JP2022001889A - Focus position evaluation device, method and program, and discriminator - Google Patents

Abstract

To enable improvement in evaluation accuracy of a focus position in a focus position evaluation device, method and program, and a discriminator.SOLUTION: A focus position evaluation device includes: a marker image detection unit; and a focus position evaluation unit. The marker image detection unit is configured to photograph a marker being a sphere, and colored to a color absorbing light transmitting an imaging optical system as an observation object by an imaging device, and thereby detect a marker image from an acquired photographing image. The focus position evaluation unit is configured to evaluate a focus position of the imaging optical system on the basis of the marker image.SELECTED DRAWING: Figure 5

Description

The techniques of the present disclosure relate to focus position evaluation devices, methods, and programs.

Conventionally, pluripotent stem cells such as ES (Embryonic Stem) cells and iPS (Induced Pluripotent Stem) cells, cells induced to differentiate, etc. are photographed with a microscope, etc., and the differentiation state of the cells is captured by capturing the characteristics of the images. A method of determination 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, elucidation of diseases, etc. There is. When photographing cells with a microscope, in order to obtain a high-magnification wide-field image, for example, the range of a culture vessel such as a well plate is scanned by an imaging optical system, and an image is taken for each observation position. It has been proposed to perform so-called tyling photography in which images for each observation position are combined.

However, when a well plate having a plurality of wells is used as a culture container, for example, the thickness of the bottom of each well varies from well to well due to manufacturing errors and the like. Therefore, when tiling photography is performed by scanning the entire well plate with an imaging optical system, it is necessary to perform autofocus control for adjusting the focus position for each observation position in order to clearly photograph the observation target. .. Therefore, for example, in Patent Document 1, a sample to be imaged is fixed by a translucent member having a mark that gives at least one of a phase change and an amplitude change to transmitted light, and an image of the sample and an image of the mark are formed. A method of acquiring a mixed captured image and evaluating the focus position based on the evaluation value calculated for the captured image and the evaluation value calculated for the reference image not including the image of the sample acquired in advance has been proposed. ..

Further, Patent Document 2 describes a method in which a sample containing beads is flowed through a microarray in which a large number of wells are formed in the flow path and the beads are accommodated in each well as an inclusion. Observing under a microscope is disclosed.

Japanese Unexamined Patent Publication No. 2013-254108 International Publication No. 2017/0435330

When evaluating the focus position using a microscope, for example, when using a spherical transparent bead as a mark, we want to obtain the center position of the bead as the focus position, but the focus position of the bead image deviates from the center position of the bead. It ends up. This is because the beads themselves function as lenses, so that light is imaged not at the center position of the beads but at a position away from the center position of the beads. Therefore, when observing the cells to be observed at the focus position determined by using the transparent bead image, the focus position cannot be evaluated accurately and the cells may be blurred, and the evaluation accuracy of the focus position may occur. Is desired to be high.

The present invention has been made in view of the above circumstances, and an object of the present invention is to improve the accuracy of evaluation of the focus position.

The focus position evaluation device of the present disclosure is a focus position evaluation device in a photographing device that photographs an observation target imaged by an imaging optical system.
A marker image detection unit that detects a marker image from a captured image acquired by photographing a marker that is spherical and colored in a color that absorbs light transmitted through the photographing optical system as an observation target by an photographing apparatus.
A focus position evaluation unit that evaluates the focus position of the photographing optical system based on the marker image,
including.

Further, in the focus position evaluation device of the present disclosure, the marker may have a light absorption rate of 10% or more.

Further, in the focus position evaluation device of the present disclosure, the color may be black.

Further, in the focus position evaluation device of the present disclosure, the marker may be colored with a pigment or a coloring material having a property of absorbing the wavelength band of the transmitted light of the photographing optical system.

Further, in the focus position evaluation device of the present disclosure, the diameter of the marker is preferably 0.5 μm or more and 10 μm or less, and more preferably 1.0 μm or more and 2.2 μm or less.

Further, in the focus position evaluation device of the present disclosure, the marker may be formed of any of polystyrene resin, glass, and silica.

Further, in the focus position evaluation device of the present disclosure, the photographed image is acquired by photographing the container containing the marker and containing the observation object with the photographing device.
It may further include a control unit that controls the image of the observation target in the container to be focused on the photographing apparatus based on the focus position evaluated by the focus position evaluation unit.

In the present disclosure, the "container" may have any form as long as it can accommodate the observation target. For example, a container having a shape having a bottom and a continuous wall portion at the bottom, such as a petri dish, a dish, a flask, or a well plate, can be used as a container. Further, a microchannel device or the like in which a fine channel is formed in a plate-shaped member can also be used as a container. Further, a container having a plate-like shape such as a slide glass can also be used as a container.

Further, in the focus position evaluation device of the present disclosure, the captured image is acquired by scanning the observation area in the container in which the observation target is housed and photographing each observation area in the container.
The control unit can control in each observation area to focus the image of the observation target in the container on the photographing apparatus based on the focus position.

Further, the focus position control method of the present disclosure is a focus position evaluation method in a photographing apparatus for photographing an observation target formed by an imaging optical system.
The marker image is detected from the captured image acquired by photographing the marker, which is spherical and colored in a color that absorbs the light transmitted through the photographing optical system, as an observation target by the photographing apparatus.
The focus position of the photographing optical system is evaluated based on the marker image.

It should be noted that the focus position evaluation method according to the present disclosure may be provided as a program for causing a computer to execute the method.

The other focus position evaluation device according to the present disclosure is a focus position evaluation device in a photographing device that photographs an observation target imaged by an imaging optical system, and has a memory for storing an instruction to be executed by a computer and a memory.
The processor comprises a processor configured to execute a stored instruction.
The marker image is detected from the captured image acquired by photographing the marker, which is spherical and colored in a color that absorbs the light transmitted through the photographing optical system, as an observation target by the photographing apparatus.
A process of evaluating the focus position of the photographing optical system is executed based on the marker image.

According to one embodiment of the present disclosure, the accuracy of the evaluation of the focus position can be improved.

