By changing the position of the imaging unit 16B in the Z direction, the position of the focal plane F2 of the imaging unit 16B is moved in the Z direction, and the depth of field DOF2 is moved in the Z direction. That is, the control unit 22 moves the depth of field DOF2 in the Z direction to change the overlapping area between the depth of field DOF1 and the depth of field DOF2, and sets the depth of field DOF in the microscope apparatus 1 to Control to change.

　なお、本実施形態において、撮像部１６Ａの被写界深度ＤＯＦ１及び撮像部１６Ｂの被写界深度ＤＯＦ２は、それぞれ±６μｍすなわち１２μｍの範囲とする。また、本実施形態において、撮像部１６Ａの焦点面Ｆ１と撮像部１６Ｂの焦点面Ｆ２とがＺ方向におけるずれ量をデフォーカス量という。例えば撮像部１６Ａの被写界深度ＤＯＦ１と撮像部１６Ｂの被写界深度ＤＯＦ２とが完全に一致している場合には、Ｚ方向におけるずれはないのでデフォーカス量は０となる。また、被写界深度ＤＯＦ１及び被写界深度ＤＯＦ２が重ならずに連続して設定されている場合には、Ｚ方向におけるずれ量は１２（＝（６－０）＋６）μｍなので、デフォーカス量は１２μｍとなる。また、被写界深度ＤＯＦ１及び被写界深度ＤＯＦ２が４μｍ重なって設定されている場合には、Ｚ方向におけるずれ量は８（＝（６－４）＋６）μｍなので、デフォーカス量は８μｍとなる。なお、図５において、距離ｄは、対物レンズ１４の横倍率が１０倍とすると、縦倍率は１０２倍となるので、距離ｄ＝デフォーカス量×１０２となる。顕微鏡装置１におけるデフォーカス量を変更する制御については、後で詳細に説明する。
In the present embodiment, the depth of field DOF1 of the imaging unit 16A and the depth of field DOF2 of the imaging unit 16B are in the range of ± 6 μm, that is, 12 μm, respectively. In the present embodiment, the amount of shift in the Z direction between the focal plane F1 of the imaging unit 16A and the focal plane F2 of the imaging unit 16B is referred to as a defocus amount. For example, when the depth of field DOF1 of the imaging unit 16A and the depth of field DOF2 of the imaging unit 16B completely match, the defocus amount is 0 because there is no shift in the Z direction. If the depth of field DOF1 and the depth of field DOF2 are set continuously without overlapping, the shift amount in the Z direction is 12 (= (6-0) +6) μm, so defocusing is performed. The amount is 12 μm. Further, when the depth of field DOF1 and the depth of field DOF2 are set to overlap by 4 μm, the shift amount in the Z direction is 8 (= (6−4) +6) μm, so the defocus amount is 8 μm. Become. In FIG. 5, if the lateral magnification of the objective lens 14 is 10 times, the distance d is 10 2 times, so the distance d is defocus amount × 10 2 . Control for changing the defocus amount in the microscope apparatus 1 will be described in detail later.

　また、顕微鏡制御装置２０には、入力装置４０と表示装置３０とがバスライン２８によって接続されている。
In addition, an input device 40 and a display device 30 are connected to the microscope control device 20 by a bus line 28.

　表示装置３０は、上述したように制御部２２によって生成された合成位相差画像を表示するものであり、一例として例えば液晶ディスプレイ等を備える。また、表示装置３０をタッチパネルによって構成し、入力装置４０と兼用してもよい。
The display device 30 displays the composite phase difference image generated by the control unit 22 as described above, and includes, for example, a liquid crystal display. Further, the display device 30 may be configured by a touch panel and may also be used as the input device 40.

　入力装置４０は、一例としてマウス及びキーボード等を備えたものであり、ユーザによる種々の設定入力を受け付ける。本実施形態の入力装置４０は、例えば位相差レンズ１４ａの倍率の変更指示及びステージの移動速度の変更指示等の設定入力を受け付ける。
The input device 40 includes a mouse and a keyboard as an example, and accepts various setting inputs by the user. The input device 40 according to the present embodiment receives setting inputs such as an instruction to change the magnification of the phase difference lens 14a and an instruction to change the moving speed of the stage.

　次に、顕微鏡装置１の本開示の技術に係る部分の作用について説明する。図６は第１実施形態に係る顕微鏡装置の作用の一例を示すフローチャート、図７は第１実施形態に係る顕微鏡装置の培養容器内における観察領域の走査位置の一例を示す図、図８は第１実施形態に係る顕微鏡装置における変位センサの配置の一例を説明するための図、図９は第１実施形態に係る顕微鏡装置のデフォーカス量調整処理の一例を示すフローチャート、図１０は第１実施形態に係る顕微鏡装置における底面の高低差の取得の一例を説明するための図である。
Next, an operation of a portion according to the technique of the present disclosure of the microscope apparatus 1 will be described. FIG. 6 is a flowchart showing an example of the operation of the microscope apparatus according to the first embodiment, FIG. 7 is a diagram showing an example of the scanning position of the observation region in the culture container of the microscope apparatus according to the first embodiment, and FIG. FIG. 9 is a flowchart for explaining an example of the arrangement of displacement sensors in the microscope apparatus according to the embodiment; FIG. 9 is a flowchart illustrating an example of defocus amount adjustment processing of the microscope apparatus according to the first embodiment; and FIG. It is a figure for demonstrating an example of acquisition of the height difference of the bottom face in the microscope apparatus which concerns on a form.

　一例として図６に示すように、先ず、ステップＳ１で、制御部２２は、水平方向駆動部１７を駆動して、観察対象である細胞が収容された培養容器５０が載置されたステージ５１を移動させることにより、結像光学系１４の観察領域を、一例として図７に示す走査開始点Ｓに位置させて、観察領域による培養容器５０の走査を開始させる。
As an example, as shown in FIG. 6, first, in step S <b> 1, the control unit 22 drives the horizontal driving unit 17 to place the stage 51 on which the culture vessel 50 containing the cells to be observed is placed. By moving, the observation region of the imaging optical system 14 is positioned at the scanning start point S shown in FIG. 7 as an example, and scanning of the culture vessel 50 by the observation region is started.

　本実施形態においては、制御部２２による制御によってステージ５１をＸ方向及びＹ方向に移動させ、結像光学系１４の観察領域を培養容器５０内において２次元状に移動させて培養容器５０を走査し、各観察領域の位相差画像を取得する。なお図７において、実線Ｍは、培養容器５０内における観察領域による走査位置を示している。
In the present embodiment, the stage 51 is moved in the X direction and the Y direction by the control of the control unit 22, and the observation region of the imaging optical system 14 is moved two-dimensionally in the culture vessel 50 to scan the culture vessel 50. Then, a phase difference image of each observation area is acquired. In FIG. 7, the solid line M indicates the scanning position of the observation region in the culture vessel 50.

　図７に示すように、結像光学系１４の観察領域は、ステージ５１の上記移動によって走査開始点Ｓから走査終了点Ｅまで実線Ｍに沿って移動する。すなわち、観察領域は、Ｘ方向の正方向（図７の右方向）に移動された後、Ｙ方向（図７の下方向）に移動し、逆の負方向（図７の左方向）に移動される。次いで、観察領域は、再びＹ方向に移動し、再び正方向に移動される。このように、観察領域のＸ方向についての往復移動とＹ方向への移動を繰り返し行うことによって、培養容器５０は２次元状に走査される。
As shown in FIG. 7, the observation region of the imaging optical system 14 moves along the solid line M from the scanning start point S to the scanning end point E by the above movement of the stage 51. That is, the observation area is moved in the positive direction of the X direction (right direction in FIG. 7), then moved in the Y direction (downward direction in FIG. 7), and moved in the opposite negative direction (left direction in FIG. 7). Is done. Next, the observation area moves again in the Y direction and is moved again in the positive direction. In this way, the culture vessel 50 is scanned two-dimensionally by repeatedly performing the reciprocating movement in the X direction and the movement in the Y direction of the observation region.

　次のステップＳ２で、制御部２２は、検出部１８に培養容器５０の底面の位置情報を検出させて、取得部２３Ａは、検出部１８により検出された位置情報を取得する。本実施形態においては、一例として図８に示すように、第１の変位センサ１８ａと第２の変位センサ１８ｂとが結像光学系１４を挟んでＸ方向に並べて設けられている。そして、結像光学系１４の観察領域Ｒは、上述したように培養容器５０内を２次元状に移動されるが、この際、制御部２２は、培養容器５０と結像光学系１４との相対的移動に応じて観察領域Ｒが移動する方向に沿って、かつ結像光学系１４よりも先行する位置に対応する培養容器５０の底面の位置を示す位置情報を検出部１８に取得させる制御を行う。
In the next step S2, the control unit 22 causes the detection unit 18 to detect the position information of the bottom surface of the culture vessel 50, and the acquisition unit 23A acquires the position information detected by the detection unit 18. In this embodiment, as shown in FIG. 8 as an example, a first displacement sensor 18a and a second displacement sensor 18b are provided side by side in the X direction with the imaging optical system 14 interposed therebetween. The observation region R of the imaging optical system 14 is moved two-dimensionally within the culture vessel 50 as described above. At this time, the control unit 22 determines whether the culture vessel 50 and the imaging optical system 14 are connected. Control that causes the detection unit 18 to acquire position information indicating the position of the bottom surface of the culture vessel 50 corresponding to the position preceding the imaging optical system 14 along the direction in which the observation region R moves in accordance with the relative movement. I do.

