WO2019093186A1

WO2019093186A1 - Microscope device and program

Info

Publication number: WO2019093186A1
Application number: PCT/JP2018/040208
Authority: WO
Inventors: 佑介和多田
Original assignee: 富士フイルム株式会社
Priority date: 2017-11-08
Filing date: 2018-10-30
Publication date: 2019-05-16
Also published as: JPWO2019093186A1; JP6830548B2

Abstract

Provided are a microscope device and a program, capable of executing autofocus control whereby data volume for storing low-value image data can be economized when capturing a wide-field image at high magnification of an observation object accommodated in a container having significant unevenness in a bottom surface thereof. Data volume consumed by data of a non-focused image is economized by subjecting data of a captured non-focused image to high compression and storing the data as a small amount of data in the case of a region in which an objective lens 14b of an imaging optical system 14 cannot be conveyed to a focal position in the optical axis direction and only a non-focused image is obtained as a result of image capture.

Description

Microscope apparatus and program

The technology of the present disclosure relates to a microscope apparatus and a program.

Conventionally, pluripotent stem cells such as ES (Embryonic Stem) cells and iPS (Induced Pluripotent Stem) cells and cells induced to differentiate are imaged with a microscope or the like, and the features of the image are captured to identify the differentiation state of the cells, etc. A method of determining 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 noted as being applicable in regenerative medicine, drug development, disease elucidation, etc. ing.

On the other hand, although high magnification images can be obtained by imaging with a microscope, the observation region of the imaging optical system of the microscope is limited to a narrow range. Therefore, it has been proposed to perform so-called tiling imaging in order to obtain a high-magnification, wide-field image of the observation target. The tiling imaging is an imaging method of imaging a plurality of narrow-field images and concatenating the plurality of imaged narrow-field images to generate a wide-field image. Specifically, the entire observation object is scanned by moving the imaging optical system of a microscope and a stage on which a culture vessel such as a well plate containing the observation object is installed two-dimensionally. By concatenating the images obtained for each, a wide field of view image can be generated that includes the entire observation object.

When a large number of observation objects are scanned in a short time and a large number of wide-field images including the entire observation object are generated, a large amount of data capacity is required. In view of the limitation on the total amount of data that can be stored in the storage device, it is not desirable to waste memory capacity on unfocused image data, which is an unfocused image.

In the microscope apparatus of Patent Document 1, the imaging condition is not normally set for the region where the imaging condition is not normally set in the process of scanning (a region where it is predicted that a non-focused image will be imaged) It is disclosed that, when displaying the wide-field image by connecting the images of the respective areas to display the wide-field image, highlight the area where the abnormal information is set.

JP, 2013-50594, A

However, it is difficult to save the memory capacity consumed for the data of the out-of-focus image by the technique according to Patent Document 1.

In one aspect, the technology of the present disclosure provides a microscope apparatus and program that can save the memory capacity consumed for data of an out-of-focus image in view of the above-described problems. In another aspect, the technology of the present disclosure provides a microscope apparatus and a program capable of suppressing vibration or the like of an imaging optical system by not performing unreasonable focus control when appropriate focus control is not possible. .

According to a first aspect of the present disclosure, an imaging optical system capable of imaging an observation target light indicating an observation target in a container containing an observation target on an imaging element is driven by being driven within a drivable range. By moving the imaging optical system in the optical axis direction, moving the imaging optical system in the plane intersecting the optical axis direction of at least one of the stage on which the container is installed and the imaging optical system, The imaging optical system is focused on a specific area before the optical axis reaches the specific area of each area in a state where the imaging device scans each area in the container by moving the area relative to the area. And a control unit configured to execute an imaging data saving process when the specific region of the optical axis reaches when the driving amount of the driving member at the time of moving to the position exceeds the drivable range.

According to the first aspect, when scanning the observation target, it is possible to save the memory capacity consumed for the data of the out-of-focus image captured when the imaging optical system is not at the in-focus position.

According to a second aspect of the present disclosure, in the first aspect, the imaging data saving process compresses the image data generated by imaging the observation target light having passed through the specific area on the imaging element higher than a predetermined amount A microscope apparatus that includes compression at a rate and storage.

According to the second aspect, the non-focused image data is consumed by compressing and storing the non-focused image data at a higher compression rate than the compression rate (predetermined amount) of the focused image data. Save memory space.

A third aspect of the present disclosure is the microscope apparatus according to the first aspect, wherein the imaging data saving process includes avoiding imaging of a specific area by the imaging optical system.

According to the third aspect, since the data of the out-of-focus image is not captured and is not stored in the memory of the storage device as a result, it is possible to save the memory capacity consumed for the data of the out-of-focus image.

A fourth aspect of the present disclosure is the microscope apparatus according to any one of the first to third aspects, wherein the drive member is a piezoelectric element that is deformable along the optical axis direction of the imaging optical system.

According to the fourth aspect, by using the piezoelectric element that can be deformed at high speed as the optical axis direction drive member, it is possible to execute autofocus control that appropriately follows the scan performed at high speed.

According to a fifth aspect of the present disclosure, in any of the first to fourth aspects, the fifth aspect further includes a detection unit that detects the position of the specific area in the optical axis direction, and the in-focus position of the specific area is detected by the detection section. It is a microscope apparatus calculated based on the position in the optical axis direction.

According to the fifth aspect, the detection result of the detection unit can be used to predict an area where an out-of-focus image is captured in the process of scanning.

According to a sixth aspect of the present disclosure, in the fifth aspect, the detection units are provided side by side across the imaging optical system in the main scanning direction with respect to each area, and each detect the position of the specific area in the optical axis direction It is a microscope apparatus which has a pair of sensors.

According to the sixth aspect, the detection unit using a pair of sensors arranged in the scanning direction can predict an area where an out-of-focus image is captured in the process of scanning according to the scanning direction.

According to a seventh aspect of the present disclosure, in any of the first to sixth aspects, the imaging optical system includes an objective lens movable in the optical axis direction, and the drive member moves the objective lens in the optical axis direction. It is a microscope device.

According to the seventh aspect, the objective lens is moved in the optical axis direction by the drive member, whereby the autofocus control is performed.

An eighth aspect of the present disclosure is the microscope apparatus according to the first to seventh aspects, wherein the container is a well plate having a plurality of wells.

According to the eighth aspect, it is possible to save the memory capacity consumed for the out-of-focus image data when scanning the observation target stored in the well plate in which the bottom surface variation easily occurs.

A ninth aspect of the present disclosure is a program for causing a computer to function as a control unit included in the microscope apparatus according to the first to eighth aspects.

According to a tenth aspect of the present disclosure, an imaging optical system capable of imaging an observation target light indicating an observation target in a container containing the observation target on an imaging element is driven by driving within a drivable range. By moving the imaging optical system in the optical axis direction, moving the imaging optical system in the plane intersecting the optical axis direction of at least one of the stage on which the container is installed and the imaging optical system, The imaging optical system is focused on a specific area before the optical axis reaches the specific area of each area in a state where the imaging device scans each area in the container by moving the area relative to the area. And a controller configured to cause the drive member to execute focus control stop processing when the drive amount of the drive member at the time of moving to the position exceeds the drivable range when the optical axis reaches a specific region.

According to the tenth aspect, vibration or the like of the imaging optical system can be suppressed by not performing excessive focus control when appropriate focus control is impossible.

In an eleventh aspect of the present disclosure, in the tenth aspect, the focus control stop processing includes moving the drive member in the optical axis direction before the optical axis reaches the specific area or after the optical axis reaches the specific area. A microscope apparatus, including stopping the

According to the eleventh aspect, in the case where appropriate focus control is impossible, movement of the drive member in the optical axis direction is stopped and kept at the current position, thereby avoiding unreasonable focus control and forming an imaging optical system. Vibration of the system can be suppressed.

According to a twelfth aspect of the present disclosure, in the tenth aspect, the focus control stopping process is performed before the optical axis reaches the specific area or after the optical axis reaches the specific area, the reference position of the drive member in the optical axis direction. A microscope apparatus, including moving it to.

