JP6969134B2 - Correction amount calculation device, microscope, correction amount calculation program, correction amount calculation method - Google Patents

Description

The present invention relates to a correction amount calculation device, a microscope, a correction amount calculation program, and a correction amount calculation method.

Conventionally, a microscope device for adjusting the influence of aberration caused by an optical system of a specimen and a microscope is known (for example, Patent Document 1).

U.S. Pat. No. 6,563,634

One aspect of the present invention is a correction amount calculating device for calculating a correction amount of aberration to an imaging apparatus including an imaging unit for imaging the imaging object, the previous SL correction amount a calculated possible conditions own device, At least one of the positions of the image pickup target and the objective lens, the degree of spherical aberration correction of the objective lens, the temperature condition, the passage of time, and the aberration on the optical path between the image pickup target and the image pickup unit. A first image acquisition unit that acquires a plurality of images captured by the same imaging target by varying the degree of imaging conditions including the lens, and a second image that acquires a second image captured by the imaging target. An acquisition unit, a third image acquisition unit that acquires a third image captured by the image pickup target at a focal position that the observer wants to observe , a plurality of the images acquired by the first image acquisition unit, and the first image. (Ii) A first evaluation value calculation unit that calculates a first evaluation value, which is an evaluation value based on the second image acquired by the image acquisition unit, and a plurality of the images acquired by the first image acquisition unit. A second evaluation value calculation unit that calculates a second evaluation value that is an evaluation value based on the third image acquired by the third image acquisition unit, and the first evaluation value calculation unit that calculates the first evaluation value. This is a correction amount calculation device including a correction amount calculation unit for calculating the correction amount based on the evaluation value of the above and the second evaluation value calculated by the second evaluation value calculation unit.

Another embodiment of the present invention, said objective lens having a spherical surface aberration correcting optical system, based on the correction amount and the correction amount calculating unit described above may be calculated, the spherical aberration correcting optical system of the objective lens It is a microscope provided with a drive unit for adjusting the amount of correction of the aberration.

Further, one aspect of the present invention includes an image pickup unit that captures an image pickup target and a photo aberration correction unit that corrects an aberration on the optical path between the image pickup target and the image pickup unit. Is a microscope that adjusts the aberration correction amount of the optical aberration correction unit based on the correction amount calculated by the correction amount calculation unit described above.

Further, one aspect of the present invention is a microscope including a driving unit that moves the relative position between the objective lens and the stage on which the image pickup target is placed based on the correction amount calculated by the correction amount calculation unit described above. Is.

Further, one aspect of the present invention is a condition in which the own device can calculate the correction amount in a computer provided with a correction amount calculation device for calculating an aberration correction amount for an image pickup device including an image pickup unit that captures an image pickup target. The positions of the image pickup target and the objective lens, the degree of spherical aberration correction of the objective lens, the temperature conditions, the passage of time, and the aberrations on the optical path between the image pickup target and the image pickup unit. Acquiring a plurality of images in which the same imaging target is captured by varying the degree of imaging conditions including at least one , acquiring a second image in which the imaging target is captured, and the observer. Is an evaluation value based on the acquisition of a third image captured by the image pickup target at the focal position desired to be observed, the plurality of acquired images, and the acquired second image. and calculating an evaluation value, a plurality of the images acquired, and calculating the second evaluation values is obtained evaluation values based on said third image, the calculated the first It is a correction amount calculation program for calculating the correction amount based on the evaluation value and the calculated second evaluation value, and executing the calculation.

Further, one aspect of the present invention is a condition in which the own device can calculate the amount of aberration correction related to the image pickup device including the image pickup unit that captures the image pickup target among the image pickup conditions, and the image pickup target and the objective lens. Position, degree of spherical aberration correction of the objective lens, temperature condition, time lapse, degree of imaging condition including at least one of aberration on the optical path between the imaging target and the imaging unit. a first image acquisition step of acquiring a plurality of images in which the same imaging target is imaged at different each other, and a second image acquisition step of the imaging target is to obtain a second image captured observer A third image acquisition step for acquiring a third image captured by the image pickup target at a focal position to be observed , a plurality of the images acquired from the first image acquisition step, and acquisition from the second image acquisition step. A first evaluation value calculation step for calculating a first evaluation value which is an evaluation value based on the second image, a plurality of the images acquired from the first image acquisition step , and the third image. The second evaluation value calculation step for calculating the second evaluation value, which is the evaluation value based on the third image acquired from the acquisition step, and the first evaluation calculated from the first evaluation value calculation step. It is a correction amount calculation method including a correction amount calculation step for calculating the correction amount based on the value and the second evaluation value calculated from the second evaluation value calculation step.

It is a figure which shows an example of the appearance composition of a microscope system. It is an enlarged view of a part of the appearance composition of a microscope which is a part of a microscope. It is a figure which shows an example of the functional structure of the correction amount calculation apparatus of 1st Embodiment. It is a figure which shows an example of ruler information. It is a flow chart which shows an example of the operation of the correction amount calculation apparatus. It is a figure which shows an example of a reference plane image, an observation image, and ruler information. It is a figure which shows an example of a reference plane correlation value, and an observation plane correlation value. It is a figure which shows an example of the functional structure of the microscope of 2nd Embodiment, and a correction amount calculation apparatus. It is a figure which shows an example of the multi-axis ruler information.

[First Embodiment]
Hereinafter, the microscope system will be described with reference to the drawings.
FIG. 1 is a diagram showing an example of the appearance configuration of the microscope system 100.
The microscope system 100 includes a correction amount calculation device 10 and a microscope 20.