A block diagram showing a schematic configuration of a microscope apparatus in a microscopy system to which the focus position evaluation apparatus of the first embodiment is applied. Schematic diagram showing the configuration of the imaging optical system Perspective view showing the configuration of the stage Schematic diagram showing the configuration of the focal length change optical system A block diagram showing a schematic configuration of a microscope observation system using the first embodiment of the focus position evaluation device of the present disclosure. The figure explaining the observation of the fine beads by a microscope device. A diagram for explaining the shooting of a marker for acquiring a teacher marker image used for learning a discriminator. Diagram showing an example of a teacher marker image The figure which shows the determination result of a focus position The figure which shows the scanning position of the observation area in a culture vessel. A flowchart showing the processing performed in the first embodiment. A flowchart showing the processing performed in the second embodiment. Diagram for explaining autofocus control

Hereinafter, a microscopic imaging system to which one embodiment of the focus position evaluation device, method, and program according to the embodiment of the present invention is applied will be described in detail with reference to the drawings. FIG. 1 is a block diagram showing a schematic configuration of a microscope apparatus in a microscopy system to which the focus position evaluation apparatus according to the first embodiment of the present invention is applied.

The microscope device 10 captures a phase difference image of the cultured cells to be observed. Specifically, as shown in FIG. 1, the microscope device 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 operating unit 15, and a photographing unit 16. .. Further, the microscope device 10 includes a focal length changing optical system 70. The imaging optical system 14 corresponds to the photographing optical system of the present disclosure, and the microscope device 10 corresponds to the photographing apparatus of the present disclosure.

The operating unit 15 includes a first operating unit 15A, a second operating unit 15B, a third operating unit 15C, a fourth operating unit 15D, a fifth operating unit 15E, a sixth operating unit 15F, and a seventh operating unit 15. It is provided with a moving unit 15G. The operations of the first to seventh operating units 15A to 15G will be described later.

The slit plate 13 is provided with a ring-shaped slit that transmits white light to a light-shielding plate that shields 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.

The imaging optical system 14 forms a phase difference image for each observation region divided within the range of the culture vessel 50 on the photographing unit 16. 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 retardation lens 14a and an imaging lens 14d. Further, the phase difference lens 14a includes an objective lens 14b and a phase plate 14c. The phase plate 14c has a phase ring formed on a transparent plate that is transparent to the wavelength of the illumination light L. The size of the slit of the slit plate 13 described above has a conjugate relationship with the phase ring of the phase plate 14c.

The phase ring is formed by forming a phase film that shifts the phase of the incident light by 1/4 wavelength and a dimming filter that dims the incident light in a ring shape. The direct light incident on the phase ring is shifted in phase by 1/4 wavelength and its brightness is weakened by passing through the phase ring. On the other hand, most of the diffracted light diffracted by the observation target passes through the transparent plate of the phase plate 14c, and its phase and brightness do not change.

The retardation lens 14a having the objective lens 14b is moved in the optical axis direction of the objective lens 14b by the fifth operating unit 15E of the operating unit 15 shown in FIG. In the present embodiment, the optical axis direction and the Z direction (vertical direction) of the objective lens 14b are the same directions. Autofocus control is performed by moving the objective lens 14b in the Z direction, and the contrast of the phase difference image acquired by the photographing unit 16 is adjusted.

Further, the magnification of the retardation lens 14a may be changed. Specifically, the retardation lens 14a or the imaging optical system 14 having different magnifications may be interchangeably configured. The phase difference lens 14a or the imaging optical system 14 may be replaced automatically or manually by the user.

Further, the objective lens 14b is made of a liquid lens whose focal length can be changed. 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 focal length of the objective lens 14b is changed by changing the applied voltage by the sixth operating unit 15F 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 also performed by changing the focal length of the objective lens 14b, and the contrast of the phase difference image acquired by the photographing unit 16 is adjusted.

A phase difference image that has passed through the phase difference lens 14a is incident on the image forming lens 14d, and this is imaged on the photographing unit 16. In the present embodiment, the imaging lens 14d comprises a liquid lens whose focal length can be changed. 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 focal length of the imaging lens 14d is changed by changing the applied voltage by the first operating unit 15A 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 image acquired by the photographing unit 16 is adjusted.

Further, the imaging lens 14d is moved in the optical axis direction of the imaging lens 14d by the second operating unit 15B in the operating unit 15 shown in FIG. In the present embodiment, the optical axis direction and the Z direction (vertical direction) of the imaging lens 14d are the same directions. Autofocus control is performed by moving the imaging lens 14d in the Z direction, and the contrast of the phase difference image acquired by the photographing unit 16 is adjusted.

The photographing unit 16 acquires a phase difference image imaged by the imaging lens 14d. The photographing unit 16 includes an image pickup element such as a CCD (Charge-Coupled Device) image sensor or a CMOS (Complementary Metal-Oxide Semiconductor) image sensor. As the image pickup element, an image pickup element provided with an RGB (Red Green Blue) color filter may be used, or a monochrome image pickup element may be used.

Further, the photographing unit 16 is moved in the Z direction by the third operating unit 15C in the operating unit 15 shown in FIG. In this embodiment, the direction perpendicular to the image pickup surface of the photographing unit 16 and the Z direction are the same directions. Autofocus control is performed by moving the photographing unit 16 in the Z direction, and the contrast of the phase difference image acquired by the photographing unit 16 is adjusted.

A stage 51 is provided between the slit plate 13 and the imaging optical system 14. A culture vessel 50 containing cells to be observed is installed on the stage 51.

The culture container 50 corresponds to the container of the present invention. As the culture vessel 50, a petri dish, a dish, a flask, a well plate, or the like can be used. In addition to these, as the container, a slide glass, a microchannel device formed by processing a fine channel, or the like can be used. The cells contained in the culture vessel 50 include pluripotent stem cells such as iPS cells and ES cells, nerve, skin, myocardial and liver cells induced to differentiate from stem cells, and skin and retina taken out from the human body. There are cells of myocardium, blood cells, nerves and organs.

The stage 51 is moved in the X direction and the Y direction orthogonal to each other by the horizontal driving unit 17 (see FIG. 5) 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 present embodiment, the X direction is the main scanning direction and the Y direction is the sub-scanning direction.

FIG. 3 is a diagram showing an example of the stage 51. A rectangular opening 51a is formed in the center of the stage 51. 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.

Further, the stage 51 is moved in the Z direction by the fourth moving unit 15D, whereby the culture vessel 50 is moved in the Z direction. The fourth operating unit 15D includes an actuator such as a piezoelectric element. In the present embodiment, the direction perpendicular to the plane on which the culture vessel 50 is installed in the stage 51 and the Z direction are the same directions. The autofocus control is also performed by moving the stage 51 in the Z direction, and the contrast of the phase difference image acquired by the photographing unit 16 is adjusted.