According to the twelfth aspect, when appropriate focus control is not possible, focus control is reset by returning the drive member to the reference position in the optical axis direction, and a new shape corresponding to the shape of the bottom surface of the container is provided. Focus control can be started.

A thirteenth aspect of the present disclosure is a program for causing a computer to function as a control unit included in the microscope apparatus according to the tenth to twelfth aspects.

According to the thirteenth aspect, it is possible to provide a program capable of suppressing the vibration or the like of the imaging optical system by avoiding impossible focus control when appropriate focus control is impossible.

According to one aspect of the present disclosure, it is possible to save a memory capacity consumed for data of an out-of-focus image. Further, according to another aspect of the present disclosure, it is possible to suppress vibration or the like of the imaging optical system by not performing excessive focus control when appropriate focus control is impossible.

It is a schematic block diagram which shows an example of a structure of the microscope apparatus which concerns on 1st Embodiment. It is a schematic diagram which shows an example of a structure of the imaging optical system contained in the microscope apparatus which concerns on 1st Embodiment. It is a schematic block diagram which shows an example of a structure of the piezoelectric element contained in the microscope apparatus which concerns on 1st Embodiment. It is a schematic block diagram which shows an example of a structure of the stage contained in the microscope apparatus which concerns on 1st Embodiment. It is a block diagram showing an example of the hardware constitutions of the electric system of the microscope device concerning a 1st embodiment. It is a block diagram showing an important section composition of a portion about a technique of this indication of a microscope device main part and a microscope control device contained in a microscope device concerning a 1st embodiment. It is a conceptual diagram which shows an example of the scanning position of the observation object area | region in the culture container installed in the stage of the microscope apparatus which concerns on 1st Embodiment. The positional relationship between the imaging optical system, the first and second displacement sensors, and the culture vessel when there is an observation target area at an arbitrary position in the culture vessel installed on the stage of the microscope apparatus according to the first embodiment It is a conceptual diagram which shows the 1st example of a form. The positional relationship between the imaging optical system, the first and second displacement sensors, and the culture vessel when there is an observation target area at an arbitrary position in the culture vessel installed on the stage of the microscope apparatus according to the first embodiment It is a conceptual diagram which shows the 2nd example of a form. It is a state transition diagram for demonstrating an example of the timing of the autofocus control in the microscope apparatus which concerns on 1st Embodiment. It is a flowchart which shows an example of the flow of the wide view image acquisition process which concerns on 1st Embodiment. It is a flowchart which shows an example of the flow of the stage movement process which concerns on 1st Embodiment. It is a conceptual diagram which shows an example of the relationship between an observation object area | region and the distance of a Z direction. It is a flow chart which shows an example of a flow of focus control preparation processing concerning a 1st embodiment. It is a flow chart which shows an example of a flow of focus control processing concerning a 1st embodiment. It is a schematic block diagram which shows an example of the voltage table which concerns on 1st Embodiment. It is a flow chart which shows an example of a flow of focus control processing concerning a 2nd embodiment. It is a schematic block diagram which shows an example of a structure of the microscope apparatus which concerns on a modification. It is a schematic block diagram which shows an example of a structure of the detection part contained in the microscope apparatus which concerns on a modification. It is a figure where it uses for explanation of change of a position of a displacement sensor in a detection part contained in a microscope device concerning a modification. It is a conceptual diagram which shows an example of the aspect by which a wide-field image acquisition program is installed in a microscope apparatus from the storage medium with which the wide-field image acquisition program concerning 1st and 2nd embodiment was stored.

Hereinafter, details of each embodiment of the present disclosure will be described with reference to the drawings.

First Embodiment
<Overview of system>
Hereinafter, a microscope apparatus 90 according to a first embodiment of the technology of the present disclosure will be described in detail with reference to the drawings. FIG. 1 is a block diagram showing a schematic configuration of the microscope apparatus 90 according to the first embodiment.

The microscope apparatus 90 includes a microscope apparatus body 10 and a microscope controller 20. The microscope device 90 may be connected to the display device 30 and the input device 40 via the microscope control device 20.

The microscope apparatus 90 is an example of the microscope apparatus of the present disclosure.

The microscope apparatus main body 10 captures a phase-contrast image of cultured cells to be observed by the microscope apparatus 90. Specifically, as shown in FIG. 1 as an example, the microscope apparatus body 10 includes a white light source 11 for emitting white light, a condenser lens 12, a slit plate 13, an imaging optical system 14, and a piezoelectric element 15. , An imaging element 16, and a detection unit 18.

Image sensor 16 is an example of an image sensor of this indication.

The slit plate 13 is provided with a ring-shaped slit for transmitting white light to a light shielding plate for shielding white light emitted from the white light source 11, and the white light passes through the slit to form a ring shape. Illumination light L is formed.

As shown in FIG. 2 as an example, the imaging optical system 14 includes a phase difference lens 14 a and an imaging lens 14 d. The phase difference lens 14a includes an objective lens 14b and a phase plate 14c. The phase plate 14 c has a phase ring formed on a transparent plate transparent to the wavelength of the illumination light L. The size of the slit of the slit plate 13 described above is in a conjugate relationship with the phase ring of the phase plate 14 c.

In the phase ring, a phase film for shifting the phase of the incident light by 1⁄4 wavelength and a light reducing filter for reducing the incident light are formed in a ring shape. The direct light incident on the phase ring has its phase shifted by 1⁄4 wavelength by passing through the phase ring, and its brightness is weakened. On the other hand, most of the diffracted light diffracted by the object of observation 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 piezoelectric element 15 shown in FIG. 1 as an example. In the first embodiment, the optical axis direction of the objective lens 14b and the Z direction (vertical direction) are the same direction. Auto focus control is performed by moving the phase difference lens 14 a to be in focus in the Z direction, and the contrast of the phase difference image captured by the image sensor 16 is adjusted.

Here, in the first embodiment, the Z-direction position of the bottom surface of the culture vessel 50 described later installed on the stage 51 described later is detected in advance and set as a reference surface. Then, in the imaging optical system 14, the reference position in the Z direction is set so as to be at the in-focus position with respect to the reference plane.

If the bottom surface of the culture vessel 50 containing the observation target described later, that is, the boundary surface between the bottom portion of the culture vessel 50 and the observation target coincides with the reference surface, the imaging optical system 14 placed at the reference position Should be in focus. However, the bottom surface of the culture vessel 50 is close to the reference surface but does not completely match. This is because the bottom of the culture vessel 50 has variations due to manufacturing errors and the like. That is, the actual in-focus position with respect to each area of the bottom of the culture container 50 often does not coincide with the reference position.

In the autofocus control in the present disclosure, the imaging optical system 14 is moved in the Z direction to correct the deviation between each area of the bottom surface of the culture vessel 50 and the reference surface, and the imaging optical system 14 is an observation target in each area. Control to be in the actual in-focus position.

Further, the magnification of the phase difference lens 14a may be changed. 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 may be performed manually by the user.

As shown in FIG. 3 as an example, the piezoelectric element 15 holds the objective lens 14b of the imaging optical system 14 and functions as a Z-direction transport device for moving the objective lens 14b in the Z direction.

The piezoelectric element 15 is an example of the drive member of the present disclosure.

The piezoelectric element 15 as the Z-direction transport device has the following differences in characteristics. The piezoelectric element 15 can move the objective lens 14b of the imaging optical system 14 in the Z direction at high speed. On the other hand, the piezoelectric element 15 has a limited drivable range.

The piezoelectric element 15 is configured to pass the phase difference light that has passed through the phase difference lens 14 a as it is.

Further, the Z-direction transport device of the objective lens 14b is not limited to the piezoelectric element 15, and may move in the Z direction at a sufficiently high speed following the scanning.

The imaging lens 14 d receives the phase difference light having passed through the phase difference lens 14 a and the piezoelectric element 15, and forms an image on the image pickup element 16.

The imaging device 16 captures a phase difference image formed by the imaging lens 14d. As the imaging device 16, a CCD (Charge-Coupled Device) image sensor, a CMOS (Complementary Metal-Oxide Semiconductor) image sensor, or the like is used. As an imaging device, an imaging device provided with a RGB (Red Green Blue) color filter may be used, or a monochrome imaging device may be used.