　すなわち、培養容器５０に対する結像光学系１４の観察領域Ｒの位置よりも観察領域Ｒの移動方向前側の位置において培養容器５０のＺ方向の位置が検出される。具体的には、観察領域Ｒが、図８に示す矢印方向（図８の右方向）に移動している場合には、第１の変位センサ１８ａ及び第２の変位センサ１８ｂのうち、観察領域Ｒの移動方向前側の第１の変位センサ１８ａによって培養容器５０のＺ方向の位置が検出される。
That is, the position of the culture container 50 in the Z direction is detected at a position in front of the movement direction of the observation region R relative to the position of the observation region R of the imaging optical system 14 with respect to the culture container 50. Specifically, when the observation region R moves in the direction of the arrow shown in FIG. 8 (the right direction in FIG. 8), the observation region of the first displacement sensor 18a and the second displacement sensor 18b. The position of the culture vessel 50 in the Z direction is detected by the first displacement sensor 18a on the front side in the R movement direction.

　一方、観察領域Ｒが、図８の矢印方向とは逆方向（図８の左方向）に移動している場合には、第１の変位センサ１８ａ及び第２の変位センサ１８ｂのうち、観察領域Ｒの移動方向前側の第２の変位センサ１８ｂによって培養容器５０のＺ方向の位置が検出される。
On the other hand, when the observation region R is moving in the direction opposite to the arrow direction in FIG. 8 (the left direction in FIG. 8), the observation region R out of the first displacement sensor 18a and the second displacement sensor 18b. The position of the culture vessel 50 in the Z direction is detected by the second displacement sensor 18b on the front side in the R movement direction.

　このように、制御部２２は、第１の変位センサ１８ａを用いた検出と第２の変位センサ１８ｂを用いた検出とを観察領域Ｒの移動方向に応じて切り替える。
As described above, the control unit 22 switches between detection using the first displacement sensor 18a and detection using the second displacement sensor 18b according to the moving direction of the observation region R.

　次にステップＳ３で、制御部２２が、取得部２３Ａによって取得された位置情報に基づいて、撮像部１６Ａと撮像部１６Ｂとの間のデフォーカス量を調整する制御を行う。
Next, in step S3, the control unit 22 performs control to adjust the defocus amount between the imaging unit 16A and the imaging unit 16B based on the position information acquired by the acquisition unit 23A.

　ここで本開示の技術に係るデフォーカス量を調整する制御処理の一例を説明する。図９は第１実施形態に係る顕微鏡装置のデフォーカス量調整処理の一例を示すフローチャート、図１０は第１実施形態に係る顕微鏡装置における底面の高低差の取得の一例を説明するための図である。一例として図９に示すように、ステップＳ２１で、取得部２３Ａが取得した底面の位置情報に基づいて、制御部２２が底面の高低差を取得する。なお、培養容器５０の底面のＺ方向の位置は、Ｘ方向及び／又はＹ方向において、検出部１８によって時系列に取得されている。
Here, an example of a control process for adjusting the defocus amount according to the technique of the present disclosure will be described. FIG. 9 is a flowchart illustrating an example of a defocus amount adjustment process of the microscope apparatus according to the first embodiment, and FIG. 10 is a diagram for explaining an example of acquisition of the height difference of the bottom surface in the microscope apparatus according to the first embodiment. is there. As an example, as illustrated in FIG. 9, in step S <b> 21, the control unit 22 acquires the height difference of the bottom surface based on the position information of the bottom surface acquired by the acquisition unit 23 </ b> A. The position of the bottom surface of the culture vessel 50 in the Z direction is acquired in time series by the detection unit 18 in the X direction and / or the Y direction.

　例えば図８に示すように、第１の変位センサ１８ａと第２の変位センサ１８ｂとが結像光学系１４を挟んでＸ方向に並べて設けられている場合には、図１０に示すように、観察領域が移動するライン、すなわち現在の観察領域の移動ラインであるＭ１及び１つ前の観察領域の移動ラインであるＭ２等の各ラインに沿って底面の位置情報が検出される。
For example, as shown in FIG. 8, when the first displacement sensor 18a and the second displacement sensor 18b are provided side by side in the X direction with the imaging optical system 14 in between, as shown in FIG. Position information of the bottom surface is detected along each line such as a line along which the observation area moves, that is, a movement line M1 of the current observation area and a movement line M2 of the previous observation area.

　従って、例えば観察領域Ｒが、一例として図８に示す位置から第１の変位センサ１８ａによって培養容器５０のＺ方向の位置が検出された位置まで移動した場合に、図８に示す第１の変位センサ１８ａの位置において前もって検出された培養容器５０のＺ方向の位置と、図８に示す観察領域Ｒの位置において前もって検出された培養容器５０のＺ方向の位置とから高低差を取得する。これにより培養容器５０のＸ方向における底面の高低差を取得することができる。
Therefore, for example, when the observation region R moves from the position shown in FIG. 8 as an example to the position where the position of the culture vessel 50 in the Z direction is detected by the first displacement sensor 18a, the first displacement shown in FIG. A height difference is acquired from the position in the Z direction of the culture vessel 50 detected in advance at the position of the sensor 18a and the position in the Z direction of the culture vessel 50 detected in advance at the position of the observation region R shown in FIG. Thereby, the height difference of the bottom face in the X direction of the culture vessel 50 can be acquired.

　なお、本実施形態においては、第１の変位センサ１８ａ及び第２の変位センサ１８ｂは結像光学系１４を挟んでＸ方向に並べて設けられているが、本発明はこれに限られるものではない。ここで、図１１に第１実施形態に係る顕微鏡装置における変位センサの配置の他の一例を説明するための図、図１２に第１実施形態に係る顕微鏡装置における変位センサの配置のさらに他の一例を説明するための図、図１３に第１実施形態に係る顕微鏡装置における底面の高低差の取得に他の一例を説明するための図をそれぞれ示す。
In the present embodiment, the first displacement sensor 18a and the second displacement sensor 18b are provided side by side in the X direction with the imaging optical system 14 interposed therebetween, but the present invention is not limited to this. . Here, FIG. 11 is a diagram for explaining another example of the arrangement of the displacement sensor in the microscope apparatus according to the first embodiment, and FIG. 12 shows still another arrangement of the displacement sensor in the microscope apparatus according to the first embodiment. FIG. 13 is a diagram for explaining an example, and FIG. 13 is a diagram for explaining another example for obtaining the height difference of the bottom surface in the microscope apparatus according to the first embodiment.

　他の一例として図１１に示すように、第１の変位センサ１８ａ及び第２の変位センサ１８ｂは、図８の実施形態と比較して結像光学系１４からＹ方向にずらした位置に設けられている。この場合、図１０に示すように、観察領域が移動するラインＬ、すなわち現在の観察領域の移動ラインであるＬ１及び１つ前の観察領域の移動ラインであるＬ２等の各ラインに沿って底面の位置情報が検出される。
As another example, as shown in FIG. 11, the first displacement sensor 18a and the second displacement sensor 18b are provided at positions shifted in the Y direction from the imaging optical system 14 as compared with the embodiment of FIG. ing. In this case, as shown in FIG. 10, the bottom surface along each line such as a line L along which the observation region moves, that is, a movement line L1 of the current observation region and a movement line L2 of the previous observation region. Position information is detected.

　従って、例えばＸ方向において観察領域Ｒが、図１１に示す位置から第１の変位センサ１８ａによって培養容器５０のＺ方向の位置が検出された位置まで移動した場合に、図１１に示す第１の変位センサ１８ａの位置において前もって検出された培養容器５０のＺ方向の位置と、図１１に示す観察領域Ｒの位置において前もって検出された培養容器５０のＺ方向の位置とからＸ方向における高低差を取得する。これにより培養容器５０のＸ方向における底面の高低差を取得することができる。
Therefore, for example, when the observation region R in the X direction moves from the position shown in FIG. 11 to the position where the position of the culture vessel 50 in the Z direction is detected by the first displacement sensor 18a, the first region shown in FIG. A difference in height in the X direction from the position in the Z direction of the culture vessel 50 detected in advance at the position of the displacement sensor 18a and the position in the Z direction of the culture vessel 50 detected in advance in the position of the observation region R shown in FIG. get. Thereby, the height difference of the bottom face in the X direction of the culture vessel 50 can be acquired.