The microscope 20 is a microscope provided with a spherical aberration correction optical system for observing an enlarged image of a sample placed on a stage 23. Specifically, the sample is a biological sample, beads, or the like to be observed. A biological sample is a sample such as a thick fluorescently stained cell. Beads are fluorescently labeled polystyrene microspheres (eg, 0.2 um in diameter). In the microscope 20, the imaging unit 25 captures this magnified image and generates an captured image. Further, the microscope 20 corrects spherical aberration with an objective lens provided with a correction ring capable of controlling the degree of correction of spherical aberration by an electric motor. Spherical aberration is an aberration in which a light ray emitted from a point light source does not focus on one focal point after passing through an optical system. Specifically, the spherical aberration changes according to the observation wavelength of the light passing through the observation target, the temperature change, the cover glass thickness, and the depth of the observation surface. The correction ring corrects this spherical aberration. In the present embodiment, the correction amount calculation device 10 calculates the correction amount of the position of the correction ring.

[About the microscope 20]
Here, the microscope 20 will be described with reference to FIG. In the following description, the case where the microscope 20 is partially configured as an inverted microscope will be described, but the present invention is not limited to this. The microscope 20 may be partially configured such as a differential interference microscope (DIC), a phase contrast microscope, a fluorescence microscope, a confocal microscope, a super-resolution microscope, and a two-photon excitation fluorescence microscope.
FIG. 2 is an enlarged view of a part of the appearance configuration of the microscope 20 which is a part of the microscope 20. In FIG. 1, the microscope 20 includes an objective lens 21, a correction ring drive unit 22, a stage 23, an objective lens drive unit 24, an image pickup unit 25, and a branch unit 26.

The objective lens driving unit 24 has a motor that moves the position of the objective lens 21 in the optical axis OA direction, and moves the objective lens in the optical axis OA direction by moving a revolver (not shown) that holds the objective lens up and down. You can move it. In FIG. 2, when the relative position ZL between the objective lens 21 and the image pickup target TP changes, the focal position of the objective lens 21 changes. In the following description, the focal position of the objective lens 21 is also referred to as a focal position OF.

In the present embodiment, the objective lens 21 includes a correction ring 21a. Spherical aberration of the objective lens 21 by adjusting the correction ring 21a and changing the position of the aberration correction lens arranged on the optical axis of the objective lens 21 in the lens barrel holding the objective lens 21 in the optical axis direction. Is corrected. The correction amount CL of the spherical aberration of the objective lens 21 corresponds to the displacement of the objective lens 21 in the optical axis OA direction. The correction amount CL is the degree of spherical aberration corrected by the correction ring 21a. That is, when the correction amount CL is adjusted, the focal position OF of the objective lens 21 moves in the optical axis OA direction. In other words, when the correction amount CL is adjusted, the focal position of the objective lens 21 changes. The correction ring 21a having such an annular structure is not indispensable, and may be configured to move the aberration correction lens in the optical axis OA direction by using a cam structure. Further, instead of the aberration correction lens, a mirror for correcting spherical aberration or an optical element such as a liquid crystal element may be used. Optical elements such as mirrors and liquid crystal elements that correct spherical aberration are arranged between the objective lens and the eyepiece lens as aberration correction optical systems, and spherical aberration is adjusted by adjusting the transmission of observation light incident on the observation optical system. Correct the aberration.

The correction ring driving unit 22 drives the correction position of the correction ring 21a. The correction ring 21a adjusts the correction amount CL of spherical aberration by driving the correction position. Specifically, the correction ring drive unit 22 is a drive device such as an electric motor, and a device that moves the holding member that holds the lens by the electric motor in the optical axis direction by a cam mechanism or the like can be used.

The image of the image pickup target TP placed on the stage 23 is imaged on the image pickup surface of the image pickup unit 25 by an image pickup lens (not shown) via the objective lens 21. In this example, the TP to be imaged is a biological sample having a thickness of TPD. The image pickup unit 25 captures an image formed by the objective lens 21. In the present embodiment, the image pickup unit 25 is a camera including a CCD sensor or a CMOS sensor as an image pickup element. In this example, the image pickup target TP is arranged on the cover glass CG. The TP to be imaged is irradiated with illumination light. The objective lens 21 receives the light transmitted through the image pickup target TP. Spherical aberration is also caused by the wavelength of the light transmitted through the TP to be imaged. The light received by the objective lens 21 is not limited to the light transmitted through the image pickup target TP. The objective lens 21 may receive the light reflected by the image pickup target TP.

The branching unit 26 branches the light from the image pickup target TP imaged by the objective lens 21 to the image pickup unit 25 and an eyepiece (not shown). Specifically, the branching unit 26 branches the light from the imaging target TP to the imaging unit 25 via the optical path OP. Specifically, the branch portion 26 is an optical element such as a dichroic prism or a half mirror.

In addition, the imaging unit 25 irradiates the cells with excitation light that excites a fluorescent substance, and images the fluorescence emitted from the fluorescent reagent added to the cells as an captured image.
In this embodiment, cells are stained with a fluorescent reagent to obtain a cell image. Specifically, in this embodiment, cells are fixed and immunostained. Cell fixation is a process of fixing cells using a reagent such as formaldehyde. The biological sample imaged by the imaging unit 25 may be, for example, a fixed specimen, a transparent specimen, or a living specimen.

Further, the microscope 20 captures the light emission or fluorescence generated from the color-developing substance itself taken into the biological substance and the light emission or fluorescence generated by the binding of the substance having a chromophore to the biological substance as the above-mentioned captured image. May be good. As a result, the microscope 20 can acquire a fluorescence image and a two-photon excitation fluorescence microscope image.
The cells in the present embodiment are, for example, primary cultured cells, established cultured cells, cells of tissue sections, and the like. In order to observe cells, the sample to be observed may be an aggregate of cells, a tissue sample, an organ, an individual (animal, etc.), and an image containing the cells may be obtained. The state of the cell is not particularly limited, and may be a living state or a fixed state, and may be either "in-vivo" or "in-vitro". Of course, a living state information and a fixed information may be combined.