The detection unit 18 detects the position in the Z direction (vertical direction) of the culture container 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 in the first embodiment are laser displacement meters, and the culture vessel 50 is irradiated with laser light, and the reflected light is detected to detect the Z direction of the bottom surface of the culture vessel 50. To detect the position of In the present disclosure, the bottom surface of the culture container 50 refers to the interface between the bottom of the culture container 50 and the cell to be observed, that is, the observation target installation surface.

The culture container 50 is an example of the container in the disclosure.

In the first embodiment, 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.

As an example, each of the first displacement sensor 18a and the second displacement sensor 18b is disposed so as to be separated from the phase difference lens 14a by a distance of 9 times the side D of the observation target region R which is a square, that is, 9D. There is. The observation target region R will be described later.

Positional information on the Z direction of the culture container 50 detected by the detection unit 18 is output to an imaging control unit 21 described later, and the imaging control unit 21 controls the piezoelectric element 15 based on the input positional information, Perform auto focus control. The detection of the position of the culture container 50 by the first displacement sensor 18a and the second displacement sensor 18b and the autofocus control by the imaging control unit 21 will be described in detail later.

A stage 51 is provided between the slit plate 13 and the phase difference lens 14 a and the detection unit 18. On the stage 51, a culture container 50 containing cells to be observed is installed.

In the microscope apparatus according to the first embodiment, a well plate having six wells W is used as the culture vessel 50 as shown in FIG. 7 as an example. However, the culture container 50 is not limited to this. For example, a well plate having 24 or 96 wells can be used as the culture vessel 50, or any well plate having any number of wells, or a vessel other than a well plate such as a petri dish or dish can be used. It is. An appropriate container can be selected as the culture container 50 depending on the observation target and the purpose of the observation.

Moreover, as cells accommodated in the culture vessel 50, pluripotent stem cells such as iPS cells and ES cells, nerves induced to differentiate from stem cells, cells of skin, myocardium and liver, skins removed from human body, retina, These include myocardium, blood cells, nerve and organ cells.

The stage 51 is moved in an X direction and a Y direction orthogonal to each other by a stage driving device 17 described later. 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 first embodiment, the X direction is a main scanning direction, and the Y direction is a sub scanning direction.

As shown in FIG. 4 as an example, a rectangular opening 51 a is formed at the center of the stage 51. The culture container 50 is placed on a member forming the opening 51a, and the phase difference light of the cells in the culture container 50 passes through the opening 51a.

The display device 30 displays the composite phase difference image generated and stored by the wide-field image acquisition processing described later, and includes, for example, a liquid crystal display. Further, the display device 30 may be configured by a touch panel and used as the input device 40.

The input device 40 includes a mouse, a keyboard, and the like, and receives various setting inputs from the user. The input device 40 receives setting inputs such as, for example, an instruction to change the magnification of the phase difference lens 14 a and an instruction to change the moving speed of the stage.

Next, the configuration of the microscope control device 20 that controls the microscope apparatus main body 10 will be described. FIG. 5 shows an example of the configuration of the electrical system of the microscope control device 20.

The microscope control device 20 includes a computer. The computer of the microscope control device 20 includes a central processing unit (CPU) 70, a primary storage unit 72, a secondary storage unit 74, an input / output interface (I / O) 76, and the like. The CPU 70, the primary storage unit 72, the secondary storage unit 74, and the I / O 76 are connected by a bus line.

The CPU 70 controls the entire microscope apparatus. The primary storage unit 72 is a volatile memory used as a work area or the like when executing various programs. An example of the primary storage unit 72 is a RAM (Random Access Memory). The secondary storage unit 74 is a non-volatile memory in which various programs, various parameters, and the like are stored in advance, and stores a wide view image acquisition program 80 which is an example of a program according to the technology of the present disclosure. Examples of the secondary storage unit 74 include an EEPROM (Electrically Erasable Programmable Read-Only Memory), a flash memory, and the like. The I / O 76 controls transmission and reception of various information between the microscope apparatus main body 10 and the microscope control apparatus 20.

The CPU 70 reads the wide field of view image acquisition program 80 from the secondary storage unit 74. Then, the CPU 70 expands the read wide-field image acquisition program 80 in the primary storage unit 72, and executes the expanded wide-field image acquisition program 80, whereby the imaging control unit 21 and the stage control unit shown in FIG. Acts as 22.

The stage control unit 22 moves the stage 51 in the X direction and the Y direction by controlling the stage driving device 17. The stage drive device 17 is, for example, an actuator having a piezoelectric element or the like.

The imaging control unit 21 performs control (autofocus control) on the piezoelectric element 15 based on the position information of the culture container 50 in the Z direction detected by the detection unit 18. The autofocus control is realized by moving the objective lens 14b of the imaging optical system 14 in the optical axis direction by driving the piezoelectric element 15.

The imaging control unit 21 is an example of a control unit according to the technology of the present disclosure.

<Wide-field image acquisition processing>
Hereinafter, the process of wide-field image acquisition by the microscope apparatus 90 according to the first embodiment of the present disclosure will be described in detail.

10, 11, 13, and 15 are flowcharts showing an example of a program for performing wide-field image acquisition processing in the first embodiment. 10, 11, 13, and 15 show an example of a program executed by the microscope control device 20.

Here, the program executed by the microscope control device 20 is specifically executed by the CPU 70 of the microscope control device 20 functioning as the imaging control unit 21 and the stage control unit 22.

In the following, for convenience of explanation, the wide-field-of-view image acquisition processing is divided into three parts of (1) start of flow, (2) scanning processing, and (3) processing after scanning processing.

Start Flow
In the microscope apparatus 90 according to the first embodiment, the microscope apparatus body 10 is disposed on the stage 51 while the microscope control device 20 performs two-dimensional movement control on the stage 51 and autofocus control on the imaging optical system 14. By continuously capturing the narrow-field image of the culture vessel 50 thus processed, a scan on the observation target is performed.

A user who desires imaging of a wide-field image of an observation target first places the culture vessel 50 containing the cells to be observed on the stage 51.

As described above, in the first embodiment, the culture vessel 50 is described as a well plate having six wells W as an example.

When the user instructs the microscope control device 20 to capture a wide-field image through the input device 40, the wide-field image acquisition process according to the first embodiment is started. Thereafter, the CPU 70 of the microscope control device 20 reads the wide field of view image acquisition program 80 from the secondary storage unit 74 and executes the process. FIG. 10 is a flowchart showing the entire wide-field image acquisition process.

[Scan process]
At step S100, the CPU 70 executes a scanning process. In step S100, stage movement processing (see FIG. 11), focus control preparation processing (see FIG. 13), and focus control processing (see FIG. 14) are performed.

Stage movement processing, focus control preparation processing, and focus control processing are performed in synchronization with each other. The processing will be described below on the premise of this synchronous control.

(Stage movement processing)
The stage moving process included in step S100 of FIG. 10 is shown as a subroutine in FIG. FIG. 11 is an example of a processing program executed by the CPU 70 of the microscope control device 20 functioning as the stage control unit 22.

In step S 202, the stage control unit 22 performs initial setting of the X-axis movement direction with respect to the stage 51. As one example, the X-axis movement direction of the stage 51 is set to a negative direction.

Next, in step S204, the stage control unit 22 causes the stage drive device 17 to start the movement of the stage 51 along the set X-axis movement direction. Immediately after the start of the subroutine of FIG. 11, since the X-axis movement direction is set to the negative direction in step S202, the stage 51 starts moving in the negative direction of the X-axis. In this case, as shown in FIG. 8A, the imaging optical system 14 of the stationary microscope apparatus main body 10 moves relative to the stage 51 in the positive direction of the X axis. In the following, relative movement of the imaging optical system 14 (and the observation target area R) relative to the stage 51 with the movement of the stage 51 by the stage drive device 17 is for convenience of the imaging optical system 14 (and the observation target area R). Described as movement.