　さらに、例えばＸ方向において観察領域Ｒが、図１１に示す位置から第１の変位センサ１８ａによって培養容器５０のＺ方向の位置が検出された位置まで移動した場合に、図１１に示す第１の変位センサ１８ａの位置において前もって検出された培養容器５０のＺ方向の位置と、Ｙ方向において１つ前の観察領域の移動ラインにおいて、つまり図１１の第１の変位センサ１８ａ－０の位置において前もって検出された培養容器５０のＺ方向の位置とからＹ方向における高低差を取得する。これにより培養容器５０のＹ方向における底面の高低差を取得することができる。
Furthermore, for example, when the observation region R in the X direction moves from the position shown in FIG. 11 to the position where the position of the culture vessel 50 in the Z direction is detected by the first displacement sensor 18a, the first region shown in FIG. The position in the Z direction of the culture vessel 50 detected in advance at the position of the displacement sensor 18a and the movement line of the observation area immediately before in the Y direction, that is, the position of the first displacement sensor 18a-0 in FIG. The height difference in the Y direction is acquired from the detected position of the culture vessel 50 in the Z direction. Thereby, the height difference of the bottom face in the Y direction of the culture vessel 50 can be acquired.

　Ｙ方向における培養容器５０の底面の高低差を取得する場合には、図８に示す態様よりも、図１１に示すように第１の変位センサ１８ａ及び第２の変位センサ１８ｂを結像光学系１４からＹ方向にずらした位置に設けることにより、図１０に示すように、１つの観察領域内において、より観察領域に近い位置で取得されたＺ方向の位置を使用することができるので、Ｙ方向における培養容器５０の底面の高低差の精度を向上させることができる。
When acquiring the height difference of the bottom surface of the culture vessel 50 in the Y direction, the first displacement sensor 18a and the second displacement sensor 18b are connected to the imaging optical system as shown in FIG. 11 rather than the embodiment shown in FIG. By providing at a position shifted in the Y direction from 14, the position in the Z direction acquired at a position closer to the observation area can be used in one observation area as shown in FIG. The accuracy of the height difference of the bottom surface of the culture vessel 50 in the direction can be improved.

　また、さらに他の一例として図１２に示すように、図１１に示す位置に設けられた第１の変位センサ１８ａ及び第２の変位センサ１８ｂの他に、さらにＹ方向において、結像光学系１４を挟んで反対側に第３の変位センサ１８ｃ及び第４の変位センサ１８ｄを設けていてもよい。この場合、図１３に示すように、観察領域の移動ラインに沿って異なるＹ方向の位置での底面の位置情報が同時に検出される。
As another example, as shown in FIG. 12, in addition to the first displacement sensor 18a and the second displacement sensor 18b provided at the position shown in FIG. A third displacement sensor 18c and a fourth displacement sensor 18d may be provided on the opposite side with respect to each other. In this case, as shown in FIG. 13, the position information of the bottom surface at different positions in the Y direction is detected simultaneously along the movement line of the observation region.

　Ｙ方向における培養容器５０の底面の高低差を取得する場合には、図１１に示す態様よりも、図１２に示すように第１の変位センサ１８ａ及び第２の変位センサ１８ｂに加えてさらに第３の変位センサ１８ｃ及び第４の変位センサ１８ｄを設けることにより、１つの観察領域内において、Ｙ方向の異なる位置で同時に取得されたＺ方向の位置を使用することができるので、Ｙ方向における培養容器５０の底面の高低差の精度をさらに向上させることができる。
When acquiring the height difference of the bottom surface of the culture vessel 50 in the Y direction, in addition to the first displacement sensor 18a and the second displacement sensor 18b as shown in FIG. By providing the third displacement sensor 18c and the fourth displacement sensor 18d, it is possible to use the Z-direction position simultaneously acquired at different positions in the Y-direction within one observation region. The accuracy of the height difference of the bottom surface of the container 50 can be further improved.

　図９に戻り、ステップＳ２２で、制御部２２は、取得した培養容器５０の底面の高低差を二次記憶部２５Ｂに記憶させる。
Returning to FIG. 9, in step S <b> 22, the control unit 22 stores the obtained height difference of the bottom surface of the culture vessel 50 in the secondary storage unit 25 </ b> B.

　次に、ステップＳ２３で、制御部２２は、二次記憶部２５Ｂに記憶された培養容器５０の底面の高低差に基づいて、撮像部１６ＢのＺ方向の位置を移動させる制御を行う。なお、本実施形態においては、デフォーカス量が０となる撮像部１６ＢのＺ方向の位置をデフォルトとする。
Next, in step S23, the control unit 22 performs control to move the position of the imaging unit 16B in the Z direction based on the height difference of the bottom surface of the culture vessel 50 stored in the secondary storage unit 25B. In the present embodiment, the position in the Z direction of the imaging unit 16B where the defocus amount is 0 is set as a default.

　そして、取得部２３Ａが取得した培養容器５０の底面の高低差が、撮像部１６Ａの被写界深度ＤＯＦ１及び撮像部１６Ｂの被写界深度ＤＯＦ２である１２μｍよりも小さい値の場合には、デフォーカス量が０、すなわち撮像部１６Ｂの位置はデフォルトの位置のままであっても、観察面は被写界深度内に位置できるので、制御部２２は、撮像部１６Ｂを移動させない。
When the height difference of the bottom surface of the culture vessel 50 acquired by the acquisition unit 23A is a value smaller than 12 μm, which is the depth of field DOF1 of the imaging unit 16A and the depth of field DOF2 of the imaging unit 16B, Even if the focus amount is 0, that is, the position of the imaging unit 16B remains the default position, the observation surface can be positioned within the depth of field, so the control unit 22 does not move the imaging unit 16B.

　一方、取得部２３Ａが取得した培養容器５０の底面の高低差が、撮像部１６Ａの被写界深度ＤＯＦ１及び撮像部１６Ｂの被写界深度ＤＯＦ２である１２μｍ以上の値の場合には、制御部２２は第３の動作部１５Ｃによって撮像部１６ＢをＺ方向に移動させる。培養容器５０の底面の高低差が１４μｍの場合には、顕微鏡装置１の被写界深度ＤＯＦが１４μｍよりも大きい値となればよい。従って、デフォーカス量を２μｍよりも大きい値にすべく、制御部２２は２μｍよりも長い距離、撮像部１６ＢをＺ方向に移動させる。
On the other hand, when the height difference of the bottom surface of the culture vessel 50 acquired by the acquisition unit 23A is a value of 12 μm or more which is the depth of field DOF1 of the imaging unit 16A and the depth of field DOF2 of the imaging unit 16B, the control unit 22 moves the imaging unit 16B in the Z direction by the third operating unit 15C. When the height difference of the bottom surface of the culture vessel 50 is 14 μm, the depth of field DOF of the microscope apparatus 1 may be a value larger than 14 μm. Accordingly, in order to set the defocus amount to a value larger than 2 μm, the control unit 22 moves the imaging unit 16B in the Z direction by a distance longer than 2 μm.

　同様にして、培養容器５０の底面の高低差が２４μｍの場合には、顕微鏡装置１の被写界深度ＤＯＦが２４μｍよりも大きい値となればよいので、デフォーカス量を１２μｍにすべく、制御部２２は１２μｍ撮像部１６ＢをＺ方向に移動させる。つまり、制御部２２は、撮像部１６Ａの被写界深度ＤＯＦ１と撮像部１６Ｂの被写界深度ＤＯＦ２を重ならせずに連続させる。
Similarly, when the difference in height of the bottom surface of the culture vessel 50 is 24 μm, the depth of field DOF of the microscope apparatus 1 only needs to be a value larger than 24 μm, so that the defocus amount is controlled to 12 μm. The unit 22 moves the 12 μm imaging unit 16B in the Z direction. That is, the control unit 22 causes the depth of field DOF1 of the imaging unit 16A and the depth of field DOF2 of the imaging unit 16B to continue without overlapping.

　以上のように、培養容器５０の底面の高低差に基づいて、制御部２２が撮像部１６ＢをＺ方向に移動させることにより、観察面を被写界深度内に位置させることができるので、観察面が被写界深度の範囲から外れることにより、焦点が合わずに撮像画像がボケた画像になるのを防止することができる。また、培養容器５０の底面の高低差が比較的小さい場合には、デフォーカス量を少なくすることによって撮像された画像の画質を維持することができる。また、培養容器５０の底面の高低差が比較的大きい場合には、デフォーカス量を多くすることによって撮像された画像の画質の低下を抑制することができる。なお、底面の高低差に基づいたデフォーカス量の調整方法を説明したが、その他の測長機や機械的な誤差要因などを加味して、デフォーカス量を調整してもよい。
As described above, since the control unit 22 moves the imaging unit 16B in the Z direction based on the height difference of the bottom surface of the culture vessel 50, the observation surface can be positioned within the depth of field. Since the surface is out of the range of the depth of field, it is possible to prevent the captured image from being out of focus and becoming a blurred image. In addition, when the height difference of the bottom surface of the culture vessel 50 is relatively small, the image quality of the captured image can be maintained by reducing the defocus amount. Moreover, when the difference in height of the bottom surface of the culture vessel 50 is relatively large, it is possible to suppress a decrease in the image quality of the captured image by increasing the defocus amount. In addition, although the adjustment method of the defocus amount based on the height difference of the bottom surface has been described, the defocus amount may be adjusted in consideration of other length measuring machines or mechanical error factors.