The first operating unit 15A and the sixth operating unit 15F include, for example, a voltage variable circuit. The first operating unit 15A changes the voltage applied to the imaging lens 14d based on the control signal output from the focus position evaluation device 30 described later. The sixth operating unit 15F changes the voltage applied to the objective lens 14b based on the control signal output from the focus position evaluation device 30 described later.

The second operating unit 15B, the third operating unit 15C, the fourth operating unit 15D, and the fifth operating unit 15E are provided with an actuator such as a piezoelectric element, and are described from the focus position evaluation device 30 described later. It is driven based on the output control signal. The moving unit 15 is configured to pass the retardation image that has passed through the retardation 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, but the imaging lens 14d, the photographing unit 16, the stage 51, and the like. Any other known configuration may be used as long as the objective lens 14b (phase difference lens 14a) can be moved in the Z direction.

FIG. 4 is a schematic view showing the configuration of the focal length changing optical system. As shown in FIG. 4, the focal length changing optical system 70 includes a circular first wedge prism 71 and a circular second wedge prism 72. The seventh moving unit 15G moves the first wedge prism 71 and the second wedge prism 72 in synchronization with each other in opposite directions. As a result, the focal position of the imaging optical system 14 is changed. Changing the focal length is synonymous with increasing or decreasing the focal length. Therefore, by changing the focal position of the imaging optical system 14, the focal length of the imaging optical system 14 is changed. In the present embodiment, changing the focal length of the imaging optical system 14 means changing the focal length of the imaging lens 14d by the first operating unit 15A, and changing the focal length of the imaging lens 14d by the sixth operating unit 15F. It also includes changing the focal length of the imaging optical system 14 by changing the focal length of the imaging optical system 14 by the seventh operating unit 15G as well as changing the focal length of the image forming optical system 14.

The first and second wedge prisms 71 and 72 are prisms in which two surfaces that can be an entrance surface and an emission surface of light are not parallel, that is, the other surface is inclined with respect to one surface. In the following description, a surface arranged perpendicular to the optical axis is referred to as a right-angled surface, and a surface arranged inclined with respect to the optical axis is referred to as a wedge surface. Wedge prisms 71 and 72 are prisms that deflect light vertically incident on a right-angled surface. The seventh operating unit 15G includes an actuator such as a piezoelectric element, and makes the first wedge prism 71 and the second wedge prism 72 at right angles based on the control signal output from the focus position evaluation device 30 described later. While keeping the planes parallel, they are moved in synchronization with each other in opposite directions. That is, when the first wedge prism 71 is moved to the right in FIG. 4, the second wedge prism 72 is moved to the left. On the contrary, when the first wedge prism 71 is moved to the left in FIG. 4, the second wedge prism 72 is moved to the right. By moving the first and second wedge prisms 71 and 72 in this way, the optical path length of the light emitted from the imaging optical system 14 is changed, thereby changing the focal length of the imaging optical system 14. You can change it to change the focal length. As a result, autofocus control is performed, and the contrast of the phase difference image acquired by the photographing unit 16 is adjusted.

Next, the configuration of the microscope control device 20 that controls the microscope device 10 will be described. FIG. 5 is a block diagram showing the configuration of the microscope observation system of the first embodiment. As for the microscope device 10, a block diagram of a part of the configuration controlled by each part of the microscope control device 20 is shown.

The microscope control device 20 controls the entire microscope device 10, and includes a focus position evaluation device 30, a scanning control unit 21, and a display control unit 22 according to the first embodiment. Further, the focus position evaluation device 30 includes a marker image detection unit 31, a discriminator 32, a focus position evaluation unit 33, an operation control unit 34, and a learning unit 35 of the discriminator 32. The motion control unit 34 corresponds to the control unit of the present disclosure.

The microscope control device 20 is composed of a computer including a CPU (Central Processing Unit) as a central processing unit, a semiconductor memory, a hard disk drive, and the like, and the hard disk drive is an embodiment of the focus position evaluation program of the present disclosure. And the microscope control program is installed. Then, when the focus position evaluation program and the microscope control program are executed by the CPU, the marker image detection unit 31, the discriminator 32, the focus position evaluation unit 33, the motion control unit 34, the learning unit 35, and the scanning unit shown in FIG. The control unit 21 and the display control unit 22 function.

In the present embodiment, the CPU executes the functions of each part by the focus position evaluation program and the microscope control program, but as a general-purpose processor that executes software and functions as various processing parts, In addition to the CPU, a programmable logic device (PLD), which is a processor whose circuit configuration can be changed after manufacturing such as FPGA (Field Programmable Gate Array), can be used. Further, the processing of each part may be executed by a dedicated electric circuit or the like which is a processor having a circuit configuration specially designed for executing a specific processing such as an ASIC (Application Specific Integrated Circuit).

One processing unit may be composed of one of these various processors, or may be a combination of two or more processors of the same type or different types (for example, a plurality of FPGAs or a combination of a CPU and an FPGA). It may be configured. Further, a plurality of processing units may be configured by one processor. As an example of configuring a plurality of processing units with one processor, first, one processor is configured by a combination of one or more CPUs and software, as represented by a computer such as a client and a server. There is a form in which the processor functions as a plurality of processing units. Secondly, as typified by System On Chip (SoC), there is a form of using a processor that realizes the functions of the entire system including a plurality of processing units with one IC (Integrated Circuit) chip. be. As described above, the various processing units are configured by using one or more of the above-mentioned various processors as a hardware-like structure.

Further, the hardware-like structure of these various processors is, more specifically, an electric circuit (Circuitry) in which circuit elements such as semiconductor elements are combined.

Here, in the present embodiment, the culture vessel 50 includes a marker in order to evaluate the focus position for performing the autofocus control. In the present embodiment, the marker is a spherical fine bead colored in a color that absorbs the light transmitted through the imaging optical system 14. Since the white light source 11 of the present embodiment irradiates the culture vessel 50 with white light, the fine beads of the present embodiment are colored black. Here, "white light" means light in which light in various wavelength ranges is uniformly mixed. For example, when a light source that irradiates light in a wavelength range other than white light is used instead of the white light source 11, fine beads colored in a color that absorbs the wavelength range are used. Further, the fine beads are, for example, made of a polystyrene resin sphere having a diameter of 1.0 μm. Such fine beads can be put into the culture vessel 50 and used as a marker.