8A and 8B show the imaging optical system 14, the first displacement sensor 18a and the second displacement sensor 18b, and the culture vessel 50 in the case where the observation target region R exists at an arbitrary position on the bottom surface of the culture vessel 50. It is the figure which showed the positional relationship on XY plane. The observation target region R is a region on the bottom surface of the culture vessel 50 where the microscope device body 10 can perform imaging and generate a phase difference image. The observation target region R also moves relative to the stage 51 as the imaging optical system 14 moves relative to the stage 51. Hereinafter, the traveling direction of the imaging optical system 14 and the observation target region R on the plane including the bottom surface of the culture container 50 along with the movement of the stage 51 in the X direction is referred to as an X-axis scanning direction. Furthermore, the XY plane including the bottom of the culture vessel 50 is referred to as a scan plane.

In the following, for convenience of explanation, the observation target region R is assumed to be a square region, but it is not limited to this.

The microscope apparatus body 10 is provided with a horizontal position detection sensor not shown in FIG. The stage control unit 22 detects the position on the scanning plane at the present time of the observation target region R on the stage 51 using the horizontal position detection sensor.

In the first embodiment, the observation target region R moves at a constant speed along the X-axis scanning direction.

When the observation target region R of the imaging optical system 14 reaches the end point position on the scanning plane, in step S206, the determination as to whether the observation target region R has reached the end point position is an affirmative determination. Here, the end point position is a position at which the scanning in the X-axis direction ends in the scanning plane, and is illustrated in FIG.

On the other hand, the stage control unit 22 continues the movement of the stage 51 along the X-axis movement direction by the stage drive device 17 until the determination in step S206 is affirmative. That is, the imaging optical system 14 continues to move along the X-axis scanning direction relative to the stage 51 until the determination in step S206 is affirmative.

In the process of moving the observation target region R in the X-axis scanning direction, the CPU 70 of the microscope control device 20 controls the microscope apparatus main body 10 by performing imaging control processing described later in synchronization with stage movement processing. A region overlapping the observation target region R is photographed at the bottom of the culture container 50, and a plurality of phase difference images are generated. That is, a phase difference image of each region on the bottom surface of the culture container 50 which is continuous along the X-axis scanning direction is generated.

By performing imaging control processing described later, the objective lens 14b of the imaging optical system 14 has its Z-direction position focused in each area on the bottom surface of the culture vessel 50 superimposed on the observation target area R. Note that it is set to position. Therefore, in the process of relative movement of the observation target region R toward the X-axis scanning direction, a phase difference image in a focused state is generated for each region on the bottom surface of the culture vessel 50 continuous along the X-axis scanning direction. Ru.

However, as will be described in detail in the description of the imaging control process described later, there are cases where the piezoelectric element 15 can not convey the objective lens 14 b to the in-focus position. If imaging is performed in this case, an out-of-focus phase difference image is generated.

Furthermore, the CPU 70 of the microscope control device 20 stores the generated plurality of phase difference images in the primary storage unit 72 as an example. The generated plurality of phase difference images may be stored in a cache memory (not shown) of the CPU 70 or the secondary storage unit 74.

If the determination in step S204 is affirmative, in step S208, the stage control unit 22 ends the movement of the stage 51 along the X-axis scanning direction by the stage drive device 17. Furthermore, the CPU 70 of the microscope control device 20 ends continuous imaging of the narrow-field image by the microscope device body 10. Next, the process proceeds to step S210.

In step S210, the stage control unit 22 determines whether the observation target region R has reached the scan end point E. The scanning end point E is a point at which the scanning process is ended in the scanning plane, and is exemplarily shown in FIG.

If the determination in step S210 is negative, the process proceeds to step S212.

In step S212, the stage control unit 22 causes the stage drive device 17 to move the stage 51 by one unit in the negative direction of the X axis. Here, one unit is a distance corresponding to the length D of one side of the observation target region R. In this regard, reference is made to FIG. Therefore, on the bottom surface of the culture container 50, the observation target region R moves by one unit in the positive direction of the Y axis. Next, the process proceeds to step S214.

In step S214, the stage control unit 22 reverses the X-axis movement direction with respect to the stage 51. Thereby, the X-axis scanning direction of the observation target area R is reversed. Then, the process of FIG. 11 returns to step S204, and the stage control unit 22 starts X-axis scanning again, and the CPU 70 of the microscope control device 20 resumes continuous imaging of the narrow-field image by the microscope device body 10.

For example, when step S214 is executed for the first time in the process of FIG. 11, the X-axis movement direction with respect to the stage 51 is set to a negative direction in step S202. It is set in the positive direction. In this case, when the process of FIG. 11 returns to step S204, the stage 51 starts moving in the positive direction of the X axis. Therefore, in this case, as shown in FIG. 8B, the imaging optical system 14 (and the observation target area R) of the stationary microscope apparatus main body 10 is in the negative direction of the X axis relative to the stage 51. It will move against.

The process of FIG. 11 is continued until the determination of step S210 is affirmative.

As described above, when the stage control unit 22 executes the stage movement processing subroutine, the stage 51 is moved in the X and Y directions, and the observation target region R of the imaging optical system 14 is the bottom surface of the culture vessel 50 Scanning in a two-dimensional manner, a phase difference image of each area is generated and stored.

The solid line M in FIG. 7 shows an example of the movement of the observation target region R on the scanning plane including the bottom surface of the culture container 50 in the scanning process.

As shown in FIG. 7, the observation target region R of the imaging optical system 14 moves along the solid line M from the scanning start point S to the scanning end point E. That is, after scanning in the positive direction of the X axis (right direction in FIG. 7), the observation target region R moves in the positive direction of the Y axis (downward direction in FIG. 7), and further moves in the negative direction of X axis (FIG. 7). Scan left). Then, the region R to be observed moves again in the positive direction of the Y axis, and scans in the positive direction of the X axis again. As described above, the observation target region R scans the bottom of the culture container 50 two-dimensionally by repeatedly performing reciprocating movement in the X direction and movement in the Y direction.

The following points should be noted regarding the setting of the end point position and the stage moving speed in the stage moving process.

In the first embodiment, in order to execute scanning on the entire bottom surface of the culture vessel 50, as shown in FIG. 7, imaging optics up to the ranges R1 and R2 outside the range of the culture vessel 50 in the X direction It is necessary to move the system 14, the first displacement sensor 18a and the second displacement sensor 18b relatively. Then, it is necessary to secure at least a distance between the first displacement sensor 18a and the imaging optical system 14 in the X direction as the width in the X direction of the range R1, and at least a second displacement sensor as the width in the X direction of the range R2. It is necessary to secure a distance between the lens 18 b and the imaging optical system 14 in the X direction. And in order to shorten the scanning time of observation object area R as much as possible, it is desirable to make the scanning range of observation object area R as narrow as possible. Therefore, the width of the range R1 in the X direction is preferably the distance between the first displacement sensor 18a and the imaging optical system 14 in the X direction, and the width of the range R2 in the X direction is smaller than that of the second displacement sensor 18b. It is desirable to set the distance in the X direction to the image optical system 14.

On the other hand, when the region R to be observed is scanned within the range of the culture container 50 by moving the stage 51 in the X direction, it is desirable that the moving speed of the region R to be observed within the range of the culture container 50 be constant. Therefore, at the start of movement of the stage 51 in the X direction, it is necessary to accelerate the stage 51 to a constant speed, and at the end of movement of the stage 51 in the X direction, the stage 51 is decelerated from a constant speed and stopped. There is a need.

In addition, when the moving speed of the stage 51 in the X direction is constant, it is possible to rapidly control the speed to a constant speed with almost no acceleration region, but when such control is performed, The liquid level of the culture solution or the like contained in the culture vessel 50 together with the cells may shake, which may lead to the deterioration of the image quality of the phase difference image. In addition, the same problem may occur when stopping the stage 51.

Therefore, in the first embodiment, the range R1 and the range R2 shown in FIG. 5 are set in the acceleration / deceleration region of the movement of the stage 51 in the X direction. Thus, by setting the acceleration / deceleration regions on both sides in the X direction of the range of the culture vessel 50, the region R to be observed is scanned at a constant speed in the range of the culture vessel 50 without wasting the scanning range. be able to. Furthermore, the fluctuation of the liquid level of the culture solution as described above can also be suppressed.