　そして、ステップＳ２３にて、制御部２２が撮像部１６Ｂを移動させると、図６に戻り、制御部２２が、ステップＳ４以降の処理を引き続き行う。
When the control unit 22 moves the imaging unit 16B in step S23, the process returns to FIG. 6 and the control unit 22 continues to perform the processing from step S4.

　ステップＳ４で、焦点調節部２４は、ステップＳ２において取得部２３Ａによって取得された位置情報に基づいてフォーカス制御量を取得する。焦点調節部２４は、上述したように、二次記憶部２５Ｂに記憶されたテーブルを参照して、結像レンズ１４ｄへの印加電圧、結像レンズ１４ｄの光軸方向の移動量、ステージ５１の光軸方向の移動量、対物レンズ１４ｂの光軸方向の移動量、及び対物レンズ１４ｂへの印加電圧をフォーカス制御量としてそれぞれ取得する。
In step S4, the focus adjustment unit 24 acquires the focus control amount based on the position information acquired by the acquisition unit 23A in step S2. As described above, the focus adjusting unit 24 refers to the table stored in the secondary storage unit 25B, applies the voltage to the imaging lens 14d, the amount of movement of the imaging lens 14d in the optical axis direction, and the stage 51. The amount of movement in the optical axis direction, the amount of movement of the objective lens 14b in the optical axis direction, and the voltage applied to the objective lens 14b are respectively acquired as focus control amounts.

　ここで、検出部１８が、図１１及び図１２の構成である場合には、図１１においては、第１の変位センサ１８ａが検出した、図１１の第１の変位センサ１８ａ－０と第１の変位センサ１８ａの位置において検出された位置情報を、取得部２３Ａが公知の技術により補間することにより結像光学系１４のＹ方向における位置と同じ位置の位置情報を取得する。また、図１２においても同様にして、第１の変位センサ１８ａと第３の変位センサ１８ｃで同時に検出された位置情報を、取得部２３Ａが公知の技術により補間することにより結像光学系１４のＹ方向における位置と同じ位置の位置情報を取得する。
Here, when the detection unit 18 has the configuration of FIGS. 11 and 12, in FIG. 11, the first displacement sensor 18a-0 and the first displacement sensor 18a-0 of FIG. The acquisition unit 23A interpolates the position information detected at the position of the displacement sensor 18a by a known technique, thereby acquiring the position information at the same position as the position of the imaging optical system 14 in the Y direction. Similarly in FIG. 12, the acquisition unit 23A interpolates the position information simultaneously detected by the first displacement sensor 18a and the third displacement sensor 18c by a known technique, so that the imaging optical system 14 The position information at the same position as the position in the Y direction is acquired.

　次にステップＳ５で、制御部２２が、ステップＳ３において焦点調節部２４が取得したフォーカス制御量を培養容器５０の底面の位置情報の検出位置のＸ－Ｙ座標上の位置と対応づけて二次記憶部２５Ｂに記憶させる。
Next, in step S5, the control unit 22 associates the focus control amount acquired by the focus adjustment unit 24 in step S3 with the position on the XY coordinate of the detection position of the position information on the bottom surface of the culture vessel 50, and performs secondary processing. It memorize | stores in the memory | storage part 25B.

　なお、ステップＳ４，Ｓ５のフォーカス制御量の取得及び記憶処理は、制御部２２によるステップＳ３のデフォーカス量を調整する制御処理と並行して行うことができる。
The focus control amount acquisition and storage processing in steps S4 and S5 can be performed in parallel with the control processing for adjusting the defocus amount in step S3 by the control unit 22.

　次に図６に戻り、ステップＳ６で、制御部２２は水平方向駆動部１７を駆動させて、第１の変位センサ１８ａによって培養容器５０の位置検出が行われた位置に向かって観察領域Ｒが移動する。
Next, returning to FIG. 6, in step S <b> 6, the control unit 22 drives the horizontal driving unit 17 so that the observation region R moves toward the position where the position of the culture vessel 50 is detected by the first displacement sensor 18 a. Moving.

　そして、ステップＳ７で、制御部２２は、培養容器５０の位置検出が行われた位置に観察領域Ｒが到達する直前において二次記憶部２５Ｂに記憶されたフォーカス制御量を取得する。
In step S7, the control unit 22 acquires the focus control amount stored in the secondary storage unit 25B immediately before the observation region R reaches the position where the position of the culture vessel 50 is detected.

　次に、ステップＳ８で、制御部２２は、取得したフォーカス制御量に基づいてオートフォーカス制御を行う。すなわち、制御部２２は、取得したフォーカス制御量に基づいて第１の動作部１５Ａ～第６の動作部１５Ｆを制御することにより、結像レンズ１４ｄ、及び対物レンズ１４ｂの焦点距離が変更し、結像レンズ１４ｄ、撮像部１６、ステージ５１、及び対物レンズ１４ｂをＺ方向に移動させる。
Next, in step S8, the control unit 22 performs autofocus control based on the acquired focus control amount. That is, the control unit 22 controls the first operation unit 15A to the sixth operation unit 15F based on the acquired focus control amount, thereby changing the focal lengths of the imaging lens 14d and the objective lens 14b. The imaging lens 14d, the imaging unit 16, the stage 51, and the objective lens 14b are moved in the Z direction.

　そして、オートフォーカス制御後、ステップＳ９で、培養容器５０の位置検出が行われた位置に観察領域Ｒが到達した時点において、撮像部１６Ａ及び撮像部１６Ｂが位相差画像の撮像を行う。観察領域Ｒの位相差画像は、撮像部１６Ａ及び撮像部１６Ｂから制御部２２に出力されて記憶される。なお、本実施形態においては、上述したように各観察領域Ｒについて、先行して培養容器５０の位置検出が行われ、その検出位置まで観察領域Ｒが到達した時点において、撮像部１６Ａ及び撮像部１６Ｂによる撮像が行われる。そして、この培養容器５０の位置検出と撮像部１６Ａ及び撮像部１６Ｂによる撮像は、観察領域Ｒを移動しながら行われ、ある位置の観察領域Ｒの撮像部１６Ａ及び撮像部１６Ｂによる撮像と、その位置よりも移動方向について前側の位置における培養容器５０の位置検出とが並行して行われる。
Then, after the autofocus control, in step S9, when the observation region R reaches the position where the position of the culture vessel 50 is detected, the imaging unit 16A and the imaging unit 16B capture a phase difference image. The phase difference image of the observation region R is output from the imaging unit 16A and the imaging unit 16B to the control unit 22 and stored. In the present embodiment, as described above, the position of the culture vessel 50 is detected in advance for each observation region R, and when the observation region R reaches the detection position, the imaging unit 16A and the imaging unit Imaging by 16B is performed. The position detection of the culture vessel 50 and the imaging by the imaging unit 16A and the imaging unit 16B are performed while moving the observation region R, and the imaging of the observation region R at a certain position by the imaging unit 16A and the imaging unit 16B, The position detection of the culture vessel 50 at the front position with respect to the movement direction with respect to the position is performed in parallel.

　すなわち、ステップＳ７～Ｓ９にてフォーカス制御及び観察領域Ｒの撮像部１６Ａ及び撮像部１６Ｂによる撮像が行われている間、上記観察領域Ｒよりも移動方向について前側の位置において検出部１８による培養容器５０の位置検出、焦点調節部２４によるフォーカス制御量の取得及び記憶、及び制御部２２によるデフォーカス量制御処理のうち培養容器５０の底面の高低差の取得が並行して行われる。なお制御部２２によりデフォーカス量制御処理のうちうち培養容器５０の底面の高低差に応じた撮像部１６Ｂの移動については、ステップＳ９の撮像部１６Ｂによる撮像が行われた後で行う。
That is, while focus control and imaging by the imaging unit 16A and the imaging unit 16B in the observation region R are performed in steps S7 to S9, the culture container by the detection unit 18 at a position ahead of the observation region R in the movement direction. Among the position detection of 50, acquisition and storage of the focus control amount by the focus adjustment unit 24, and the defocus amount control processing by the control unit 22, the height difference of the bottom surface of the culture vessel 50 is acquired in parallel. Of the defocus amount control processing by the control unit 22, the movement of the imaging unit 16B according to the height difference of the bottom surface of the culture vessel 50 is performed after the imaging by the imaging unit 16B in step S9.