An immersion space i is provided between the cover glass CG and the objective lens 21. The immersion space i is filled with the liquid. By filling the immersion space i with liquid, the numerical aperture of the objective lens 21 is improved, and the resolution of the objective lens 21 such as resolution, depth of focus and brightness can be improved. Specifically, the liquid that fills the immersion space i is oil, water, or the like. When the immersion space i is filled with oil, the spherical aberration changes due to the temperature change. In the following description, the fact that the immersion space i is filled with oil is also described as oil immersion. When the immersion space i is filled with water, the spherical aberration changes depending on the thickness of the cover glass. In the following description, the fact that the immersion space i is filled with water is also described as water immersion.
Further, the distance between the cover glass CG and the focal position OF changes as the observation position changes inside the image pickup target TP. When the objective lens 21 is an oil-immersion objective lens, spherical aberration occurs due to a change in the distance between the cover glass CG and the focal position OF.

Returning to FIG. 1, the correction amount calculation device 10 calculates the correction amount of the spherical aberration of the image.
In the present embodiment, the correction amount calculation device 10 includes a display unit 11 and a control unit 12. The control unit 12 calculates the degree CL of the correction of spherical aberration of the objective lens 21. The display unit 11 displays the captured image captured by the microscope 20. The display unit 11 displays the result calculated by the control unit 12. The correction amount calculation device 10 is not limited to this, and may be a tablet-type device capable of transmitting and receiving information by a personal computer or wirelessly.

[Functional configuration of correction amount calculation device 10]
Next, the functional configuration of the correction amount calculation device 10 will be described with reference to FIG.
FIG. 3 is a diagram showing an example of the functional configuration of the correction amount calculation device 10 of the first embodiment.

The control unit 12 includes an image acquisition unit 13, a reference focus plane information acquisition unit 16, an operation detection unit 17, a storage unit ST, a ruler information generation unit 18, an evaluation value calculation unit 14, and a degree calculation unit 15. To prepare for. The image acquisition unit 13 is an example of a first image acquisition unit, a second image acquisition unit, and a third image acquisition unit. The evaluation value calculation unit 14 is an example of the first evaluation value calculation unit and the second evaluation value calculation unit. The degree calculation unit 15 is an example of a correction amount calculation unit.

The image acquisition unit 13 acquires an image captured as an image signal captured by the image pickup unit 25. The image acquisition unit 13 acquires a condition parameter indicating a condition from which the correction amount of the imaging conditions of the captured image can be calculated from the microscope 20. The condition parameter is a condition in which the own device can calculate the correction amount among the imaging conditions.
The captured image includes a plurality of images in which the same image pickup target TP is captured by making controllable condition parameters different from each other, and a captured image in which the image pickup target TP being observed is captured. In this embodiment, the condition parameter is the relative position ZL. As described above, the relative position ZL is the relative position between the objective lens 21 described above and the image pickup target TP.
The image acquisition unit 13 acquires a plurality of images in which the same imaging target TP is captured by making the relative positions ZL different from each other. Further, the captured image in which the image-taking target TP being observed is captured is an example of the second image. In the following description, the captured image captured by the image pickup target TP under observation is also referred to as an observation image. The image acquisition unit 13 outputs the acquired captured image to the evaluation value calculation unit 14.

The image acquisition unit 13 acquires an image from the microscope 20 in association with each other and the condition parameters when the image is captured. The image acquisition unit 13 associates the image acquired from the microscope 20 with the condition parameters when the image is captured, and outputs the image to the ruler information generation unit 18. The condition parameter may be given by the user who operates the correction amount calculation device 10. In this case, the operation detection unit 17 detects an operation for inputting the user's condition parameter.

The reference focus plane information acquisition unit 16 acquires a reference plane image. The reference plane image is an image in which the image pickup target TP at the focal position OF that the observer wants to observe is captured in this embodiment. This reference plane image is an example of a third image. The observer is a person who observes the image pickup target TP by operating the correction amount calculation device 10 and the microscope 20. The reference focus plane information acquisition unit 16 outputs the acquired reference plane image to the evaluation value calculation unit 14.

The operation detection unit 17 detects the input operation of the observer. Specifically, the operation detection unit 17 detects the basic parameters input by the input operation from the observer. The basic parameters are information such as a target region ROI (Region of Interest), an adjustment range of a correction ring, ruler information used for adjusting the correction ring, and a control target.

The target area ROI is a part of the captured pixels of the captured image. The target area ROI is, in this example, an area of 90% or less of the size of the captured image. The target region ROI may be the entire region of the size of the captured image.

The adjustment range of the correction ring is a range in which the correction ring can be moved in this embodiment. The range in which this correction ring can be moved is set for each objective lens. The range in which the correction ring can be moved is, specifically, information indicating the minimum value and the maximum value of the degree of correction of the correction ring.

The information of the control target is the information of the target to be controlled based on the correction amount calculated by the correction amount calculation device 10. Here, the control target is the correction ring drive unit 22, the objective lens drive unit 24, the stage drive unit 27, and the like. In this embodiment, the information indicates the correction ring 21a and the correction ring driving unit 22 for driving the correction ring 21a.
When the observer wants to change the degree of correction by adjusting the correction position of the correction ring 21a, the observer inputs the information indicating the correction ring 21a and the correction ring drive unit 22 as the information to be controlled in the basic parameter. do. The correction ring drive unit 22, the objective lens drive unit 24, and the stage drive unit 27 are driven based on the correction amount calculated by the correction amount calculation device 10 to adjust the image observed by the microscope 20. Note that the basic parameters do not have to include information to be controlled. In this case, the correction amount calculation device 10 may calculate the correction amount of the control target corresponding to the condition indicated by the ruler information RPI based on the ruler information RPI designated by the observer.
The operation detection unit 17 outputs the detected operation result to the evaluation value calculation unit 14.

Ruler information RPI is stored in the storage unit ST. Here, the ruler information RPI will be described with reference to FIG.
FIG. 4 is a diagram showing an example of ruler information RPI.

The ruler information RPI is information in which a plurality of ruler images and condition parameters when the ruler images are captured are associated with each other. In the following description, a plurality of ruler images will also be referred to as a ruler image group. The conditional parameter is, in the present embodiment, the relative position ZL, as described above.
The ruler information RPI1 is information in which a plurality of captured images captured by making the relative positions ZL different from each other and the relative positions ZL when the captured images are captured are associated with each other. In the present embodiment, the ruler information RPI1 includes ruler image RP11 to ruler image RP15 captured with relative positions ZL different from each other. The ruler image RP11 is an image captured by capturing the image pickup target TP in the state of the relative position ZL1. Similarly, in the ruler image RP12 to the ruler image RP15, the relative positions ZL2 to the relative positions ZL5 are associated with each other.