(Imaging control processing)
The imaging control process included in step S100 of FIG. 10 is shown in FIG. 13 and FIG. 14 as a subroutine.

As described above, in the first 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 as shown in FIGS. 8A and 8B. . Therefore, when scanning each area on the bottom surface of the culture vessel 50 in which the observation target area R of the imaging optical system 14 continues along the X direction in the stage movement processing described above, the detection unit 18 selects the observation target area R The position in the Z direction of the bottom surface of the culture vessel 50 can be detected with respect to a region located forward in the X-axis scanning direction than the position of.

Specifically, when the observation target area R moves in the direction of the arrow shown in FIG. 8A (the right direction in FIG. 8A), the observation target area R of the first displacement sensor 18a and the second displacement sensor 18b. The first displacement sensor 18a located forward in the X-axis scanning direction detects the position in the Z direction of the bottom surface of the culture vessel 50. Then, when the observation target region R further moves in the right direction in FIG. 8A, the position in the Z direction of the bottom surface of the culture container 50 detected by the first displacement sensor 18a and the reference surface height of the culture container 50 detected in advance. By performing focus control based on parameters including the distance in the Z direction position of the objective lens 14b of the imaging optical system 14 in each region on the bottom surface of the culture vessel 50 along the X axis scanning direction. It can be adjusted. That is, by performing focus control, when the observation target area R moves in the right direction of FIG. 7, each area is kept in focus while keeping the imaging optical system 14 in focus with respect to the bottom surface of the culture vessel 50. It can be taken.

On the other hand, when the observation target area R is moving in the arrow direction of FIG. 8B (left direction in FIG. 8B), the observation target area R of the first displacement sensor 18a and the second displacement sensor 18b. The second displacement sensor 18 b located forward in the X-axis scanning direction detects the position of the bottom surface of the culture vessel 50 in the Z direction. Then, as described with reference to FIG. 8A, by performing focus control, the imaging optical system 14 is focused on the bottom surface of the culture vessel 50 when the observation target region R moves in the left direction of FIG. 8B. Each region can be photographed while keeping the

As described above, the Z direction position detection of the bottom surface of the culture container 50 using the first displacement sensor 18 a and the Z direction position detection of the bottom surface of the culture container 50 using the second displacement sensor 18 b By switching in accordance with the above, it is possible to always obtain positional information of the culture container 50 in the Z direction at the position of the observation target region R prior to imaging of the observation target region R.

The imaging control unit 21 performs focus control by adjusting the Z-direction position of the objective lens 14 b of the imaging optical system 14 using the piezoelectric element 15. Specifically, the imaging control unit 21 performs focus control by appropriately changing the amount of voltage applied to the piezoelectric element 15.

However, since the piezoelectric element 15 has a limit in the drivable range, a situation may occur where the objective lens 14 b can not be moved by the distance necessary for focus control.

This point will be described with reference to FIG. For convenience, in the following description, it is assumed that the observation target region R moves in the positive direction of the X-axis as shown in FIG. 8A.

For convenience of explanation, it is considered to divide the X-axis direction of the bottom surface of the culture vessel 50 by one side D of the region R to be observed. When the observation target area R is in the I-th area, the observation planned area is arranged in the (I + 10) -th area. That is, the region to be observed is located 10 units ahead of the region to be observed R in the X-axis scanning direction. FIG. 9 is a diagram showing the above relationship.

Here, the observation planned area is an area on the bottom of the culture vessel 50 located vertically above the first displacement sensor 18a, and is an area where the observation target area R is overlapped after a predetermined time has elapsed. As described above, as an example, the first displacement sensor 18a is disposed so as to be separated from the phase difference lens 14a in the X direction by a distance nine times the side D of the region R to be observed.

The region to be observed is an example of the specific region of the present disclosure.

FIG. 12 schematically shows the in-focus position of the objective lens 14b of the imaging optical system 14 for each of the I-th to I + 10-th regions shown in FIG. In FIG. 12, the observation target region R is located in the I-th region on the current scan plane, and the observation scheduled region is the I + 10-th region.

In FIG. 12, the lower broken line shows the height corresponding to the reference plane of the bottom surface of the culture vessel 50 (the Z direction position of the objective lens 14b to be in focus with respect to the reference plane), and the central broken line shows the objective lens 14b. The current Z direction position is shown, and the upper broken line represents the upper limit of the drivable range of the piezoelectric element 15. The horizontal bars indicate the in-focus positions of the I-th to I + 10-th regions. In the preceding scan, the Z-direction position of the bottom surface of the culture vessel 50 has already been detected by the first displacement sensor 18a for each of the I-th to I + 10-th regions. Then, the in-focus position in each area can be derived by subtracting the Z-direction position of each area detected from the Z-direction position of the reference surface of the culture vessel 50.

With the stage movement process being synchronized, the observation target area R is the I + 10th area in the X-axis scanning direction on the scanning plane, such as the Ith area to the I + 1th area and the I + 2th area. That is, it moves to the observation scheduled area). The imaging control unit 21 controls the piezoelectric element 15 to align the Z-direction position of the objective lens 14 b with each focusing position in each region.

However, in the situation shown in FIG. 12, the distance between the (I + 10) th region (that is, the region to be observed) and the reference surface of the in-focus position exceeds the drivable range of the piezoelectric element 15. This situation is called range over. The drivable range of the piezoelectric element 15 is an example of the drivable range of the present disclosure.

Therefore, the imaging control unit 21 can not cause the imaging optical system 14 to reach the in-focus position when the observation target region R reaches the observation target region by control using only the piezoelectric element 15. The narrow-field image captured in the (I + 10) th region is out of focus. Out-of-focus narrow-field images are of low value in generating wide-field images for the observed object. In view of the limitation of the total amount of data that can be stored in the storage device, out-of-focus narrow-field image data is less valuable to store.

Therefore, when the observation target area R reaches the observation target area, the imaging control unit 21 according to the first embodiment performs imaging in an out-of-focus state, and combines the generated data of the out-of-focus narrow-field image. The data is compressed at a compression rate higher than that at the time of storing the focused narrow-field image, and stored in the storage device such as the primary storage unit 72 or the like. Hereinafter, compression of data at a compression rate higher than that at the time of storing a narrow-field image in focus state is called high compression. By storing the low-value non-focused image data in a highly compressed manner, it is possible to save storage memory.

The compression rate at the time of storing the in-focus narrow-field image is an example of the predetermined amount of the present disclosure. In addition, storing image data in a highly compressed manner is an example of the imaging data saving process of the present disclosure.

Although not shown in FIG. 12, the objective lens 14b can be transported vertically downward from the reference surface by appropriately arranging the position of the piezoelectric element 15 in the Z direction. That is, it is not necessary to align the lowest position of the Z-direction deformation of the piezoelectric element 15 with the position corresponding to the reference plane (the broken line in FIG. 12), and set the lower limit of the drivable range below the reference plane in the Z direction. It is possible.

The imaging control process according to the first embodiment includes the focus control preparation process subroutine shown in FIG. 13 and the focus control process subroutine shown in FIG. The imaging control unit 21 executes the focus control preparation processing subroutine and the focus control processing subroutine in synchronization with each other to scan the bottom surface of the culture container 50, and the objective lens 14b is at the out-of-focus position. The compressed narrow-field image data is highly compressed and stored.

In the focus control preparation processing subroutine, an area where the in-focus position exceeds the drivable range of the piezoelectric element 15 is detected, and a flag is stored. In the focus control processing subroutine, based on whether the flag for each area is off or on, it is determined whether to store the captured image data at a normal compression ratio or to store it at high compression. Ru.

First, the focus control preparation processing subroutine of FIG. 13 will be described. For convenience, as shown in FIG. 12, description will be made using a situation where the observation target area R is located in the I-th area on the scanning plane.

In step S302, the imaging control unit 21 detects the position in the Z direction of the region to be observed on the bottom surface of the culture container 50 by the first displacement sensor 18a.

Next, in step S304, the imaging control unit 21 derives Z-direction position information of the region to be observed, and stores the information in the storage device. The Z-direction position information includes information of the detected Z-direction position and the in-focus position of the scheduled observation area determined based on the detected Z-direction position. As an example, it is stored in a cache memory (not shown) of CPU 70. Also, the detected Z-direction position may be stored in the primary storage unit 72 or the secondary storage unit 74.