　そして、ステップＳ１０で、制御部２２が、水平方向駆動部１７を駆動させることにより観察領域Ｒを図７に示す加減速域の範囲Ｒ２まで移動させていない場合には、判定が否定されて、図６に示すステップＳ２へ移行する。
In step S10, if the control unit 22 has not moved the observation region R to the acceleration / deceleration region range R2 shown in FIG. 7 by driving the horizontal direction driving unit 17, the determination is negative, Control proceeds to step S2 shown in FIG.

　一方、ステップＳ１０で、制御部２２が、水平方向駆動部１７を駆動させることにより観察領域Ｒを図７に示す加減速域の範囲Ｒ２まで移動させ、Ｙ方向に移動させた後、Ｘ方向について逆方向に移動させる場合には、すなわち、制御部２２により観察領域Ｒの移動方向が、図８の矢印方向から矢印方向の反対方向に変更された場合には、判定が肯定されて、図６に示すステップＳ１１へ移行する。
On the other hand, in step S10, the control unit 22 drives the horizontal direction driving unit 17 to move the observation region R to the acceleration / deceleration region range R2 shown in FIG. In the case of moving in the reverse direction, that is, when the moving direction of the observation region R is changed by the control unit 22 from the arrow direction in FIG. 8 to the direction opposite to the arrow direction, the determination is affirmed and FIG. The process proceeds to step S11 shown in FIG.

　ステップＳ１１で、制御部２２は使用する変位センサを第１の変位センサ１８ａから第２の変位センサ１８ｂに切り替える。
In step S11, the control unit 22 switches the displacement sensor to be used from the first displacement sensor 18a to the second displacement sensor 18b.

　本実施形態においては、上述したように各観察領域Ｒについてそれぞれ前もって培養容器５０のＺ方向の位置が検出されるため、各観察領域Ｒの培養容器５０の位置の検出タイミングと、位相差画像の撮像タイミングとが時間的にずれる。したがって、オートフォーカス制御は、第１の変位センサ１８ａ又は第２の変位センサ１８ｂによって培養容器５０の位置の検出が行われた後、その検出位置に観察領域Ｒが到達するまでの間に行われる。
In this embodiment, since the position in the Z direction of the culture vessel 50 is detected in advance for each observation region R as described above, the detection timing of the position of the culture vessel 50 in each observation region R and the phase difference image The imaging timing is shifted in time. Accordingly, the autofocus control is performed after the position of the culture vessel 50 is detected by the first displacement sensor 18a or the second displacement sensor 18b and before the observation region R reaches the detection position. .

　そして、ステップＳ１２で、処理部２３Ｂが、全ての走査が終了したか否かを判定する。ステップＳ１２において、全ての走査が終了していない場合には、判定が否定されて、図６に示すステップＳ２へ移行する。そして制御部２２が観察領域Ｒを加減速域の範囲Ｒ１，Ｒ２まで移動させる度に、制御部２２は使用する変位センサを切り替えて、全ての走査が終了するまでステップＳ２～ステップＳ１１までの処理が繰り返して行われる。
In step S12, the processing unit 23B determines whether or not all scanning has been completed. If all the scans are not completed in step S12, the determination is negative and the process proceeds to step S2 shown in FIG. Each time the control unit 22 moves the observation region R to the acceleration / deceleration range R1, R2, the control unit 22 switches the displacement sensor to be used, and the processing from step S2 to step S11 is performed until all scanning is completed. Is repeated.

　そして、ステップＳ１２において、全ての走査が終了した場合には、すなわち制御部２２が観察領域Ｒを図７に示す走査終了点Ｅの位置に到達させた場合には、判定が肯定されて制御部２２は全ての走査を終了させる。
In step S12, when all the scans are completed, that is, when the control unit 22 causes the observation region R to reach the position of the scan end point E shown in FIG. 7, the determination is affirmed and the control unit 22 terminates all scanning.

　制御部２２が全ての走査が終了させた後、ステップＳ１３で、処理部２３Ｂは、各観察領域Ｒの位相差画像を結合して合成位相差画像を生成する。本実施形態においては、撮像部１６Ａと撮像部１６Ｂにより同一の観察領域Ｒの位相差画像が取得される。例えば１枚の位相差画像が、５１２０×５１２０ピクセルで表示される場合には、処理部２３Ｂは、６４×６４ピクセル毎に位相差画像を分割し、各分割領域毎に画質のより良い方を選択して合成する。このとき、例えば画像中のコントラストが高い方を画質の良い画像として選択することができる。また、より高精細な画像を合成するために、撮像部１６Ａと撮像部１６Ｂの相対位置を公知のカメラキャリブレーション技術を用いて補正してもよい。
After the control unit 22 finishes all scanning, the processing unit 23B combines the phase difference images of the observation regions R to generate a combined phase difference image in step S13. In the present embodiment, phase difference images of the same observation region R are acquired by the imaging unit 16A and the imaging unit 16B. For example, when one phase difference image is displayed with 5120 × 5120 pixels, the processing unit 23B divides the phase difference image into 64 × 64 pixels, and selects the better image quality for each divided region. Select and synthesize. At this time, for example, the higher contrast in the image can be selected as an image with good image quality. Further, in order to synthesize a higher-definition image, the relative positions of the imaging unit 16A and the imaging unit 16B may be corrected using a known camera calibration technique.

　次に、ステップＳ１４で、制御部２２は、処理部２３Ｂが生成した合成位相差画像を表示装置３０に表示させて、顕微鏡装置１による一連の処理が終了する。
Next, in step S14, the control unit 22 causes the display device 30 to display the combined phase difference image generated by the processing unit 23B, and the series of processes by the microscope device 1 is completed.

　このように、本実施形態においては、制御部２２が、取得部２３Ａにより取得された位置情報に基づいて撮像部１６Ｂの位置を変更させて、撮像部１６Ａと撮像部１６Ｂの各々に、焦点面が異なる光学像を結像させることにより位相差画像を取得させるので、培養容器５０の底面の形状に応じてデフォーカス量を変更することができる。これにより、観察面を被写界深度内に位置させることができるので、観察面が被写界深度の範囲から外れることによって、焦点が合わずに撮像画像がボケた画像になるのを防止することができる。

また、培養容器５０の底面の高低差が比較的小さい場合には、デフォーカス量を少なくすることによって撮像された画像の画質を維持することができる。また、培養容器５０の底面の高低差が比較的大きい場合には、デフォーカス量を多くすることによって撮像された画像の画質の低下を抑制することができる。
Thus, in the present embodiment, the control unit 22 changes the position of the imaging unit 16B based on the position information acquired by the acquisition unit 23A, and causes each of the imaging unit 16A and the imaging unit 16B to have a focal plane. Since the phase difference image is acquired by forming different optical images, the defocus amount can be changed according to the shape of the bottom surface of the culture vessel 50. As a result, since the observation surface can be positioned within the depth of field, it is possible to prevent the captured image from being out of focus and becoming a blurred image due to the observation surface being out of the range of the depth of field. be able to.

In addition, when the height difference of the bottom surface of the culture vessel 50 is relatively small, the image quality of the captured image can be maintained by reducing the defocus amount. Moreover, when the difference in height of the bottom surface of the culture vessel 50 is relatively large, it is possible to suppress a decrease in the image quality of the captured image by increasing the defocus amount.

　なお、本実施形態においては、ステップＳ１３にて、処理部２３Ｂが上述したようにして、合成位相差画像を生成したが、本発明はこれに限られない。ここで、図１４に第２実施形態に係る顕微鏡装置の構成の一例を示すブロック図を示す。なお、図１４は、図４の上記実施形態の顕微鏡装置１にさらに選択部２３Ｃを備えた装置であり、その他の構成は、上記実施形態と同様であるため、ここでの説明は省略し、異なる箇所についてのみ詳細に説明する。
In the present embodiment, in step S13, the processing unit 23B generates the composite phase difference image as described above, but the present invention is not limited to this. FIG. 14 is a block diagram showing an example of the configuration of the microscope apparatus according to the second embodiment. Note that FIG. 14 is an apparatus that further includes the selection unit 23C in the microscope apparatus 1 of the above-described embodiment of FIG. 4, and other configurations are the same as in the above-described embodiment. Only different parts will be described in detail.

　本実施形態の顕微鏡装置は、図１４に示すように、選択部２３Ｃを備えている。選択部２３Ｃは、撮像部１６Ａ及び撮像部１６Ｂによって取得した２枚の画像から、適切な画像を選択する。すなわち、より焦点のあった方の画像を選択する。具体的には、画像中のコントラストが高い方の画像を選択してもよいし、撮像部１６Ａ及び撮像部１６Ｂの画像を取得した際の合焦位置を取得して比較することにより、より焦点のあった方の画像を選択してもよい。
As shown in FIG. 14, the microscope apparatus of the present embodiment includes a selection unit 23C. The selection unit 23C selects an appropriate image from the two images acquired by the imaging unit 16A and the imaging unit 16B. That is, the more focused image is selected. Specifically, an image with a higher contrast in the image may be selected, or by obtaining and comparing the in-focus positions when the images of the imaging unit 16A and the imaging unit 16B are acquired, the focus is increased. You may select the image that has.