Further, the ruler information RPI2 is information in which a plurality of captured images captured with different temperature TEs and the temperature TE when the captured images are captured are associated with each other. Specifically, the ruler information RPI2 includes ruler image RP21 to ruler image RP25 captured at different temperatures TE. The ruler image RP21 is an image captured by capturing the image target TP at the temperature TE1. Similarly, in the ruler image RP22 to the ruler image RP25, the temperatures TE2 to TE5 are associated with each other.
These ruler information RPIs are stored in the storage unit ST.

Returning to FIG. 3, the ruler information generation unit 18 acquires an captured image from the image acquisition unit 13. The ruler information generation unit 18 acquires the condition parameters associated with the captured image from the image acquisition unit 13. The ruler information generation unit 18 generates the ruler information RPI based on the captured image acquired from the image acquisition unit 13 and the condition parameters. Specifically, the ruler information generation unit 18 stores the ruler image, which is an image captured from the image acquisition unit 13, in the storage unit ST in a state in which the ruler image and the condition parameters when the ruler image is imaged are associated with each other. ..

The evaluation value calculation unit 14 acquires an observation image from the image acquisition unit 13. The evaluation value calculation unit 14 acquires ruler information from the ruler information generation unit 18. The evaluation value calculation unit 14 acquires a reference plane image from the reference focus plane information acquisition unit 16. The evaluation value calculation unit 14 acquires basic parameters from the operation detection unit 17.

The evaluation value calculation unit 14 calculates the evaluation value based on the observation image and the ruler image included in the ruler information. Here, the evaluation value is a value indicating the degree of correlation between a certain two captured images. In the following description, the evaluation value of correlation is also referred to as a correlation value. The evaluation value calculation unit 14 calculates the correlation value between the observation image acquired from the image acquisition unit 13 and the ruler image group included in the ruler information acquired from the ruler information generation unit 18, respectively. In the following description, a plurality of correlation values between the observation image and the ruler image are also referred to as observation surface correlation values. In this embodiment, the evaluation value calculation unit 14 calculates a higher correlation value in the case of an image in which two captured images are close to each other than in the case of an image in which the two captured images are not close to each other. If the basic parameter includes the designation of the target area ROI, the evaluation value calculation unit 14 calculates the correlation value of the designated area.

In the present embodiment, the evaluation value calculation unit 14 interpolates the calculated observation surface correlation values to calculate the peak observation surface correlation value. The peak observation surface correlation value is a correlation value in which the correlation value is higher than other correlation values in the range of the calculated observation surface correlation value. This peak observation surface correlation value is an example of the first evaluation value.
Specifically, the evaluation value calculation unit 14 interpolates the values between the correlation values and estimates the influence of the difference in the condition parameters on the calculated correlation value. The evaluation value calculation unit 14 estimates that the condition parameter corresponding to the estimated peak observation surface correlation value is the condition parameter corresponding to the observation image. The peak observation surface correlation value is, in other words, a condition parameter under the imaging conditions when the observation image is captured.

Further, the evaluation value calculation unit 14 calculates the correlation value based on the reference plane image and the ruler image included in the ruler information. Here, the evaluation value calculation unit 14 uses the same ruler information for calculating the observation surface correlation value and the reference surface correlation value. Specifically, the evaluation value calculation unit 14 has a plurality of correlation values between the reference surface image acquired from the reference focus surface information acquisition unit 16 and the ruler image included in the ruler information acquired from the ruler information generation unit 18. calculate. In the following description, a plurality of correlation values between the reference plane image and the ruler image are also referred to as reference plane correlation values. The ruler information generation unit 18 may generate ruler information. In this case, the ruler information generation unit 18 acquires an image taken by the same image pickup target TP with different degrees of correction amount among the image pickup conditions for the conditions that can be calculated by the own device. The ruler information generation unit 18 generates ruler information in which a plurality of images captured by the same image pickup target TP and conditions under which the correction amount when the image is captured can be calculated by the own device are associated with each other.

In the present embodiment, the evaluation value calculation unit 14 interpolates the calculated reference plane correlation values to calculate the peak reference plane correlation value. The peak reference plane correlation value is a correlation value in which the correlation value is higher than other correlation values in the range of the calculated reference plane correlation value. This peak reference plane correlation value is an example of the second evaluation value.
The evaluation value calculation unit 14 estimates that the condition parameter corresponding to the estimated peak reference plane correlation value is the condition parameter corresponding to the reference plane image. The peak reference plane correlation value is, in other words, a condition parameter under the imaging conditions when the reference plane image is captured.
The evaluation value calculation unit 14 outputs the calculated peak observation surface correlation value, the peak reference surface correlation value, and the basic parameter to the degree calculation unit 15.

The degree calculation unit 15 calculates the correction amount to be controlled based on the peak observation surface correlation value, the peak reference surface correlation value, and the basic parameters. Specifically, the degree calculation unit 15 acquires the peak observation surface correlation value and the peak reference surface correlation value from the evaluation value calculation unit 14. The degree calculation unit 15 calculates a correction amount for the microscope 20 based on the peak observation surface correlation value acquired from the evaluation value calculation unit 14, the peak reference surface correlation value, and the basic parameters. This correction amount may be a correction amount of a controlled object that can directly control the condition indicated by the condition parameter, and the degree of correction calculated by applying the condition indicated by the condition parameter to each term of the Zernike polynomial. It may be the correction amount of the control target corresponding to. The Zernike polynomial is an equation for calculating the aberration of the optical system.