FIG. 6 is a diagram illustrating observation of fine beads by the microscope device 10. As shown in FIG. 6, when observing the fine beads as the marker M housed in the culture vessel 50, the fine beads are aligned with the upper surface of the bottom of the culture vessel 50, that is, the center position of the fine beads installed on the observation target installation surface P1. When the fine beads are transparent, the fine beads themselves function as a lens when they are to be charred. Therefore, the fine beads themselves are not at the center position but at a position ΔZ away from the center position of the fine beads, for example, the culture vessel 50. If the thickness d of the bottom portion of the bead is sufficiently thick, light is formed between the observation target installation surface P1 and the lower surface P2 of the bottom portion of the culture vessel 50.

Therefore, when observing the cells to be observed at the focus position determined by using the fine beads, the cells may be blurred, and it is desired to improve the accuracy of the evaluation of the focus position.

Therefore, in the present embodiment, by coloring the fine beads in black, the function of the fine beads as a lens is reduced, and the value of the distance ΔZ between the position where the light is actually formed and the center position of the fine beads is set. Make it smaller. This makes it possible to improve the accuracy when evaluating the focus position by using the fine beads as described later. Therefore, when the cells are photographed at the focus position determined by using the fine beads, the blurring of the cells on the image can be reduced.

In the present embodiment, the fine beads are used as the marker, but the technique of the present disclosure is not limited to this, and it is sufficient that the lens function of the fine beads themselves can be reduced. The fine beads can reduce the lens function, for example, by setting the light absorption rate to 10% or more. Further, the fine beads may be colored with a pigment or a coloring material having a property of absorbing the wavelength band of light of the imaging optical system 14. The wavelength band of light used in a normal biological microscope device is generally 380 to 780 nm, which is the visible range. Further, when a camera having a wide spectral sensitivity range is used, the wavelength band of light is used at 300 to 1100 nm. As the pigment, for example, OPLAS BLACK 838 of OPLAS (registered trademark) COLORS can be used. Also, as dyes, for example, NUBIAN (registered trademark) 6807-25 and NUBIAN (registered trademark) 6807-40 of NUBIAN (registered trademark) BLACK, and NUBIAN (registered trademark) 7807-25 and NUBIAN (registered trademark) 7807-40. Can be used.

Further, the fine beads are not limited to those made of polystyrene resin, and may be made of either glass or silica. The diameter of the fine beads is not limited to 1 μm, and may be 0.5 μm or more and 10 μm or less, more preferably 1.0 μm or more and 2.2 μm or less. Since the larger the diameter of the fine beads, the larger the bead image, it is desirable that the diameter of the fine beads is as small as possible within the observable range in the microscope device 10.

In the present embodiment, fine beads are used as markers, but the present disclosure is not limited to this, and for example, magnetic beads may be used.

Generally, fluorescent beads or polystyrene beads are used as the fine beads used to evaluate the focus position in the microscope device. On the other hand, magnetic beads are used as fine beads used for cell separation and protein and nucleic acid separation. Magnetic beads are mainly used for adsorbing proteins to beads and separating the proteins together with the beads with a strong magnetic force device. The magnetic beads have a structure dispersed or embedded in a polymer such as polystyrene in order to prevent aggregation of the magnetic beads. Magnetic beads sold for biological use are difficult to use because they are more expensive as a material for absorbing light than fine beads made of polystyrene. Since the structure of the magnetic particle mass contained in the magnetic beads is not a smooth circle but an uneven circle, it becomes noise when recognizing an image in order to quantitatively determine the focus position, and the accuracy is deteriorated. May cause you to. Therefore, as the marker, the above-mentioned fine beads are preferable to the magnetic beads.

In the present embodiment, in order to evaluate the focus position, an image for evaluating the focus position (hereinafter referred to as a captured image G0) is acquired by the photographing unit 16 prior to the acquisition of the phase difference image.

The marker image detection unit 31 detects a marker image from the captured image G0 for focus position evaluation acquired by the photographing unit 16. In the present embodiment, the captured image G0 is a phase difference image, and the above-mentioned marker is represented by a contrast different from that of the background image in the phase difference image. Therefore, the marker image detection unit 31 detects the marker image from the captured image G0 by performing the threshold value processing.

The discriminator 32 is learned by using a plurality of teacher marker images taken at different focus positions, that is, features of a plurality of teacher marker images taken at different focus positions, and inputs the marker images. Determines the focus position of the marker image input by.

Hereinafter, learning of the discriminator 32 will be described. The learning unit 35 performs learning of the discriminator 32. FIG. 7 is a diagram for explaining shooting of a marker for acquiring a teacher marker image used for learning of the discriminator 32. In addition, in FIG. 7, photography of one marker M will be described. As shown in FIG. 7, in order to acquire the teacher marker image, the marker M is photographed at a plurality of focus positions. That is, first, the imaging optical system 14 is adjusted, focus control is performed so as to focus on the position P0 of the marker M, and an image focused on the marker M is acquired. Further, focus control is performed so as to focus on the positions P1 and P2 in front of the marker M, and an image defocused in the plus direction is acquired. Further, focus control is performed so as to focus on the positions P3 and P4 behind the marker M, and an image defocused in the negative direction is acquired. In FIG. 7, the marker M is photographed at the five focus positions P0 to P4, but the image is not limited to this, and the marker M is photographed at more focus positions or less focus positions. You may do it.

Then, the learning unit 35 extracts a region containing the marker from the image acquired by photographing the marker M at a plurality of focus positions as described above, and generates a teacher marker image. FIG. 8 is a diagram showing an example of a teacher marker image. Note that FIG. 8 shows teacher marker images T0, T1, and T2 generated from an image acquired by focusing on the positions P0, P1, and P2. A large number (for example, 1000) of teacher marker images are prepared at each focus position.

Further, the learning unit 35 associates the defocus amount with the teacher marker image. For example, the teacher marker image acquired at the focus position P0 is associated with 0 as the focus position, the teacher marker image acquired at the focus position P1 is associated with +6 μm as the focus position, and the teacher marker image acquired at the focus position P2 is associated with the teacher image. The marker image is associated with +12 μm as the focus position. Further, the teacher marker image acquired at the focus position P3 is associated with −6 μm as the focus position, and the teacher marker image acquired at the focus position P4 is associated with −12 μm as the focus position.

The learning unit 35 learns the discriminator 32 so as to discriminate the focus position of the input marker image by using the teacher marker image. In the present embodiment, when the marker image to be discriminated is input, the discriminator 32 discriminates the focus position of the marker image. Specifically, the discriminator 32 calculates the probability of having a plurality of focus positions for the marker image to be discriminated, and discriminates the focus position having the highest probability from the focus position of the input marker image. do. Therefore, the learning unit 35 acquires a feature amount in a region of a predetermined size (for example, 3 × 3 or the like) from the teacher marker image, inputs the acquired feature amount to the discriminator 32, and inputs the feature amount. Learning of the discriminator 32, that is, machine learning is performed so as to output the discriminant result which is the focus position corresponding to the teacher marker image.