　本実施形態においては、処理部２３ＢがステップＳ１３にて合成位相差画像を生成する際に、観察領域毎に選択部２３Ｃにより選択された画像を採用する。
In the present embodiment, when the processing unit 23B generates a composite phase difference image in step S13, an image selected by the selection unit 23C for each observation region is employed.

　また、上述した実施形態においては、第３の動作部１５Ｃは、撮像部１６ＢをＺ方向に移動させるものとしたが、本発明はこれに限られるものではなく、培養容器５０の底面の傾きに合わせて撮像部１６Ｂの姿勢を変更させてもよい。ここで、図１５に第３実施形態に係る顕微鏡装置におけるデフォーカス量の調整を説明する図を示す。
In the above-described embodiment, the third operation unit 15C moves the imaging unit 16B in the Z direction. However, the present invention is not limited to this, and the inclination of the bottom surface of the culture vessel 50 is determined. In addition, the posture of the imaging unit 16B may be changed. Here, FIG. 15 is a diagram illustrating adjustment of the defocus amount in the microscope apparatus according to the third embodiment.

　図１５に示すように、第３の動作部１５Ｃは、一例として２つの圧電素子及び高電圧を印加させる駆動源を含み、制御部２２から出力された制御信号に基づいて駆動する。２つの圧電素子は、撮像部１６Ｂの底面に各々距離を有して配設される。培養容器５０の底面の傾きに合わせて、少なくとも一方の圧電素子が制御部２２により駆動されることにより、撮像部１６Ｂの姿勢が変更される。撮像部１６Ｂの姿勢を変更するためには、２つの圧電素子を相対的に駆動すればよい。なお、第３の動作部１５Ｃを構成する圧電素子は、２つに限られず２つ以上個であってもよい。
As illustrated in FIG. 15, the third operation unit 15 </ b> C includes, as an example, two piezoelectric elements and a drive source that applies a high voltage, and is driven based on a control signal output from the control unit 22. The two piezoelectric elements are disposed at a distance from the bottom surface of the imaging unit 16B. At least one piezoelectric element is driven by the control unit 22 in accordance with the inclination of the bottom surface of the culture vessel 50, whereby the posture of the imaging unit 16B is changed. In order to change the posture of the imaging unit 16B, the two piezoelectric elements may be driven relatively. The number of piezoelectric elements constituting the third operating unit 15C is not limited to two and may be two or more.

　なお、高低差と底面の傾きとの関係を示すテーブルを予め二次記憶部２５Ｂに記憶しておき、制御部２２は、このテーブルを参照して培養容器５０の底面の傾きを取得する。
Note that a table indicating the relationship between the height difference and the bottom surface inclination is stored in advance in the secondary storage unit 25B, and the control unit 22 acquires the bottom surface inclination of the culture vessel 50 by referring to this table.

　ここで、高低差と底面の傾きとの関係を示すものは、テーブルに限定されるものではなく、例えば式であってもよい。上記関係を示すものは、高低差から底面の傾きを導出できれば何れの方法を使用してもよい。
Here, what shows the relationship between a height difference and the inclination of a bottom face is not limited to a table, For example, a formula may be sufficient. Any method showing the above relationship may be used as long as the inclination of the bottom surface can be derived from the height difference.

　本実施形態においては、上述した実施形態の図９に示すフローチャートにおいて、培養容器５０の底面の高低差のかわりに、底面の傾きを取得及び記憶する。また、制御部２２は、培養容器５０の底面の高低差ではなく培養容器５０の底面の傾きに合わせて撮像部１６Ｂを傾ける。
In this embodiment, in the flowchart shown in FIG. 9 of the above-described embodiment, the inclination of the bottom surface is acquired and stored instead of the height difference of the bottom surface of the culture vessel 50. In addition, the control unit 22 tilts the imaging unit 16B according to the inclination of the bottom surface of the culture vessel 50, not the height difference of the bottom surface of the culture vessel 50.

　本実施形態においては、制御部２２が培養容器５０の底面の傾きに合わせて撮像部１６Ｂを傾けることにより、培養容器５０の底面の傾きと撮像部１６Ｂの傾きとを一致させる制御を行う。これにより、培養容器５０の底面の傾きに焦点面の傾きを合わせることができるので、観察面を被写界深度内に位置させることができる。従って、観察面が被写界深度の範囲から外れることによって、焦点が合わずに撮像画像がボケた画像になるのを防止することができる。
In the present embodiment, the control unit 22 controls the tilt of the bottom surface of the culture vessel 50 and the tilt of the image capturing unit 16B by tilting the image capturing unit 16B in accordance with the tilt of the bottom surface of the culture vessel 50. Thereby, since the inclination of the focal plane can be matched with the inclination of the bottom surface of the culture vessel 50, the observation surface can be positioned within the depth of field. Accordingly, it is possible to prevent the captured image from being out of focus and becoming a blurred image due to the observation surface being out of the range of the depth of field.

　なお、本実施形態の第３の動作部１５Ｃは、培養容器５０の底面の傾きに合わせて撮像部１６Ｂの姿勢を変更させるものとしたが、本発明はこれに限られず、さらに、撮像部１６ＢをＺ方向に移動させる機能を備えてもよい。すなわち、培養容器５０の底面の傾き等の形状に合わせて撮像部１６Ｂの姿勢及びＺ方向の位置を変更させてもよい。なお、本実施形態においては、撮像部の姿勢を変更させることにより撮像部の受光面の傾きを変更しているが、撮像部の姿勢を変更させることなく、撮像部の受光面の傾きのみを変更させる構成であっても構わない。
In addition, although the 3rd operation | movement part 15C of this embodiment shall change the attitude | position of the imaging part 16B according to the inclination of the bottom face of the culture container 50, this invention is not limited to this, Furthermore, the imaging part 16B May be provided with a function of moving the Z in the Z direction. That is, the posture of the imaging unit 16B and the position in the Z direction may be changed according to the shape such as the inclination of the bottom surface of the culture vessel 50. In the present embodiment, the inclination of the light receiving surface of the imaging unit is changed by changing the attitude of the imaging unit, but only the inclination of the light receiving surface of the imaging unit is changed without changing the attitude of the imaging unit. The configuration may be changed.

　この場合、上記実施形態の構成において、制御部２２が、２つの圧電素子を同じ駆動量で駆動させることにより、撮像部１６ＢをＺ方向に移動させることができる。なお、２つの圧電素子とは別に、撮像部１６ＢをＺ方向に移動させるための１つ以上の圧電素子をさらに設けてもよい。
In this case, in the configuration of the above embodiment, the control unit 22 can move the imaging unit 16B in the Z direction by driving the two piezoelectric elements with the same driving amount. In addition to the two piezoelectric elements, one or more piezoelectric elements for moving the imaging unit 16B in the Z direction may be further provided.

　本実施形態のように、制御部２２が撮像部１６ＢのＺ方向の位置と姿勢とを変更させることにより、上述した実施形態と比較して培養容器５０の底面の形状にあったより細かい制御を行うことができるので、撮像部１６Ａ及び撮像部１６Ｂが取得した画像の画質の低下をより抑制することができる。
As in this embodiment, the control unit 22 changes the position and orientation of the imaging unit 16B in the Z direction, thereby performing finer control that matches the shape of the bottom surface of the culture vessel 50 as compared to the above-described embodiment. Therefore, it is possible to further suppress deterioration in image quality of images acquired by the imaging unit 16A and the imaging unit 16B.

　なお、上述した実施形態において、取得部２３Ａが取得した培養容器５０の底面の高低差及び／又は傾きが予め定められた閾値よりも大きい場合に、制御部２２は撮像部１６ＢのＺ方向位置及び撮像部１６Ｂ姿勢の少なくとも一方を変更する制御を行う。
In the above-described embodiment, when the height difference and / or inclination of the bottom surface of the culture vessel 50 acquired by the acquisition unit 23A is larger than a predetermined threshold, the control unit 22 determines the position of the imaging unit 16B in the Z direction and Control to change at least one of the postures of the imaging unit 16B is performed.

　具体的には、例えば顕微鏡装置１の最大の被写界深度である２４μｍよりも大きい値の場合には、撮像部１６Ａ及び撮像部１６Ｂによる１回の撮影では観察面を被写界深度内に位置させることができないので、例えば制御部２２は、１つの観察領域Ｒを観察面が被写界深度内に収まる複数の観察領域に分けて、かつ撮影間隔を上記実施形態よりも短くして撮像部１６Ａ及び撮像部１６Ｂに複数回の撮像を行わせる制御を行う。
Specifically, for example, when the value is larger than 24 μm, which is the maximum depth of field of the microscope apparatus 1, the observation surface is within the depth of field in one shooting by the imaging unit 16A and the imaging unit 16B. For example, the control unit 22 divides one observation region R into a plurality of observation regions in which the observation surface is within the depth of field, and the imaging interval is shorter than that in the above embodiment. Control for causing the unit 16A and the imaging unit 16B to perform imaging a plurality of times is performed.