More specifically, when the control target is specified in the basic parameter, the degree calculation unit 15 calculates the correction amount of the designated control target. In this embodiment, the degree calculation unit 15 calculates the correction amount of the relative position ZL of the objective lens 21 and the aberration correction amount of the correction ring 21a. That is, the degree calculation unit 15 calculates the drive direction and drive amount in which the objective lens drive unit 24 drives the objective lens 21, and the drive direction and drive amount in which the correction ring drive unit 22 drives the correction ring 21a.
The degree calculation unit 15 outputs the calculated correction amount corresponding to the focus surface OF of the reference focus surface to the display unit 11 and the correction ring drive unit 22.

In the present embodiment, the display unit 11 displays the correction amount calculated by the degree calculation unit 15. The correction ring driving unit 22 drives the correction ring 21a based on the correction amount supplied from the degree calculation unit 15.

[Outline of operation of correction amount calculation device 10]
Next, with reference to FIG. 5, an outline of the operation of the correction amount calculation device 10 will be described.
FIG. 5 is a flow chart showing an example of the operation of the correction amount calculation device 10. The processing procedure shown here is an example, and the processing procedure may be omitted or added.
The operation detection unit 17 acquires the basic parameters input by the observer's operation (step S110). The operation detection unit 17 supplies the acquired basic parameters to the evaluation value calculation unit 14. The operation detection unit 17 supplies the acquired basic parameters to the ruler information generation unit 18.

The reference focus plane information acquisition unit 16 acquires a reference plane image by the operation of the observer (step S120). Specifically, the observer focuses the objective lens on the portion to be observed while looking at the image of the TP to be imaged (the image visually confirmed or the image captured). At this time, the observer does not need to correct the aberration. The reference focus plane information acquisition unit 16 supplies the acquired reference plane image to the evaluation value calculation unit 14.

The evaluation value calculation unit 14 acquires basic parameters from the operation detection unit 17. The evaluation value calculation unit 14 acquires a reference plane image from the reference focus plane information acquisition unit 16. The evaluation value calculation unit 14 acquires a ruler image group from the ruler information generation unit 18 (step S130). Specifically, the evaluation value calculation unit 14 acquires ruler information from the ruler information generation unit 18.

The imaging unit 25 captures an image of the observation object as a pre-observation image. The pre-observation image is an observation image captured at the correction position CL of a certain correction ring. The image pickup unit 25 supplies the pre-observation image to the image acquisition unit 13. The image acquisition unit 13 acquires a pre-observation image from the image pickup unit 25 (step S140). The image acquisition unit 13 supplies the pre-observation image acquired from the image pickup unit 25 to the evaluation value calculation unit 14.

The evaluation value calculation unit 14 calculates the maximum correlation value between the pre-observation image acquired from the image acquisition unit 13 and the ruler image group RP included in the ruler information RPI (step S142). Specifically, the evaluation value calculation unit 14 acquires a pre-observation image from the image acquisition unit 13. The evaluation value calculation unit 14 calculates the correlation value between the pre-observation image acquired from the image acquisition unit 13 and the ruler image group RP included in the ruler information RPI. The evaluation value calculation unit 14 calculates the above-mentioned peak observation surface correlation value based on the calculated correlation value.

The evaluation value calculation unit 14 supplies the calculated peak observation surface correlation value to the degree calculation unit 15. The degree calculation unit 15 acquires the peak observation surface correlation value from the evaluation value calculation unit 14. The degree calculation unit 15 calculates the position of the relative position ZL corresponding to the peak observation surface correlation value.
The degree calculation unit 15 calculates the drive direction and the drive amount for driving the objective lens 21 at the position of the relative position ZL corresponding to the calculated peak observation surface correlation value. The degree calculation unit 15 supplies the calculated drive direction and drive amount of the objective lens 21 to the objective lens drive unit 24.

The objective lens driving unit 24 acquires the driving direction and driving amount of the objective lens 21 from the degree calculation unit 15. The objective lens driving unit 24 drives the objective lens 21 based on the driving direction and driving amount of the objective lens 21 acquired from the degree calculation unit 15.

The image acquisition unit 13 acquires an observation image group at the relative position ZL of the maximum correlation value between the pre-observation image and the ruler image group RP included in the ruler information RPI from the imaging unit 25 (step S144). The observation image group is an observation image of the relative position ZL corresponding to the peak observation surface correlation value, and a plurality of observation surface images taken by changing the relative position ZL.
The image acquisition unit 13 supplies the observation image group acquired from the image pickup unit 25 to the evaluation value calculation unit 14.

The evaluation value calculation unit 14 acquires an observation image group from the image acquisition unit 13. The evaluation value calculation unit 14 calculates the maximum correlation value between the observation image group acquired from the image acquisition unit 13 and the reference image (step S146). Specifically, the evaluation value calculation unit 14 calculates the correlation value between the reference plane image and the plurality of observation images included in the observation image group, respectively. In the following description, the correlation value between the reference plane image and the plurality of observation images included in the observation image group is also described as a reference comparison correlation value. The evaluation value calculation unit 14 calculates the maximum correlation value based on the calculated reference comparison correlation value. In the present embodiment, the evaluation value calculation unit 14 interpolates the reference comparison correlation values to calculate the peak reference comparison correlation value.
The evaluation value calculation unit 14 supplies the calculated peak reference comparison correlation value to the degree calculation unit 15. The degree calculation unit 15 acquires a peak reference comparison correlation value from the evaluation value calculation unit 14.

The degree calculation unit 15 selects the relative position ZL of the maximum correlation value between the observation image group and the reference image (step S148). Specifically, the degree calculation unit 15 calculates the position of the relative position ZL corresponding to the peak reference comparison correlation value.
The degree calculation unit 15 calculates the drive direction and the drive amount for driving the objective lens 21 at the position of the relative position ZL corresponding to the calculated peak reference comparison correlation value. The degree calculation unit 15 supplies the calculated drive direction and drive amount of the objective lens 21 to the objective lens drive unit 24.
The objective lens driving unit 24 acquires the driving direction and driving amount of the objective lens 21 from the degree calculation unit 15. The objective lens driving unit 24 drives the objective lens 21 based on the driving direction and driving amount of the objective lens 21 acquired from the degree calculation unit 15.