The discriminator 32 includes a support vector machine (SVM (Support Vector Machine)), a deep neural network (DNN (Deep Neural Network)), a convolutional neural network (CNN (Convolutional Neural Network)), and a recurrent neural network (RNN (RNN). It can be configured by Recurrent Neural Network)).

Further, as the feature quantity of the teacher marker image, a simultaneous occurrence matrix related to the teacher marker image may be used. The simultaneous occurrence matrix is a matrix showing the distribution of the signal values of the pixels in the image, and represents the frequency of the signal values of the pixels adjacent to the pixels having a certain signal value as a matrix. Here, when the focus position of the marker image is 0, that is, when the marker image is in focus, the contrast of the marker image is high, so that the pixels adjacent to the pixels with high brightness (that is, low density) have low brightness (that is, that is). High concentration). Therefore, when the focus position of the marker image is 0, the frequency of adjacent pixels with high signal values increases for high-luminance pixels. On the other hand, when the marker image is out of focus, the pixels adjacent to the high-luminance pixels do not have so low brightness. Therefore, when the marker image is blurred, the contrast of the marker image is low, so that the frequency of adjacent pixels with signal values having similar brightness increases for high-luminance pixels. Therefore, the simultaneous occurrence matrix for the teacher marker image becomes a characteristic matrix according to the degree of blurring of the marker image. Therefore, by using the simultaneous occurrence matrix as a feature quantity, the discriminator 32 can be learned so that the focus position can be discriminated with high accuracy.

The discriminator 32 learned in this way determines the focus position of the marker included in the captured image G0 acquired by the photographing unit 16. FIG. 9 is a diagram showing the determination result of the focus position. In the photographed image G0 shown in FIG. 9, a nucleolus of a cell is used as a marker, and in FIG. 9, the marker image is indicated by a white circle. As shown in FIG. 9, the discriminator 32 discriminates the focus position for each of the plurality of marker images included in the captured image G0. In FIG. 9, for the sake of explanation, a numerical value (μm) indicating the focus position for each marker image is shown in the vicinity of each marker image.

The focus position evaluation unit 33 evaluates the statistical value of the focus position of the plurality of marker images determined by the discriminator 32 as the focus position of the captured image G0 for one captured image G0, and determines the focus position. As the statistical value, the average value, the median value, the mode value, and the like of the focus positions of the plurality of marker images can be used. For example, when the mode value is the mode, the statistical value of the focus position is determined to be 7 μm for the captured image G0 whose focus position is determined as shown in FIG.

The operation control unit 34 operates the operation unit 15 to perform autofocus control based on the focus position determined by the focus position evaluation unit 33 as described above. Specifically, a control signal is output to each of the first operating unit 15A to the seventh operating unit 15G based on the focus position. As a result, the focal length of the imaging lens 14d is changed by the first moving unit 15A, and the focal length of the imaging optical system 14 is changed. Further, the imaging lens 14d is moved in the optical axis direction by the second moving unit 15B. Further, the photographing unit 16 is moved in the optical axis direction by the third operating unit 15C. Further, the stage 51 is moved in the optical axis direction by the fourth moving unit 15D. Further, the objective lens 14b is moved in the optical axis direction by the fifth moving unit 15E. The focal length of the objective lens 14b is changed by the sixth moving unit 15F, and the focal length of the imaging optical system 14 is changed. Further, the focal position of the imaging optical system 14 is changed by the seventh operating unit 15G, and the focal length of the imaging optical system 14 is changed. Autofocus control is performed by these seven operations.

The scanning control unit 21 drives and controls the horizontal drive unit 17, thereby moving the stage 51 in the X and Y directions and moving the culture vessel 50 in the X and Y directions. The horizontal drive unit 17 is composed of an actuator such as a piezoelectric element.

Hereinafter, the movement control of the stage 51 by the scanning control unit 21 and the autofocus control by the operation control unit 34 will be described in detail.

In the present embodiment, the stage 51 is moved in the X direction and the Y direction under the control of the scanning control unit 21, and the observation area of the imaging optical system 14 is moved two-dimensionally in the culture vessel 50 to move the culture vessel 50. Scan and image each observation area to obtain a phase difference image. FIG. 10 is a diagram showing the scanning position by the observation area in the culture vessel 50 by the solid line J. In this embodiment, a well plate having 6 wells W is used as the culture container 50.

As shown in FIG. 10, the observation area of the imaging optical system 14 moves along the solid line J from the scanning start point S to the scanning end point E. That is, the observation area R is moved in the positive direction in the X direction (right direction in FIG. 10), then in the Y direction (downward in FIG. 10), and in the opposite negative direction (left direction in FIG. 10). Will be moved. Then, the observation area R moves in the Y direction again and then moves in the positive direction again. In this way, the culture vessel 50 is scanned in a two-dimensional manner by repeatedly reciprocating the observation area R in the X direction and moving in the Y direction.

Further, in the present embodiment, the stage 51 is once stationary in each observation area R. In this state, the photographing unit 16 acquires the captured image G0 for focusing position evaluation, determines the focus position, performs autofocus control based on the focus position, captures the observation area R, and produces a phase difference image. To be acquired. When the phase difference image is acquired, the stage 51 moves, autofocus control is performed in the next observation area R, and the phase difference image is acquired. By repeating this operation, a plurality of phase difference images representing the entire culture vessel 50 are acquired, and the plurality of phase difference images are combined to generate a composite phase difference image.

That is, the motion control unit 34 performs autofocus control by driving and controlling the motion unit 15 based on the focus position determined in the observation area R. Specifically, the motion control unit 34 includes the focus position, the voltage applied to the imaging lens 14d for changing the focal distance of the imaging lens 14d, the amount of movement of the imaging lens 14d in the optical axis direction, and imaging. The amount of movement of the unit 16 in the optical axis direction, 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, the voltage applied to the objective lens 14b for changing the focal distance of the objective lens 14b, And the relationship with the movement amount of the focal distance changing optical system 70 is stored in advance as a table. This table is referred to as a first table.