Next, in step S306, the imaging control unit 21 derives a distance that is a shift of the in-focus position of the scheduled observation region from the Z direction position (broken line in the lower part of FIG. 12) corresponding to the reference plane. As described above, it is assumed that the in-focus position of each area is derived by the preceding process.

The calculated distance is recorded in, for example, a cache memory (not shown) of the CPU 70. The distance may be stored in the primary storage unit 72 or the secondary storage unit 74.

In order to appropriately photograph the region to be observed, it is necessary to move the objective lens 14b of the imaging optical system 14 by a distance in the Z direction when the region to be observed R moves to the current region to be observed.

Then, in step S308, the imaging control unit 21 determines whether the distance is smaller than a threshold. As described above, the threshold is, for example, the upper limit of the movable distance of the piezoelectric element 15.

If the determination in step S308 is affirmative, the process of FIG. 13 proceeds to step S310. In step S310, the imaging control unit 21 sets a flag on the region to be observed (turns on the flag). The flag indicates that the in-focus position of the region to be observed is not reachable by the piezoelectric element 15. The flag is recorded in a cache memory (not shown) of the CPU 70 as an example.

Then, when the observation target region R moves to the adjacent (I + 1) th region by the stage movement processing synchronized, the determination in step S312 becomes positive, and the processing in FIG. 13 proceeds to step S314. In this case, the observation target region R is located in the (I + 1) th region, and the (I + 11) th region is a new observation scheduled region.

In step S314, the imaging control unit 21 determines whether or not a new observation planned region has reached the scanning end point E in the stage moving process performed by the stage control unit 22. The determination in step S314 corresponds to step S210 of the stage moving subroutine shown in FIG.

If the determination in step S324 is negative, the process of FIG. 13 returns to step S302 again, and a new process is started. That is, with respect to the (I + 1) th area in which the observation target area R is currently arranged, the processing in step S302 and subsequent steps is performed with the (I + 11) th area as a new observation scheduled area.

On the other hand, if the determination in step S308 is negative, the flag is not set (is not turned on) for the observation scheduled region (I + the tenth region). That is, the flag for the (I + 10th) region is off). Then, when the observation target region R moves to the adjacent (I + 1) th region by the stage movement processing synchronized, the determination in step S312 becomes positive, and the processing in FIG. 13 proceeds to step S314. The subsequent processes are the same as in the case where the determination in step S308 is positive.

Thus, in the focus control preparation process, the imaging control unit 21 determines whether the deviation from the reference plane is within the threshold value or less for the area 10 units ahead of the observation target area R in the X-axis scanning direction, Repeat storing the flag on or off.

Next, the focus control process of FIG. 14 will be described. For convenience, as shown in FIG. 12, description will be made using a situation where the observation target area R is located in the I-th area.

In the preceding focus control preparation process, whether or not the Z direction position information and the flag are off is stored in a cache memory (not shown) of the CPU 70 for each of the (I + 10) th areas.

In step S402, the imaging control unit 21 performs the Z direction of the (I + 1) th area (that is, an area adjacent to the current position of the observation target area R in the X-axis scanning direction) stored in the preceding focus control preparation process. Get location information. As described above, the Z direction position information includes information of the in-focus position of the (I + 1) th region. As described above, as an example, the Z direction position information of the (I + 1) th area is stored in a cache memory (not shown) of the CPU 70.

Next, in step S404, the imaging control unit 21 determines whether or not the flag is off for the (I + 1) th region (that is, the region where the observation target region R reaches next). As described above, the flag for the (I + 1) th area is stored in the cache memory (not shown) of the CPU 70 as an example in the preceding focus control preparation process.

If the determination in step S404 is affirmative, from the reference plane of the in-focus position of the next area (that is, the (I + 1) -th area) that precedes the observation target area R by one unit in the X-axis scanning direction in the scanning plane. The deviation of the above is within the drivable range of the piezoelectric element 15. In this case, the imaging control unit 21 can adjust the objective lens 14b of the imaging optical system 14 to the in-focus position in the next area by controlling the piezoelectric element 15.

Then, when the observation target region R reaches the adjacent (I + 1) th region by the stage movement processing synchronized, the determination in step S406 is positive. Then, the process of FIG. 14 proceeds to step S408.

In step S408, the imaging control unit 21 executes control to apply a voltage to the piezoelectric element 15 to deform the piezoelectric element 15 in the Z direction, and the objective lens 14b of the imaging optical system 14 held by the piezoelectric element. Is moved to the in-focus position with respect to the (I + 1) th region.

Here, the piezoelectric element 15 of the first embodiment is controlled based on the voltage table 500 as an example. An example of the voltage table 500 is shown in FIG. The voltage table 500 includes information of the voltage to be applied to the piezoelectric element 15 with respect to the amount of displacement in the Z direction. Voltage table 500 represents the performance value of piezoelectric element 15, and is stored in primary storage unit 72 as an example.

In step S402 described above, Z direction position information of the (I + 1) th region (that is, the current position of the observation target region R on the scan plane) is acquired. Further, in the preceding process, Z direction position information of the I-th area (the position of the observation target area R at the start time of the focus control processing subroutine of FIG. 14) is also acquired. A voltage value to be applied is derived in step S408 as the difference between the in-focus position of the I-th area and the in-focus position of the (I + 1) -th area.

For example, assuming that the distance in the Z direction between the in-focus position of the immediately preceding area (I-th area) and the in-focus position of the current position (I + 1-th area) is Z3, the voltage value V3 is applied to the piezoelectric element 15 Then, the objective lens 14b can be brought to the in-focus position in the region of the (I + 1) th region.

Derivation of the applied voltage by the voltage table 500 is an example, and the contents described above can be expressed by a functional expression.

Now, it is considered that the displacement of the in-focus position of the (I + 1) th region from the reference plane falls within the drivable range of the piezoelectric element 15, so the piezoelectric element 15 to which the voltage V3 is applied is in the Z direction. In deformation, the objective lens 14b is moved to the in-focus position with respect to the (I + 1) th region.

Thus, in step S408, the imaging control unit 21 brings the objective lens 14b of the imaging optical system to the in-focus position.

Next, in step S410, the imaging control unit 21 images the (I + 1) th area on the scanning plane to generate a phase difference image. Since appropriate focus control is performed in step S408, a focused narrow-field image of the (I + 1) th region is generated in step S410.

In step S412, the imaging control unit 21 stores the generated narrow-field image in the in-focus state in the primary storage unit 72 as an example. The narrow-field image may be stored in a not-shown cache memory or secondary storage unit 74 of the CPU.

When the process of step S412 is completed, the process of FIG. 14 proceeds to step S422. In step S422, the imaging control unit 21 determines whether the observation target region R has reached the scanning end point E in the stage moving process performed by the stage control unit 22. The determination in step S422 corresponds to step S210 of the stage moving subroutine shown in FIG.

If the determination in step S422 is negative, the process of FIG. 14 returns to step S402, and the above-described process is repeated again. Thus, while the stage control unit 22 executes the stage movement process, the imaging control unit 21 continues to generate and store the narrow-field images of the respective areas on the scanning plane.

On the other hand, the case where the determination in step S404 is negative will be described.

In this case, in step S310 of the focus control preparation process of FIG. 13 preceding, a flag is set (turned on) to I + 1, and is recorded in a cache memory (not shown) of the CPU 70 as an example.

If a negative determination is made in step S404, a shift from the reference plane of the in-focus position of the next area (that is, the (I + 1) th area) preceding the observation target area R by one unit in the X-axis scanning direction in the scanning plane However, the drivable range of the piezoelectric element 15 is exceeded. Therefore, even if the voltage applied to the piezoelectric element 15 is appropriately adjusted, the imaging control unit 21 can not align the objective lens 14b of the imaging optical system 14 at the in-focus position in the next region.

When the observation target region R reaches the adjacent I + 1th region by the stage movement processing synchronized, the determination in step S414 is positive. Then, the process of FIG. 14 proceeds to step S416.