The image acquisition unit 13 acquires an observation image captured at the relative position ZL of the maximum correlation value between the observation image group and the reference image from the imaging unit 25. In the following description, the observation image captured at the relative position ZL of the maximum correlation value between the observation image group and the reference image is also referred to as an observation reference plane image. This observation reference plane image is an image obtained by capturing a position close to the focal position captured in the reference plane image at the correction position CL of a certain correction ring. The image acquisition unit 13 supplies the observation reference plane image to the evaluation value calculation unit 14.
The evaluation value calculation unit 14 acquires an observation reference plane image from the image acquisition unit 13. The evaluation value calculation unit 14 calculates the evaluation value of the observation reference surface image and the reference surface image (step S150). In the following description, the evaluation value of the observation reference plane image and the reference plane image is also described as the observation reference plane correlation value.

The evaluation value calculation unit 14 determines whether or not the evaluation values of the observation reference plane images of the correction positions CL of all the correction rings have been calculated (step S160).
When the evaluation value calculation unit 14 determines that the evaluation values of the observation reference plane images of the correction positions CL of all the correction rings have not been calculated, the relative position ZL selected in step S148 is left as it is. The process is repeated from step S140 by changing the position of the correction position CL of the correction ring (step S160; NO). The position of the correction position CL of the correction ring is, for example, a position where the correction amount adjusted by the correction ring 21a is equally divided and changed by the equally divided correction amount. That is, the evaluation value calculation unit 14 repeatedly performs the processes from step S140 to step S160 while changing the correction position of the correction ring 21a.

When the evaluation value calculation unit 14 determines that the evaluation values of the observation reference plane image have been calculated for all the correction ring correction position CLs (step S160; YES), the evaluation value calculation unit 14 observes the correction ring for each correction position CL. The optimum correction amount is calculated based on the surface correlation value (step S170). In the present embodiment, the evaluation value calculation unit 14 interpolates the observation reference plane correlation values for each correction position CL of the correction ring to calculate the peak observation reference plane correlation value. This peak observation reference plane correlation value is a correlation value indicating the correction position CL of the correction ring to which the observation reference plane image and the reference plane image are approximated.

The evaluation value calculation unit 14 supplies the peak observation reference plane correlation value to the degree calculation unit 15. The degree calculation unit 15 acquires the peak observation reference plane correlation value from the evaluation value calculation unit 14. The degree calculation unit 15 calculates the drive direction and the drive amount for driving the correction ring 21a at the correction position CL of the correction ring indicated by the peak observation reference plane correlation value acquired from the evaluation value calculation unit 14 as the optimum correction amount. (Step S180).

[Example of peak observation surface correlation value and peak reference surface correlation value]
Here, with reference to FIGS. 6 and 7, the peak observation surface correlation value calculated by the evaluation value calculation unit 14 and the peak reference surface correlation value will be described.
FIG. 6 is a diagram showing an example of the reference plane image TFI1, the observation image NP1, and the ruler information RPI1.
As described above, the ruler information RPI1 includes the ruler image RP11 and the relative position ZL as a conditional parameter in a state of being associated with each other. The ruler information RPI1 also includes the ruler image RP12 to the ruler image RP15 in a state in which the relative positions ZL2 to the relative positions ZL5 are associated with each other. In this embodiment, the relative position ZL is sequentially increased from the relative position ZL1 to the relative position ZL5. That is, the relative position ZL5 has a larger degree of correction CL than the relative position ZL1.

FIG. 7 is a diagram showing an example of the reference plane correlation value CRV and the observation plane correlation value NRV.
FIG. 7A is a graph showing an example of the reference plane correlation value CRV and the peak reference plane correlation value CRP.
The graph of FIG. 7A is a graph showing the relative position ZL, which is a conditional parameter included in the ruler information RPI1, on the horizontal axis and the reference plane correlation value CRV on the vertical axis.
The reference plane correlation value CRV1 is a correlation value between the reference plane image TFI1 and the ruler image RP11. Similarly, the reference plane correlation value CRV2 to the reference plane correlation value CRV5 are the correlation values of the reference plane image TFI1 and the ruler image RP12 to the ruler image RP15, respectively. The evaluation value calculation unit 14 calculates the curve L1 by complementing the reference plane correlation values CRVs. In this curve L1, the correlation value of the peak portion is the peak reference plane correlation value CRP. The relative position CZL corresponding to the peak reference plane correlation value CRP is a relative position between the objective lens 21 and the image target TP when the reference plane image is captured.

FIG. 7B is a graph showing an example of the observation surface correlation value NRV and the peak observation surface correlation value NRP.
The graph of FIG. 7B is a graph showing the relative position ZL, which is a conditional parameter included in the ruler information RPI1, on the horizontal axis and the observation surface correlation value NRV on the vertical axis.
The observation surface correlation value NRV1 is a correlation value between the observation image NP1 and the ruler image RP11. Similarly, the observation surface correlation value NRV2 to the observation surface correlation value NRV5 are correlation values between the observation image NP1 and each image from the ruler image RP12 to the ruler image RP15. The evaluation value calculation unit 14 calculates the curve L2 by complementing the observation surface correlation values NRVs. In this curve L2, the correlation value of the peak portion is the peak observation surface correlation value NRP. The relative position NZL corresponding to the peak observation surface correlation value NRP is a relative position between the position of the objective lens 21 when the observation image is imaged and the position of the image pickup target TP.

The degree calculation unit 15 calculates the drive direction and the drive amount of the objective lens 21 based on the difference DL between the relative position CZL calculated by the evaluation value calculation unit 14 and the relative position NZL.

Here, the calculation method of the curves L1 and L2 will be described. In the following description, when the reference plane correlation value and the observation plane correlation value are not distinguished, they are also described as the correlation value RV.
The evaluation value calculation unit 14 fits the correlation values RVs to each other by a quadratic curve. Specifically, the evaluation value calculation unit 14 fits the correlation values RVs to each other by a quadratic curve fitting method by the least squares method. The evaluation value calculation unit 14 may fit the correlation values RV to each other by linear function fitting or exponential function fitting.
The interpolation when calculating the peak reference comparison correlation value and the peak observation reference plane correlation value described above may be performed by quadratic curve fitting in the same manner as the method of fitting the correlation values RVs to each other.