The motion control unit 34 refers to the first table based on the determined focus position, and refers to the voltage applied to the imaging lens 14d for changing the focal distance of the imaging lens 14d, and the imaging lens 14d. To change the amount of movement in the optical axis direction, the amount of movement in the optical axis direction of the photographing unit 16, the amount of movement in the optical axis direction of the stage 51, the amount of movement in the optical axis direction of the objective lens 14b, and the focal distance of the objective lens 14b. The voltage applied to the objective lens 14b and the amount of movement of the focal distance changing optical system 70 are obtained. In the following description, the voltage applied to the imaging lens 14d for changing the focal distance of the imaging lens 14d, the amount of movement of the imaging lens 14d in the optical axis direction, and the movement of the imaging unit 16 in the optical axis direction. The amount, 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, the voltage applied to the objective lens 14b for changing the focal distance of the objective lens 14b, and the focal distance changing optical system 70. The amount of movement is called the focus control amount.

Then, in order to control the operation unit 15, the operation control unit 34 outputs a control signal according to the focus control amount to the first operation unit 15A to the seventh operation unit 15G. Specifically, the motion control unit 34 refers to the first table based on the defocus amount, acquires the focus control amount, and outputs the focus control amount to the first motion unit 15A to the seventh motion unit 15G.

The operating unit 15, that is, the first operating unit 15A to the seventh operating unit 15G is driven based on the input control signal. As a result, focus control is performed according to the focus position of the observation area R.

Returning to FIG. 5, the display control unit 22 generates one composite phase difference image by combining the phase difference images of each observation area R captured by the microscope device 10, and displays the composite phase difference image. Displayed on the device 23.

The display device 23 displays a composite phase difference image generated by the display control unit 22 as described above, and includes, for example, a liquid crystal display or the like. Further, the display device 23 may be configured by a touch panel and may also be used as the input device 24.

The input device 24 includes a mouse, a keyboard, and the like, and accepts various setting inputs by the user. The input device 24 of the present embodiment receives setting inputs such as an instruction to change the magnification of the retardation lens 14a and an instruction to change the moving speed of the stage 51.

Next, the operation of the microscope observation system to which the focus position evaluation device of the first embodiment is applied will be described with reference to the flowchart shown in FIG. First, the culture vessel 50 containing the cells to be observed is placed on the stage 51 (step ST10). Next, the stage 51 moves, the observation area R of the imaging optical system 14 is set at the position of the scanning start point S shown in FIG. 10, and scanning by the observation area R is started (step ST12).

Here, in the present embodiment, as described above, for each observation area R, the captured image G0 for focusing position evaluation is acquired, the marker image is detected, the focus position is evaluated, the focus position is determined, and the focus is set. The control amount is calculated, autofocus control is performed, and a phase difference image is acquired. These operations are performed while moving the observation area R, and acquisition of a captured image G0 for the observation area R at a certain position, detection of a marker image, determination of a focus position, determination of a focus position, calculation of a focus control amount, After the autofocus control and the acquisition of the phase difference image are performed, in the next observation area R, acquisition of the captured image G0, detection of the marker image, determination of the focus position, determination of the focus position, calculation of the focus control amount, Autofocus control and phase difference image acquisition are performed.

Therefore, in the first observation area R, the captured image G0 for focusing position evaluation is acquired by the photographing unit 16 (step ST14), and the marker image detection unit 31 detects the marker image from the captured image G0 (step ST16). .. Next, the discriminator 32 determines the focus position of the marker image included in the captured image G0 (step ST18), and the focus position evaluation unit 33 determines the focus position in the observation area R (step ST20). Then, the operation control unit 34 calculates the focus control amount based on the determined focus position (step ST22), and performs autofocus control based on the focus control amount (step ST24). That is, the motion control unit 34 drives and controls the motion unit 15 based on the movement amount stored in advance, changes the focal length of the imaging lens 14d, and Zs the imaging lens 14d, the photographing unit 16 and the objective lens 14b. Move in the direction. Then, after the autofocus control, the photographing unit 16 takes an image of the observation area R and acquires a phase difference image of the observation area R (step ST26). The acquired phase difference image is output from the photographing unit 16 to the display control unit 22 and stored.

Then, when all the scans are not completed (step ST28; NO), the observation area R moves in the X direction or the Y direction, and until all the scans are completed, the above-mentioned captured image G0 is acquired and the marker is used. Image detection, focus position determination, focus position determination, focus control amount calculation, autofocus control, and phase difference image acquisition are repeated (steps ST14 to ST26). Then, when the observation area R reaches the position of the scanning end point E shown in FIG. 10, all scanning ends (step ST28; YES).

After all the scans are completed, the display control unit 22 combines the phase difference images of each observation area R to generate a composite phase difference image (step ST30), and displays the generated composite phase difference image on the display device 23. (Step ST32).

As described above, in the present embodiment, by coloring the fine beads in black, the function of the fine beads as a lens is reduced, and the distance between the position where the light is actually formed and the center position of the fine beads ΔZ. Make the value of. This makes it possible to improve the accuracy when evaluating the focus position using the fine beads. Therefore, when the cells are photographed at the focus position determined by using the fine beads, the blurring of the cells on the image can be reduced.

Further, in the present embodiment, a plurality of captured images G0 for focus position evaluation including a marker to be evaluated for the focus position are acquired, a marker image is detected from the captured image G0, and a plurality of images are captured at different focus positions. Learning was performed using the feature amount related to the teacher marker image of the above, and the focus position was discriminated by the discriminator 32 for discriminating the focus position of the input marker image. Therefore, the defocus amount can be determined at high speed with a small amount of calculation.

Further, by discriminating the focus position for each of the plurality of marker images included in the captured image G0 and determining the statistical value of the plurality of focus positions at the focus position of the observation area R in which the captured image G0 is acquired, the discriminator is used. It is possible to accurately determine the focus position by absorbing the variation in the discrimination result due to 32.

Further, by focusing the image of the observation target in the culture vessel 50 on the photographing unit 16 based on the focus position, the focus position can be determined at high speed, so that the autofocus control can be performed at high speed. ..

In the first embodiment, the focus position evaluation device 30 according to the first embodiment is applied to the microscopic imaging system, and while moving the observation region R, the captured image G0 is acquired in each observation region R, and a marker is used. Image detection, focus position determination, focus position determination, focus control amount calculation, autofocus control, and phase difference image acquisition are performed, but the present invention is not limited thereto. For example, for a certain culture vessel 50, acquisition of a photographed image G0, detection of a marker image, determination of a focus position, determination of a focus position, and a focus control amount in each observation area R in the culture vessel 50 without accommodating cells. May be calculated. In this case, after the focus position is determined in the observation area R of all the culture vessels 50, the cells housed in the culture vessel 50 of the same type as the culture vessel 50 for which the focus position is determined are to be observed, and the phase difference image is acquired. Is done. Hereinafter, this will be described as a second embodiment.