　これにより、観察面を被写界深度内に位置させることができるので、観察面が被写界深度の範囲から外れることにより、焦点が合わずに撮像画像がボケた画像になるのを防止することができる。
As a result, the observation surface can be positioned within the depth of field, and thus the captured image is prevented from being out of focus and being out of focus due to the observation surface being out of the range of the depth of field. be able to.

　なお、上述した実施形態においては、制御部２２は、撮像部１６ＢのみのＺ方向の位置及び／又は姿勢を変更する制御を行ったが、本発明はこれに限られず、撮像部１６Ａのみ、もしくは撮像部１６Ａと撮像部１６Ｂの両方の移動を制御してもよい。撮像部１６Ａの位置を変更する場合には、新たにＸ軸方向、つまり光路分割部１９で反射した後の光の光軸方向に撮像部１６Ａを移動させる動作部を設ける。また、撮像部１６Ａの姿勢を変更する場合には、新たに撮像部１６Ａの例えば図１５中左側の側面に２つの圧電素子を、撮像部１６ＡがＸ方向に移動可能に設ける。なお、この動作部は、第３の動作部１６Ｃと同様の構成にすることができる。
In the above-described embodiment, the control unit 22 performs control to change the position and / or orientation in the Z direction of only the imaging unit 16B. However, the present invention is not limited to this, and only the imaging unit 16A or The movement of both the imaging unit 16A and the imaging unit 16B may be controlled. When the position of the imaging unit 16A is changed, an operation unit that moves the imaging unit 16A in the X-axis direction, that is, the optical axis direction of the light reflected by the optical path dividing unit 19 is newly provided. When changing the orientation of the imaging unit 16A, two piezoelectric elements are newly provided on the left side of the imaging unit 16A, for example, in FIG. 15, so that the imaging unit 16A can move in the X direction. Note that this operation unit can have the same configuration as the third operation unit 16C.

　また、上述した実施形態の顕微鏡装置は、撮像部を２つ備えているが、本発明はこれに限られず、例えば３つ以上の複数の撮像部を備えていてもよい。例えば、顕微鏡装置が３つの撮像部を備える場合には、光路分割部１９として２つのビームスプリッタを使用すればよい。
Moreover, although the microscope apparatus of the embodiment described above includes two imaging units, the present invention is not limited to this, and may include, for example, three or more imaging units. For example, when the microscope apparatus includes three imaging units, two beam splitters may be used as the optical path dividing unit 19.

　なお、上記実施形態は、本発明を位相差顕微鏡に適用したものであるが、本発明は、位相差顕微鏡に限らず、微分干渉顕微鏡及び明視野顕微鏡等のその他の顕微鏡の観察に適用することができる。
In the above embodiment, the present invention is applied to a phase contrast microscope. However, the present invention is not limited to the phase contrast microscope, and is applied to observation of other microscopes such as a differential interference microscope and a bright field microscope. Can do.

　また、上記各実施形態では、観察制御プログラム２６を二次記憶部２５Ｂから読み出す場合を例示したが、必ずしも最初から二次記憶部２５Ｂに記憶させておく必要はない。例えば、図１６に示すように、ＳＳＤ（Ｓｏｌｉｄ　Ｓｔａｔｅ　Ｄｒｉｖｅ）、ＵＳＢ（Ｕｎｉｖｅｒｓａｌ　Ｓｅｒｉａｌ　Ｂｕｓ）メモリ、又はＤＶＤ－ＲＯＭ（Ｄｉｇｉｔａｌ　ｖｅｒｓａｔｉｌｅ　ｄｉｓｃ－Ｒｅａｄ　Ｏｎｌｙ　Ｍｅｍｏｒｙ）等の任意の可搬型の記憶媒体２５０に先ずは観察制御プログラム２６を記憶させておいてもよい。この場合、記憶媒体２５０の観察制御プログラム２６が顕微鏡制御装置２０にインストールされ、インストールされた観察制御プログラム２６がＣＰＵ２１によって実行される。
Further, in each of the above embodiments, the case where the observation control program 26 is read from the secondary storage unit 25B is exemplified, but it is not always necessary to store the observation control program 26 in the secondary storage unit 25B from the beginning. For example, as shown in FIG. 16, first, an arbitrary portable storage medium 250 such as an SSD (Solid State Drive), a USB (Universal Serial Bus) memory, or a DVD-ROM (Digital versatile disc-Read Only Memory) is used. The observation control program 26 may be stored. In this case, the observation control program 26 of the storage medium 250 is installed in the microscope control apparatus 20, and the installed observation control program 26 is executed by the CPU 21.

　また、通信網（図示省略）を介して顕微鏡装置１に接続される他のコンピュータ又はサーバ装置等の記憶部に観察制御プログラム２６を記憶させておき、観察制御プログラム２６が顕微鏡装置本体１０の要求に応じてダウンロードされるようにしてもよい。この場合、ダウンロードされた観察制御プログラム２６はＣＰＵ２１によって実行される。
The observation control program 26 is stored in a storage unit such as another computer or server device connected to the microscope apparatus 1 via a communication network (not shown), and the observation control program 26 requests the microscope apparatus body 10. It may be downloaded according to. In this case, the downloaded observation control program 26 is executed by the CPU 21.

　また、上記各実施形態で説明した観察制御処理はあくまでも一例である。従って、主旨を逸脱しない範囲内において不要なステップを削除したり、新たなステップを追加したり、処理順序を入れ替えたりしてもよいことは言うまでもない。
In addition, the observation control process described in the above embodiments is merely an example. Therefore, it goes without saying that unnecessary steps may be deleted, new steps may be added, and the processing order may be changed within a range not departing from the spirit.

　本明細書に記載された全ての文献、特許出願及び技術規格は、個々の文献、特許出願及び技術規格が参照により取り込まれることが具体的かつ個々に記された場合と同程度に、本明細書中に参照により取り込まれる。
All documents, patent applications and technical standards mentioned in this specification are to the same extent as if each individual document, patent application and technical standard were specifically and individually stated to be incorporated by reference. Incorporated by reference in the book.

１　　　顕微鏡装置

１０　　顕微鏡装置本体

１１　　白色光源

１２　　コンデンサレンズ

１３　　スリット板

１４　　結像光学系

１４ａ　位相差レンズ

１４ｂ　対物レンズ

１４ｃ　位相板

１４ｄ　結像レンズ

１５　　動作部

１５Ａ　第１の動作部

１５Ｂ　第２の動作部

１５Ｃ　第３の動作部

１５Ｄ　第４の動作部

１５Ｅ　第５の動作部

１５Ｆ　第６の動作部

１６Ａ，１６Ｂ　　撮像部

１７　　水平方向駆動部

１８　　検出部

１８ａ　第１の変位センサ

１８ｂ　第２の変位センサ

１８ｃ　第３の変位センサ

１８ｄ　第４の変位センサ

１９　　光路分割部

２０　　顕微鏡制御装置

２２　　制御部

２３Ａ　取得部

２３Ｂ　処理部

２３Ｃ　選択部

２４　　焦点調節部

２５Ａ　一次記憶部

２５Ｂ　二次記憶部

２５０　記憶媒体

２６　　観察制御プログラム

２７　　位置情報

２８　　バスライン

２８Ａ　外部Ｉ／Ｆ３０　　表示装置

４０　　入力装置

５０　　培養容器

５１　　ステージ

５１ａ　開口

ｄ　　　距離

Ｓ　　　走査開始点

Ｅ　　　走査終了点

Ｌ　　　照明光

Ｍ　　　実線

Ｒ　　　観察領域
1 Microscope device

10 Microscope unit

11 White light source

12 condenser lens

13 Slit plate

14 Imaging optical system

14a phase difference lens

14b Objective lens

14c phase plate

14d imaging lens

15 Operation part

15A First operation unit

15B 2nd operation | movement part

15C 3rd operation part

15D 4th operation part

15E Fifth operating part

15F 6th operation part

16A, 16B Imaging unit

17 Horizontal drive

18 Detector

18a First displacement sensor

18b Second displacement sensor

18c Third displacement sensor

18d Fourth displacement sensor

19 Optical path divider

20 Microscope control device

22 Control unit

23A acquisition unit

23B processing section

23C selector

24 Focus adjustment unit

25A primary storage

25B Secondary storage unit

250 storage media

26 Observation control program

27 Location information

28 Bus line

28A External I / F30 display device

40 input devices

50 culture vessels

51 stages

51a opening

d distance

S Scan start point

E Scan end point

L Illumination light

M Solid line

R observation area

Claims (13)


  1. 　観察対象を収容する容器の底面の位置を示す位置情報を取得する取得部と、

    　各々に焦点面が異なる光学像を結像可能な複数の撮像部と、

    前記容器に収容された前記観察対象を示す光学像を結像させる結像光学系に対し、前記複数の撮像部の中の少なくとも１つの撮像部の位置及び姿勢の少なくとも一方を変更可能な少なくとも１つの動作部と、

    　前記取得部により取得された前記位置情報に基づいて、前記動作部を駆動して前記複数の撮像部の中の少なくとも一つの撮像部の前記位置及び前記姿勢の少なくとも一方を変更し、前記複数の撮像部の各々に、焦点面が異なる光学像を結像させる制御を行う制御部と、

    　を含む観察装置。
    An acquisition unit that acquires position information indicating the position of the bottom surface of the container that accommodates the observation target;

    A plurality of imaging units each capable of forming an optical image having a different focal plane;

    At least one that can change at least one of the position and orientation of at least one of the plurality of imaging units with respect to an imaging optical system that forms an optical image representing the observation object accommodated in the container. Two operating parts,

    Based on the position information acquired by the acquisition unit, the operation unit is driven to change at least one of the position and the posture of at least one of the plurality of imaging units, and the plurality of the plurality of imaging units A control unit that performs control to form optical images with different focal planes on each of the imaging units;

    An observation device.