The correction amount calculation device 10 may reacquire the ruler information including the position of the correction ring after correction in step S160. Further, the correction amount calculation device 10 may generate ruler information including a new ruler image including the position of the correction ring after correction.

[How to calculate the correlation value]
Next, the evaluation value calculation unit 14 will explain a method of calculating the correlation value. The evaluation value calculation unit 14 correlates by comparing the statistical value of the brightness of the captured image, the brightness distribution of the captured image, the contrast of the captured image, the frequency component of the captured image, the point image distribution function of the captured image, and the spatial distribution of the brightness. There is a way to calculate the value. In the following description, when the reference plane image TFI and the observation image NP are not distinguished, they are also described as comparative images.

Specifically, when the evaluation value calculation unit 14 calculates the correlation value based on the brightness value of the captured image, the evaluation value calculation unit 14 calculates the correlation value by comparing the maximum value of the brightness included in the captured image.
Further, the evaluation value calculation unit 14 determines that the larger the contrast, the larger the luminance dispersion value, or the larger the luminance differential value with the adjacent pixel, the better the spherical aberration is corrected, and the correlation value. Is calculated.

When the evaluation value calculation unit 14 calculates the correlation value based on the frequency component of the spatial frequency of the image of the captured image 8, the value of the frequency component obtained by Fourier transform or the like is used as the correlation value. This is because the better the spherical aberration is corrected, the larger the value of the high frequency component becomes.
When the evaluation value calculation unit 14 calculates the correlation value based on the point image distribution function, the evaluation value calculation unit 14 sets the point image distribution function as the correlation value. The better the spherical aberration is corrected, the smaller the point spread function.

When calculating an image having a close spatial distribution of luminance, there is a method of calculating the difference between the comparison image and the reference image in addition to calculating the correlation between the comparison image and the reference image. In this example, the combination of the comparative image and the reference image includes the combination of the above-mentioned "ruler image group" and "pre-observation image", and the combination of "observation image group" and "reference plane image". .. This comparative image is an example of a plurality of images acquired by the first image acquisition unit.
Specifically, the ruler image obtained by capturing the same focal position OF as the comparative image is an image having a close spatial distribution. An image having a similar spatial distribution is an image in which the shape and arrangement of the captured observation objects are similar.
The evaluation value calculation unit 14 may calculate the luminance correlation between the comparison image and the ruler image, respectively. This luminance correlation is calculated by normalization cross-correlation and the like.
The evaluation value calculation unit 14 may calculate the correlation between the comparison image and the plurality of ruler images, and compare the calculated correlations with each other. In this case, the evaluation value calculation unit 14 determines that the ruler image showing a higher correlation was captured under conditions close to the imaging conditions captured in the comparative image.

[Summary of the first embodiment]
As described above, the correction amount calculation device 10 includes an image acquisition unit 13, an evaluation value calculation unit 14, a degree calculation unit 15, a reference focus plane information acquisition unit 16, and a ruler information generation unit 18. The image acquisition unit 13 acquires an observation image. The evaluation value calculation unit 14 acquires ruler information from the ruler information generation unit 18. The evaluation value calculation unit 14 acquires an observation image from the image acquisition unit 13.
The evaluation value calculation unit 14 has a reference surface correlation value based on the reference surface image supplied from the reference focus surface information acquisition unit 16 and a plurality of ruler images included in the ruler information supplied from the ruler information generation unit 18. Is calculated. The evaluation value calculation unit 14 calculates the observation surface correlation value based on the observation image supplied from the image acquisition unit 13 and the plurality of ruler images included in the ruler information supplied from the ruler information generation unit 18. The degree calculation unit 15 acquires the reference plane correlation value and the observation plane correlation value from the evaluation value calculation unit 14. The degree calculation unit 15 calculates the correction amount of the controlled object based on the reference plane correlation value and the observation plane correlation value. As a result, the correction amount calculation device 10 can adjust the control target based on the captured images captured by a plurality of the same imaging targets. That is, the correction amount calculation device 10 can adjust the control target based on the correlation between the comparison image and the reference image.

Further, the correction amount calculation device 10 can generate ruler information by including the ruler information generation unit 18. This ruler information may be generated in advance. As a result, the correction amount calculation device 10 can suppress the number of imaging times when observing a sample or the like whose fluorescence brightness is reduced by imaging.

Further, the correction amount calculation device 10 includes an operation detection unit 17. The correction amount calculation device 10 can designate a part of the captured image as a range to be evaluated based on the target region ROI included in the basic parameters input from the operation detection unit 17. By this designation, the correction amount calculation device 10 can reduce the time required for calculating the correlation value as compared with the case where the range is not specified.

In the above description, the case where the objective lens 21 is an oil-immersed objective lens or a liquid-immersed objective lens such as a water-immersed objective lens has been described, but any objective lens may be used. That is, the objective lens 21 may be a dry objective lens that does not correspond to immersion.

When the number of vertical and horizontal pixels of the target area ROI described above is an integral multiple of 32 pixels, the evaluation value calculation unit 14 calculates the spatial frequency in the target area ROI by a fast Fourier transform. can do. In this case, the evaluation value calculation unit 14 can calculate the correlation value RV at high speed.

In the above description, the evaluation value calculation unit 14 is based on the luminance value of the captured image, the brightness distribution of the captured image, the contrast of the captured image, the frequency component of the spatial frequency of the captured image, the point image distribution function of the captured image, and the like. The case of calculating the correlation value RV has been described.
When the evaluation value calculation unit 14 calculates the correlation value RV based on the spatial frequency component, the spatial frequency indicated by the comparative image and a certain image among the ruler images indicate each ruler image having a different correction amount CL. The evaluation value RV is calculated based on the spatial frequency. Specifically, the evaluation value calculation unit 14 calculates the correlation value RV based on the spatial frequency of a predetermined band among the spatial frequencies. In this example, the evaluation value calculation unit 14 calculates the correlation value RV based on the ratio of the values of the low frequency component and the high frequency component.