FIG. 12 is a flowchart showing a process performed in the second embodiment for determining the defocus amount prior to the acquisition of the phase difference image. First, the culture vessel 50 containing the fine beads as markers is installed on the stage 51 (step ST40). Next, the stage 51 moves, the observation area R of the imaging optical system 14 is set at the position of the scanning start point S shown in FIG. 7, and scanning by the observation area R is started (step ST42).

Then, in the first observation area R, the captured image G0 for focusing position evaluation is acquired by the photographing unit 16 (step ST44), and the marker image detection unit 31 detects the marker image from the captured image G0 (step ST46). Next, the discriminator 32 determines the focus position of the marker image included in the captured image G0 (step ST48), and the focus position evaluation unit 33 determines the focus position in the observation area R (step ST50). Then, the motion control unit 34 calculates the focus control amount based on the determined focus position (step ST52), and stores the focus control amount in association with the position on the XY coordinates of the detection position of the culture vessel 50. (Step ST54).

Then, when all the scans are not completed (step ST56; NO), the observation area R moves in the X direction or the Y direction, and until all the scans are completed, the above-mentioned captured image G0 is acquired and the marker is used. Image detection, focus position determination, focus position determination, focus control amount calculation, and focus control amount storage are repeated (steps ST44 to ST54). Then, when the observation area R reaches the position of the scanning end point E shown in FIG. 10, all scanning ends (step ST56; YES).

In the second embodiment, when the phase difference image is acquired, the culture vessel 50 is scanned in the same manner as when the focus position is determined, and when the phase difference image is acquired in each observation area R. The motion control unit 34 performs autofocus control using the focus control amount stored in association with the XY coordinates of the culture vessel 50 corresponding to the observation area R. As a result, the phase difference image is acquired while performing focus control in each observation area R. In this case, it is necessary to scan the culture vessel 50 for storing the focus control amount in advance, but when the same type of culture vessel 50 is used, when the phase difference image is acquired, in each observation area R. It is not necessary to stop the stage 51 once to acquire the captured image G0, detect the marker image, determine the focus position, determine the focus position, calculate the focus control amount, perform autofocus control, and acquire the phase difference image. As a result, the observation area R can be continuously operated on the culture vessel 50, so that the phase difference image can be acquired at a higher speed.

In the second embodiment, the motion control unit 34 stores the focus control amount in each observation area R, but the determined focus position may be stored. In this case, when the phase difference image is acquired in each observation area R, the focus control amount is calculated based on the stored focus position, and the observation area R is photographed and the phase difference image is acquired.

Further, in the second embodiment, when the focus position determined in step ST50 is a focus position that cannot be set in the microscope device 10, that is, the focus control amount cannot be calculated, an error display is displayed on the display device 23, for example. It may be output, or it may be notified that the focus control amount cannot be calculated by a notification unit (not shown). Here, in the present disclosure, the "notifying unit" is recorded on a recording medium such as a display for visually displaying a message or the like, a voice reproducing device for displaying an audible voice by outputting a voice, or a recording medium such as paper for permanent visibility. It means a printer for displaying, a communication means such as a mail or a telephone, an indicator light, and the like, and at least two or more of the display, the voice reproduction device, the printer, the communication means, and the indicator light may be combined.

By the way, as the teacher marker image used in the learning of the discriminator 32, both the image defocused in the plus direction and the image defocused in the minus direction are used. However, when the image defocused in the positive direction and the image defocused in the negative direction are similar, the focus position can be obtained even by using the discriminator 32 that has been trained using such a teacher marker image. It may be difficult to determine whether is the focus position on the plus side or the focus position on the minus side.

However, in the present embodiment, even if the plus and minus of the focus position are erroneously determined, the autofocus control can be performed at high speed. FIG. 13 is a diagram for explaining autofocus control. Note that FIG. 13 shows autofocus control when the imaging lens 14d is moved in the Z direction. As shown in FIG. 13, it is assumed that the focus position when the imaging lens 14d is at the position P10 is determined to be + α. In this case, if the actual focus position is positive (that is, the focus is farther than the observation target), the observation target is moved in a direction away from the observation target, for example, the position P11. Can be focused on. However, when the focus is actually closer to the observation target and the focus position is −α, moving the imaging lens 14d to the position P11 causes the focus to be more out of focus.

In this case, when the imaging lens 14d is moved to the position P11, the captured image G0 for the focus position evaluation is acquired again and the focus position is determined. If the determined focus position is not 0, the plus and minus of the focus position are incorrect, so that the motion control unit 34 moves the imaging lens 14d toward the observation target, for example, from the position P11 to the position P12. Determine the focus control amount to move.

Here, when the autofocus control is performed by determining the contrast of the image as in the conventional case, it is necessary to repeat the acquisition of the captured image G0 and the determination of the focus control amount until the image is focused on the observation target. On the other hand, in the present embodiment, even if the plus and minus of the focus control amount are erroneously discriminated, the accurate focus control amount can be determined only by performing the operation of determining the focus position again. Therefore, in the present embodiment, even if the plus and minus of the focus control amount are erroneously discriminated, the autofocus control can be performed at high speed.

If the image defocused in the plus direction and the image defocused in the minus direction are similar, only one of the image defocused in the plus direction and the image defocused in the minus direction is taught. The discriminator 32 may be trained by using it as a marker image. For example, when the discriminator 32 is trained using only the image defocused in the positive direction as the teacher marker image, the discriminated focus position has a positive value. In this case, when the actual focus position is negative, as shown in FIG. 13, if the imaging lens 14d is moved to the position P11 as in the case where the focus position is positive, the focus becomes more out of focus.

In this case, when the imaging lens 14d is moved to the position P11, the captured image G0 for the focus position evaluation is acquired again and the focus position is determined. If the determined focus position is not 0, it is determined that the focus position is actually negative, and the motion control unit 34 sets the focus control amount so as to move the imaging lens 14d from the position P11 to P12. decide. As a result, an accurate focus control amount can be determined only by performing the operation of determining the focus position once again, as in the case where the plus and minus of the focus position are mistaken. Therefore, even if the discriminator 32 is trained by using only one of the image defocused in the positive direction and the image defocused in the negative direction as the teacher marker image, the autofocus control can be performed at high speed. It can be carried out.