As described in step S408, the voltage to be applied to the piezoelectric element 15 for focus control is derived by referring to the acquired Z-direction position information and the voltage table 500. However, since the in-focus position at the current position (I + 1st region) on the scanning plane of the observation target region R exceeds the drivable range of the piezoelectric element 15, the derived voltage value is applied to the piezoelectric element 15 However, the objective lens 14b of the imaging optical system 14 can not be transported to the in-focus position.

For example, it is assumed that the distance in the Z direction between the in-focus position of the immediately preceding area (I-th area) and the in-focus position of the current position (I + 1-th area) is Z3 and a voltage value V3 is applied to the piezoelectric element 15 Do. In this case, it is considered that the Z direction position of the objective lens 14b moves to the drivable range upper limit (or drivable range lower limit) illustrated in FIG.

That is, in step S416, the imaging control unit 21 makes the Z-direction position of the objective lens 14b follow the variation of the bottom surface of the culture container 50 as much as possible within the drivable range of the piezoelectric element 15. However, in this case, the objective lens 14b can not be brought into focus at the current position (the (I + 1) th region).

Next, the process of FIG. 14 proceeds to step S418. In step S418, the imaging control unit 21 images the (I + 1) th region on the scanning plane to generate a phase difference image. At this time, since the objective lens 14 b is not at the in-focus position, in step S 418, an out-of-focus narrow-field image of the (I + 1) th region is generated. Then, the process proceeds to step S420.

As mentioned above, the out-of-focus narrow-field image is of low value in generating a wide-field image for the observation object. Therefore, in step S420, the imaging control unit 21 highly compresses the out-of-focus narrow-field image generated in step S418 to reduce the data amount, and then stores the data. This can save memory of the storage device consuming less valuable images. The highly compressed image data is stored in the primary storage unit 72 as an example.

When the process of step S420 is completed, the process of FIG. 14 proceeds to step S422. Step S422 has already been described.

As described above, by repeating the focus control process of FIG. 14, the narrow-field image of each area of the bottom of the culture vessel 50 is generated and stored. Then, if the narrow-field image is out of focus, it is stored as highly compressed small data.

Processing after scan processing
At the end of the scanning process in step S100 of FIG. 10, a narrow-field image is generated for each area on the bottom of the culture vessel 50, and the primary storage unit 72, a cache memory (not shown) of the CPU 70, or a secondary storage unit 74. Is stored in In particular, the out-of-focus narrow-field image is stored as highly compressed small data.

The process of FIG. 10 proceeds to step S102.

In step S102, the CPU 70 of the microscope control device 20 reads out and combines the stored narrow-field images, thereby providing a single composite phase-contrast image (ie, wide-field image) showing the entire bottom surface of the culture vessel 50. Generate

In the wide-field-of-view image to be generated, the region where the range-over of the piezoelectric element 15 has occurred can be represented as an out-of-focus narrow-field image in which the image quality is deteriorated due to high compression. Alternatively, in a wide-field image, an out-of-focus narrow-field image may not be displayed, and may be replaced with some simple image. For example, it is possible to paint the area where the range over of the piezoelectric element 15 has occurred using a certain color. As an example, it can be black and solid.

Next, in step S104, the CPU 70 of the microscope control device 20 stores the generated composite phase difference image, and ends the wide-field image acquisition process. The generated composite phase difference image can be stored, for example, in the secondary storage unit 74.

The stored wide-field image can be displayed on the display device 30.

According to the first embodiment of the technology of the present disclosure, in the process of scanning the bottom surface of the culture vessel 50 containing the observation target, the non-focused image is highly compressed and stored as small data. The data capacity of the device can be reduced.

Second Embodiment
Next, a microscope apparatus using a second embodiment according to the technology of the present disclosure will be described in detail with reference to the drawings. The microscope apparatus of the second embodiment is the same as the microscope apparatus of the first embodiment in that the wide-field image acquisition program 80 stored in the secondary storage unit 74 controls the focus control shown in FIG. It differs in that it includes a module related to processing. The other configuration of the microscope apparatus of the second embodiment is the same as that of the first embodiment, and therefore, focus control processing of the second embodiment will be mainly described below. In addition, about the structure which is common in 1st Embodiment, it demonstrates using the same code | symbol.

As described in the first embodiment, the displacement of the in-focus position from the reference plane exceeds the drivable range of the piezoelectric element 15 in the region on the bottom surface of the culture vessel 50 to be imaged of the phase difference image. In some cases, even if the element 15 is controlled, the imaging optical system 14 can not be brought into focus. In this case, in the focus control process of FIG. 14 according to the first embodiment, the memory capacity of the storage device is saved by highly compressing and storing low-value unfocused image data in step S420.

On the other hand, in the second embodiment, the shift of the in-focus position from the reference plane beyond the drivable range of the piezoelectric element 15 makes the imaging optical system 14 in focus even if the piezoelectric element 15 is controlled. When it is not possible, the imaging by the imaging optical system 14 is not performed in the first place. Hereinafter, description will be made with reference to FIG.

FIG. 16 shows a focus control processing subroutine of the second embodiment. The processes in steps S602 to S614 in FIG. 16 are the same as the processes in steps S402 to S422 in FIG. 14 according to the first embodiment, and thus the description thereof is omitted.

If the determination in step S604 corresponding to step S404 in FIG. 14 is negative, the process in FIG. 16 proceeds to step S616.

When the observation target area R reaches the adjacent I + 1th area by the stage movement processing synchronized, the determination in step S614 is positive. Then, the process of FIG. 16 proceeds to step S614.

Therefore, in a region where the deviation of the in-focus position from the reference plane exceeds the drivable range of the piezoelectric element 15, the imaging control unit 21 according to the second embodiment does not apply a voltage to the piezoelectric element 15, and The imaging of the narrow-field image by the imaging optical system 14 is not performed. Not performing imaging of a narrow-field image by the imaging optical system 14 is an example of an imaging data saving process of the present disclosure.

Since no voltage is applied to the piezoelectric element 15, the objective lens 14b moves to the (I + 1) th region while maintaining the Z direction position in the Ith region. That is, when the observation target region R moves one unit in the X-axis scanning direction by the synchronized stage movement processing, the objective lens 14 b moves parallel to the horizontal plane (XY plane). Stopping the movement of the objective lens 14b in the Z direction is an example of the focus control stop process of the present disclosure.

Instead of moving the objective lens 14 b in parallel to the horizontal plane (XY plane) without applying a voltage to the piezoelectric element 15, processing may be performed to restore the objective lens 14 b to the initial position corresponding to the reference plane. In this case, the imaging control unit 21 according to the second embodiment returns the objective lens 14 b to the initial position by applying a voltage of an appropriate value to the piezoelectric element based on the Z direction position information and the voltage table 500. Can. The return of the objective lens 14 b to the initial position is an example of the focus control stop process of the present disclosure.

In addition, imaging is not performed in the (I + 1) th region, and a narrow-field image is not generated. It should be noted that this area is an area where an in-focus image can not be generated even if imaging is performed.

At the end of the scanning process (see FIG. 10) in step S100 according to the second embodiment, a narrow-field image is generated for each area on the bottom surface of the culture vessel 50 in which range over has not occurred. It is stored in the storage unit 72, a cache memory (not shown) of the CPU 70, or the secondary storage unit 74. On the other hand, no narrow-field image is stored in the region where the range over of the piezoelectric element 15 has occurred.

The scanning process of step S100 according to the second embodiment and the process of FIG. 10 shift to step S102, and the generation of a wide-field image is performed as in the first embodiment. In the wide-field-of-view image generated in the second embodiment, it is possible to paint using a certain color the region where the range over of the piezoelectric element 15 where imaging is not performed has occurred. As an example, it can be black and solid.

The other configuration and operation of the microscope apparatus of the second embodiment are the same as those of the microscope apparatus of the first embodiment.

According to the second embodiment of the technology of the present disclosure, in the process of scanning the bottom of the culture vessel 50 containing the observation target, imaging is not performed in a situation where only low-value out-of-focus images can be generated. Since the data is not stored, the data capacity of the storage device required for the entire scan can be reduced.