  2. 　前記観察対象を透過した光の光路を複数の光路に分割する光路分割部を含み、

    　前記複数の撮像部の各々は、前記光路分割部により分割された複数の光路の各々に配置されている請求項１に記載の観察装置。
    An optical path dividing unit that divides an optical path of light transmitted through the observation object into a plurality of optical paths;

    The observation apparatus according to claim 1, wherein each of the plurality of imaging units is disposed in each of a plurality of optical paths divided by the optical path dividing unit.

  3. 　前記少なくとも１つの撮像部は、前記結像光学系に対する前記結像光学系の光軸上の位置が変更可能であり、

    　前記制御部は、前記取得部により取得された位置情報に基づいて、前記少なくとも１つの撮像部の前記光軸上の位置を変更する制御を行う請求項１又は２に記載の観察装置。
    The at least one imaging unit is capable of changing a position on the optical axis of the imaging optical system with respect to the imaging optical system,

    The observation apparatus according to claim 1, wherein the control unit performs control to change a position on the optical axis of the at least one imaging unit based on position information acquired by the acquisition unit.

  4. 　前記少なくとも１つの撮像部は、前記結像光学系に対する受光面の光軸上における傾きが変更可能であり、

    　前記制御部は、前記取得部により取得された位置情報に基づく前記底面の傾きに合わせて、前記少なくとも１つの撮像部の受光面の前記光軸上における傾きを変更する制御を行う請求項１又は２に記載の観察装置。
    The at least one imaging unit can change the inclination of the light receiving surface with respect to the imaging optical system on the optical axis,

    The control unit performs control to change an inclination of the light receiving surface of the at least one imaging unit on the optical axis in accordance with the inclination of the bottom surface based on the position information acquired by the acquisition unit. 2. The observation apparatus according to 2.

  5. 　前記少なくとも１つの撮像部は、前記結像光学系に対する前記結像光学系の光軸上の位置、及び前記結像光学系に対する受光面の光軸上における傾きが変更可能であり、

    　前記制御部は、前記取得部により取得された位置情報に基づいて、前記少なくとも１つの撮像部の前記光軸上の位置を変更する制御、及び前記取得部により取得された位置情報に基づく前記底面の傾きに合わせて、前記少なくとも１つの撮像部の受光面の前記光軸上における傾きを変更する制御を行う請求項１又は２に記載の観察装置。
    The at least one imaging unit can change a position on the optical axis of the imaging optical system with respect to the imaging optical system and an inclination of the light receiving surface with respect to the imaging optical system on the optical axis,

    The control unit is configured to control the position of the at least one imaging unit on the optical axis based on the position information acquired by the acquisition unit, and the bottom surface based on the position information acquired by the acquisition unit. The observation apparatus according to claim 1, wherein control is performed to change an inclination of the light receiving surface of the at least one imaging unit on the optical axis in accordance with the inclination of the image pickup unit.

  6. 　前記制御部は、前記取得部により取得された位置情報に基づく前記底面の高低差が予め定められた閾値よりも大きい場合に、前記少なくとも１つの撮像部の前記位置及び前記姿勢の少なくとも一方を変更する制御を行う請求項１から５のいずれか１項に記載の観察装置。
    The control unit changes at least one of the position and the posture of the at least one imaging unit when a difference in height of the bottom surface based on the position information acquired by the acquisition unit is larger than a predetermined threshold. The observation apparatus according to any one of claims 1 to 5, wherein control is performed.

  7. 　前記結像光学系及び前記撮像部と、前記容器との少なくとも一方を前記結像光学系の光軸に交差する特定の交差面において相対的に移動させる駆動部とをさらに含み、

    　前記取得部は、前記結像光学系及び前記撮像部と、前記容器との相対的移動に応じて前記結像光学系の観察領域が移動する方向に沿って、かつ前記結像光学系よりも先行する位置に対応する前記容器の底面の位置を示す位置情報を取得する請求項１から６のいずれか１項に記載の観察装置。
    A drive unit that relatively moves at least one of the imaging optical system and the imaging unit and the container on a specific intersection plane that intersects the optical axis of the imaging optical system;

    The acquisition unit is along a direction in which an observation region of the imaging optical system moves in accordance with relative movement of the imaging optical system, the imaging unit, and the container, and more than the imaging optical system. The observation apparatus according to claim 1, wherein position information indicating a position of a bottom surface of the container corresponding to a preceding position is acquired.

  8. 　前記駆動部が、前記結像光学系及び前記撮像部と、前記容器との少なくとも一方を、前記交差面において主走査方向及び前記主走査方向に直交する副走査方向に移動させ、

    　前記取得部は、先に取得した主走査方向における位置情報と、副走査方向に移動させた後の主走査方向における位置情報とに基づいて前記底面の形状情報を取得する請求項７に記載の観察装置。
    The drive unit moves at least one of the imaging optical system, the imaging unit, and the container in a main scanning direction and a sub-scanning direction orthogonal to the main scanning direction on the intersecting plane,

    The said acquisition part acquires the shape information of the said bottom face based on the positional information in the main scanning direction acquired previously, and the positional information in the main scanning direction after moving to a subscanning direction. Observation device.

  9. 　前記駆動部が、前記結像光学系及び前記撮像部と、前記容器との少なくとも一方を、前記交差面において主走査方向及び前記主走査方向に直交する副走査方向に移動し、

    　前記取得部が、前記主走査方向において前記結像光学系よりも先行する位置に対応する前記容器の底面を示す位置情報であって、かつ、前記副走査方向において異なる２つ以上の位置情報を取得する請求項７に記載の観察装置。
    The drive unit moves at least one of the imaging optical system, the imaging unit, and the container in a main scanning direction and a sub-scanning direction orthogonal to the main scanning direction on the intersecting plane,

    The acquisition unit is position information indicating a bottom surface of the container corresponding to a position preceding the imaging optical system in the main scanning direction, and two or more pieces of position information different in the sub-scanning direction. The observation apparatus according to claim 7 to be acquired.

  10. 　前記複数の撮像部によって取得した複数の画像をそれぞれ合成して１枚の画像を生成する画像処理部を備える請求項１から９のいずれか１項に記載の観察装置。
    The observation apparatus according to claim 1, further comprising an image processing unit that generates a single image by combining a plurality of images acquired by the plurality of imaging units.

  11. 　前記複数の撮像部によって取得した複数の画像から、適切な画像を選択する選択部を備える請求項１から９のいずれか１項に記載の観察装置。
    The observation device according to claim 1, further comprising a selection unit that selects an appropriate image from a plurality of images acquired by the plurality of imaging units.

  12. 　請求項１に記載の観察装置を動作させる観察装置の作動方法であって、

    前記観察対象を収容する容器の底面の位置を示す前記位置情報を前記取得部で取得し、

    前記取得部により取得された前記位置情報に基づき、前記動作部を駆動することにより、前記容器に収容された前記観察対象を示す光学像を結像させる前記結像光学系に対して、前記複数の撮像部の中の少なくとも１つの撮像部の前記位置及び前記姿勢の少なくとも一方を変更し、

    前記複数の撮像部の各々に、前記焦点面が異なる光学像を結像させる、観察装置の作動方法。
    An operation method of an observation apparatus for operating the observation apparatus according to claim 1,

    The position information indicating the position of the bottom surface of the container that accommodates the observation target is acquired by the acquisition unit,

    Based on the position information acquired by the acquisition unit, by driving the operation unit, the plurality of imaging optical systems that form an optical image indicating the observation object stored in the container Changing at least one of the position and the posture of at least one of the imaging units of

    An operation method of an observation apparatus, wherein an optical image having a different focal plane is formed on each of the plurality of imaging units.

  13. 　コンピュータを、

    　請求項１から請求項１１の何れか１項に記載の観察装置に含まれる前記取得部及び前記制御部として機能させるための観察制御プログラム。
    Computer

    The observation control program for functioning as the said acquisition part and the said control part which are contained in the observation apparatus of any one of Claims 1-11.
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