In each of the above embodiments, a marker image having a known focus position is used as a teacher marker image for learning the discriminator 32, but the present invention is not limited to this. For example, a marker image whose focus position is unknown may be used as a teacher marker image. In this case, for the marker image whose focus position is unknown, the learning unit 35 learns the discriminator 32 so as to discriminate that the focus position is unknown. As the marker image whose focus position is unknown, a marker image in which the focus position is erroneously determined as a result of input to the discriminator 32 can be used. Therefore, the learning unit 35 first learns from the discriminator 32 so as not to discriminate that the focus position is unknown. Then, when the focus position is determined by the discriminator 32 at a stage where learning has progressed to some extent, the marker image for which the focus position is erroneously determined is determined as the marker image whose focus position is unknown. Then, using such a marker image again, the discriminator 32 is learned so as to discriminate that the focus position is unknown. This makes it possible to generate a discriminator 32 capable of discriminating that the focus position is unknown. Therefore, it is possible to reduce the possibility of acquiring an erroneous focus position determination result.

In each of the above embodiments, the operating unit 15 performs autofocus control by the first to seventh operating units 15A to 15G, but any one of the first to seventh operating units 15A to 15G. The autofocus control may be performed using only one or a plurality of these. Further, only one of the first to seventh operating units 15A to 15G or a plurality of these may be provided.

Further, in each of the above embodiments, the focal length changing optical system 70 is arranged between the imaging optical system 14 and the photographing unit 16, but is arranged between the imaging optical system 14 and the stage 51. You may.

Further, in each of the above embodiments, the culture vessel 50 is moved in the optical axis direction by moving the stage 51 in the optical axis direction by the fourth operating unit 15D. However, instead of moving the stage 51 in the optical axis direction, a mechanism for moving the culture vessel 50 in the optical axis direction may be provided so that only the culture vessel 50 is moved in the optical axis direction.

Further, in each of the above embodiments, the focus position evaluation unit 33 evaluates and determines the focus position based on the focus position determined by the discriminator 32, but the technique of the present disclosure is not limited to this. The focus position evaluation device may not include the discriminator 32 and the learning unit 35. In this case, the focus position evaluation unit 33 detects and stores the reference marker image from the captured images captured and acquired in advance for each different focus position. Then, the focus position evaluation unit 33 calculates the correlation value for all the combinations of the marker image detected by the marker image detection unit 31 in the captured observation image and the reference marker image for each different focus position. When the correlation value is equal to or higher than a preset threshold value, the focus position evaluation unit 33 determines that the combination of the marker image having the correlation value and the reference marker image is a combination to be associated with the reference marker image. The focus position from which the marker image is acquired is determined as the focus position of the observation image. As a method for calculating the correlation value, for example, normalized cross-correlation (ZNCC) may be used for calculation. However, the present invention is not limited to this, and other calculation methods may be used.

Further, in each of the above embodiments, the focus position evaluation apparatus according to the present invention is applied to a phase-contrast microscope, but the present disclosure is not limited to the phase-contrast microscope, but other microscopes such as a differential interference microscope and a bright-field microscope. May be applied to.

10 Microscope device (imaging device)
11 White light source 12 Condenser lens 13 Slit plate 14 Imaging optical system (photographing optical system)
14a Phase difference lens 14b Objective lens 14c Phase plate 14d Imaging lens 15 Operating unit 15A 1st operating unit 15B 2nd operating unit 15C 3rd operating unit 15D 4th operating unit 15E 5th operating unit 15F 6th 15G 7th operating unit 16 Imaging unit 17 Horizontal drive unit 20 Microscope control device 21 Scan control unit 22 Display control unit 23 Display device 24 Input device 30 Focus position evaluation device 31 Marker image detector 32, 32A Discriminator 33 Focus position evaluation unit 34 Motion control unit 35 Learning unit 50 Culture container 51 Stage 51a Opening 70 Focal length change optical system 71 First wedge prism 72 Second wedge prism J Scanning position by observation area S Scanning start point E Scanning end Point M Marker, Fine beads L Illumination light R Observation area W Well

Claims (11)

It is a focus position evaluation device in a photographing device that photographs an observation target formed by an imaging optical system.
A marker image detection unit that detects a marker image from a captured image acquired by photographing a marker that is spherical and colored in a color that absorbs light transmitted through the photographing optical system as the observation target by the photographing apparatus.
A focus position evaluation unit that evaluates the focus position of the photographing optical system based on the marker image, and a focus position evaluation unit.
Focus position evaluation device including. The focus position evaluation device according to claim 1, wherein the marker has a light absorption rate of 10% or more. The focus position evaluation device according to claim 1 or 2, wherein the color is black. The focus position evaluation device according to any one of claims 1 to 3, wherein the marker is colored with a pigment or a coloring material having a characteristic of absorbing the wavelength band of transmitted light of the photographing optical system. The focus position evaluation device according to any one of claims 1 to 4, wherein the marker has a diameter of 0.5 μm or more and 10 μm or less. The focus position evaluation device according to claim 5, wherein the marker has a diameter of 1.0 μm or more and 2.2 μm or less. The focus position evaluation device according to any one of claims 1 to 6, wherein the marker is made of any one of polystyrene resin, glass, and silica. The photographed image is acquired by photographing the container including the marker and accommodating the observation object with an imaging apparatus.
Any one of claims 1 to 7, further comprising a control unit that controls the image of the observation target in the container to be focused on the photographing apparatus based on the focus position evaluated by the focus position evaluation unit. The focus position evaluation device according to the section. The photographed image is acquired by scanning the observation area in the container in which the observation object is housed and photographing each observation area in the container.
The focus position evaluation device according to claim 8, wherein the control unit controls to focus the image of the observation target in the container on the photographing device based on the focus position in each observation area. It is a focus position evaluation method in an imaging device that photographs an observation target imaged by an imaging optical system.
The marker image is detected from the captured image acquired by photographing the marker, which is spherical and colored in a color that absorbs the light transmitted through the photographing optical system, as the observation target by the photographing apparatus.
A focus position evaluation method for evaluating a focus position of the photographing optical system based on the marker image. It is a focus position evaluation program in a photographing device that photographs an observation target imaged by an imaging optical system.
A procedure for detecting a marker image from a captured image acquired by photographing a marker that is spherical and colored in a color that absorbs light transmitted through the photographing optical system with the photographing apparatus as the observation target.
A procedure for evaluating the focus position of the photographing optical system based on the marker image, and
Focus position evaluation program that causes the computer to execute.

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