Further, according to the second embodiment, it is possible to suppress the vibration of the imaging optical system by not performing unreasonable focus control in a situation where the objective lens 14 b can not be transported to the in-focus position.

Furthermore, according to the second embodiment, the focus control is reset by returning the objective lens 14b to the initial position in a situation where the objective lens 14b can not be transported to the in-focus position, and the shape of the bottom surface of the culture vessel 50 New focus control can be started according to

<Modification>
Hereinafter, modifications of the present disclosure will be described. Although the present modification will be described below based on the first embodiment, the present modification can also be applied to the second embodiment. The microscope apparatus according to the present modification differs from the microscope apparatus according to the first embodiment in the configuration of the detection unit. The other configuration of the microscope apparatus of this modification is the same as that of the first embodiment, and therefore, the configuration of the detection unit of the microscope apparatus of this modification will be mainly described below.

The detection unit 18 of the first embodiment includes the two

displacement sensors

18a and 18b, and switches the displacement sensor to be used according to the change in the X-axis scanning direction of the observation target region R, but the second embodiment The detection unit 19 has a single displacement sensor, and switches the position of the displacement sensor in accordance with the change of the X-axis scanning direction of the observation target region R. An example of the microscope apparatus main body 10 which has a detection part 19 in FIG. 17 is shown.

FIGS. 18 and 19 are diagrams showing a specific configuration of the detection unit 19 of the present modification. The detection unit 19 includes a displacement sensor 19a and a guide mechanism 19b for guiding the displacement sensor 19a and moving the position as shown in FIGS.

The displacement sensor 19a is the same as the first and

second displacement sensors

18a and 18b of the first embodiment, and is constituted by a laser displacement sensor.

The guide mechanism 19 b includes a semicircular guide member, and moves the displacement sensor 19 a along the guide member. The guide member moves the displacement sensor 19a from one side to the other side in the X direction across the imaging optical system 14 (the objective lens 14b).

FIG. 18 is a view showing the position of the displacement sensor 19a when the X-axis scanning direction of the observation region R is the arrow direction of FIG. 18 (the right direction of FIG. 18). On the other hand, FIG. 19 is a view showing the position of the displacement sensor 19a when the X-axis scanning direction of the observation target region R is the arrow direction of FIG. 19 (left direction in FIG. 19). When the X-axis scanning direction of the observation region R is changed from the arrow direction of FIG. 18 to the arrow direction of FIG. 19, the displacement sensor 19a moves along the guide member of the guide mechanism 19b from the position shown in FIG. , Is switched to the position shown in FIG.

In the second embodiment, the guide mechanism 19b described above is provided as a displacement sensor moving mechanism for moving the position of the displacement sensor, but the configuration of the displacement sensor moving mechanism is not limited to this, and the position of the displacement sensor If it is the structure which can be changed similarly, you may use another structure.

In the first and second embodiments, the wide-field-of-view image acquisition program 80 is read from the secondary storage unit 74 by way of example, but it is not always necessary to store the wide-field image acquisition program 80 in the secondary storage unit 74 from the beginning. For example, as shown in FIG. 22, first, any portable storage medium 800 such as a solid state drive (SSD), a universal serial bus (USB) memory, or a digital versatile disc-read only memory (DVD-ROM) may be used. The wide view image acquisition program 80 may be stored. In this case, the wide-field image acquisition program 80 of the storage medium 800 is installed in the microscope apparatus 90, and the installed wide-field image acquisition program 80 is executed by the CPU 70.

Further, the wide-field image acquisition program 80 is stored in a storage unit such as another computer or a server device connected to the microscope apparatus 90 via a communication network (not shown), and the wide-field image acquisition program 80 It may be downloaded according to 90 requests. In this case, the downloaded wide-field image acquisition program 80 is executed by the CPU 70.

Moreover, the wide-field-of-view image acquisition processing described in the first and second embodiments is merely an example. Therefore, needless to say, unnecessary steps may be deleted, new steps may be added, or the processing order may be changed without departing from the scope of the present invention.

Further, in the first and second embodiments, the case where the wide view image acquisition processing is realized by the software configuration using a computer is exemplified, but the technology of the present disclosure is not limited to this. For example, instead of the software configuration using a computer, the wide-field image acquisition processing may be executed only by a hardware configuration such as a field-programmable gate array (FPGA) or an application specific integrated circuit (ASIC). . The wide view image acquisition processing may be performed by a combination of a software configuration and a hardware configuration.

DESCRIPTION OF SYMBOLS 10 microscope apparatus main body 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 piezoelectric element 16 imaging element 17 stage drive device 18 detection unit

18a displacement sensor

18b displacement Sensor 19 Detection unit

19a Displacement sensor

19b Guide mechanism 20 Microscope control device 21 Imaging control unit 22 Stage control unit 30 Display device 40 Input device 50 Culture vessel 51 Stage 51a Opening 72 Primary storage unit 74 Secondary storage unit 80 Wide field of view image acquisition program 90 microscope apparatus 500 voltage table 800 storage medium D one side E scan end point L illumination light M solid line Pd detection position Pr position R1 range R2 range R observation object area S scan start point V3 voltage value W well

Claims

An imaging optical system capable of imaging an observation target light indicating the observation target in a container in which the observation target is accommodated on an imaging element;
A driving member for moving the imaging optical system in the optical axis direction by being driven within a drivable range;
By moving the imaging optical system with respect to each region in the container by moving the stage on which the container is installed and at least one of the imaging optical system in a plane intersecting the optical axis direction. The imaging optical system is moved to the in-focus position with respect to the specific area before the optical axis reaches the specific area of the respective areas in a state where the imaging element scans each area in the container. A control unit that executes an imaging data saving process when the optical axis reaches the specific region when the drive amount of the drive member at the time of movement exceeds the drivable range;
Microscope equipment.
The imaging data saving process includes compressing and storing image data generated by focusing the observation target light having passed through the specific area on the imaging device at a compression rate higher than a predetermined amount.
The microscope apparatus according to claim 1.
The imaging data saving process includes avoiding imaging of the specific area by the imaging optical system.
The microscope apparatus according to claim 1.
The driving member is a piezoelectric element that can be deformed along the optical axis direction of the imaging optical system.
The microscope apparatus according to any one of claims 1 to 3.
It further includes a detection unit that detects the position of the specific area in the optical axis direction,
The in-focus position of the specific area is calculated based on the position in the optical axis direction detected by the detection unit.
The microscope apparatus as described in any one of Claims 1-4.
The detection unit includes a pair of sensors provided side by side across the imaging optical system in the main scanning direction with respect to each of the areas, and detecting the position of the specific area in the optical axis direction.
The microscope apparatus according to claim 5.
The imaging optical system has an objective lens movable in the optical axis direction,
The drive member moves the objective lens in the optical axis direction.
The microscope apparatus according to any one of claims 1 to 6.
The container is a well plate having a plurality of wells,
The microscope apparatus according to any one of claims 1 to 7.
Computer,
The program for functioning as said control part contained in the microscope apparatus as described in any one of Claims 1-8.
An imaging optical system capable of imaging an observation target light indicating the observation target in a container in which the observation target is accommodated on an imaging element;
A driving member for moving the imaging optical system in the optical axis direction by being driven within a drivable range;
By moving the imaging optical system with respect to each region in the container by moving the stage on which the container is installed and at least one of the imaging optical system in a plane intersecting the optical axis direction. The imaging optical system is moved to the in-focus position with respect to the specific area before the optical axis reaches the specific area of the respective areas in a state where the imaging element scans each area in the container. A control unit that causes the drive member to execute focus control stop processing when the optical axis reaches the specific region when the drive amount of the drive member at the time of movement exceeds the drivable range;
Microscope equipment.
The focus control stop process includes stopping the movement of the drive member in the optical axis direction before the optical axis reaches the specific area or after the optical axis reaches the specific area.
The microscope apparatus according to claim 10.
The focus control stop processing includes moving the drive member to a reference position in the optical axis direction before the optical axis reaches the specific area or after the optical axis reaches the specific area.
The microscope apparatus according to claim 10.
Computer,
The program for functioning as said control part contained in the microscope apparatus as described in any one of Claims 10-12.