In the above description, the case where the second image is a captured image in which the image pickup target TP being observed is captured is not limited to this. The second image may be a captured image observed in the past.
Further, in the above description, the case where the third image is an image different from the ruler image has been described, but the present invention is not limited to this. The third image may be one of the ruler images included in the ruler information. In this case, the evaluation value calculation unit 14 may use the value indicated by the condition parameter associated with the third image as the peak reference plane correlation value.

In the above description, the case where the number of ruler image RPs and condition parameters included in the ruler information is 5 each has been described, but the present invention is not limited to this. Further, the evaluation value calculation unit 14 may calculate the correlation value RV between a part of the ruler image included in the ruler information and the comparison image.

In the above description, the evaluation value calculation unit 14 has described a configuration in which the peak observation surface correlation value is calculated by interpolating the observation surface correlation values, but the present invention is not limited to this. For example, the evaluation value calculation unit 14 may calculate the highest correlation value among the observation surface correlation values as the peak observation surface correlation value. Further, the configuration of calculating the peak reference plane correlation value by interpolating the reference plane correlation values has been described, but the present invention is not limited to this.

[Second Embodiment]
Up to this point, the configuration of the correction amount calculation device 10 for calculating the correction amount based on one ruler information has been described. Next, a configuration of a correction amount calculation device that calculates a correction amount based on a plurality of ruler information will be described. The same configurations and operations as those of the first embodiment are designated by the same reference numerals, and the description thereof will be omitted.

Next, the functional configuration of the correction amount calculation device 10a will be described with reference to FIGS. 8 and 9.
FIG. 8 is a diagram showing an example of the functional configuration of the microscope 20a of the second embodiment and the correction amount calculation device 10a.
The microscope 20a includes a light modulation unit 28a. The optical modulation unit 28a is arranged on the optical path OP shown in FIG. The optical modulation unit 28a modulates the light on the optical path OP. Modulation is to change the phase and amplitude of light. The optical modulation unit 28a corrects aberrations by modulating the light. Specifically, the optical modulation unit 28a is an optical element such as a spatial light modulator or a liquid crystal element.

The correction amount calculation device 10a includes a control unit 12a. The control unit 12a includes an evaluation value calculation unit 14a and a degree calculation unit 15a.
The evaluation value calculation unit 14a acquires an observation image from the image acquisition unit 13. The evaluation value calculation unit 14a acquires a plurality of ruler information from the ruler information generation unit 18. The evaluation value calculation unit 14a acquires a reference plane image from the reference focus plane information acquisition unit 16. The evaluation value calculation unit 14a acquires basic parameters from the operation detection unit 17.

The evaluation value calculation unit 14a calculates the peak observation surface correlation value based on the observation image and the ruler image included in the ruler information. The evaluation value calculation unit 14a calculates this peak observation surface correlation value for each of a plurality of ruler information. The evaluation value calculation unit 14a supplies the calculated plurality of peak observation surface correlation values to the degree calculation unit 15a.
Further, the evaluation value calculation unit 14a calculates the peak reference plane correlation value based on the reference plane image and the ruler image included in the ruler information. The evaluation value calculation unit 14a calculates this peak reference plane correlation value for each ruler information. The evaluation value calculation unit 14a supplies the calculated plurality of peak reference plane correlation values to the degree calculation unit 15a.

Here, with reference to FIG. 9, a plurality of ruler information acquired by the evaluation value calculation unit 14a will be described.
FIG. 9 is a diagram showing an example of a multi-axis ruler information RPC.

FIG. 9A is an example of the observation image ONP. In the observation image ONP, a living cell OB, which is a TP to be imaged, and a plurality of beads BE are imaged. FIG. 9B is a graph in which the multi-axis ruler information RPC, the reference plane image TFI2, and the observation image NP2 are arranged in order of the imaging time. Here, the multi-axis ruler information RPC is a plurality of ruler information RPIs. In this embodiment, the multi-axis ruler information RPC includes ruler information RPI91, ruler information RPI92, and ruler information RPI93. The multi-axis ruler information RPC includes a plurality of ruler image RPs captured around time t = 0. Conditional parameters related to the change in the refractive index of the substance in the optical path OP of the microscope 20a and the change in the aberration are set in the ruler information included in the multi-axis ruler information RPC. Specifically, this conditional parameter is a parameter corresponding to each term of the Zernike polynomial described above. The ruler information included in the multi-axis ruler information RPC is set so that the types of the condition parameters are different from each other. The types of condition parameters are, for example, the correction position CL and the temperature TE of the correction ring described above.

Here, the same bead BE is imaged in each of the plurality of ruler images, the reference plane image TFI2, and the observation image NP2 included in the multi-axis ruler information RPC. Here, the reference plane image TFI2 and the observation image NP2 are images of the region where the beads BE included in the observation image ONP are captured. Living cell OBs may change shape over time. The correction amount calculation device 10a calculates the correction amount in a shorter time by calculating the correlation value RV between the images captured by the images to be imaged whose shape and position are unlikely to change according to the time change such as the bead BE. can do.

Returning to FIG. 8, the degree calculation unit 15a acquires a plurality of peak reference plane correlation values from the evaluation value calculation unit 14a. The degree calculation unit 15a acquires a plurality of peak observation surface correlation values from the evaluation value calculation unit 14a. The degree calculation unit 15a calculates the correction amount of the aberration corresponding to this condition parameter based on the peak reference plane correlation value having the same condition parameter and the peak observation plane correlation value. The degree calculation unit 15a calculates the correction amount of the aberration corresponding to the condition parameter based on at least two of the acquired plurality of peak reference plane correlation values and peak observation plane correlation values. In this embodiment, the degree calculation unit 15a calculates a correction amount for correcting the degree of modulation of the optical modulation unit 28a. The correction amount for correcting the degree of modulation of the optical modulation unit 28a is a correction amount based on the aberration correction amount calculated from the Zernike polynomial.

[Summary of the second embodiment]
As described above, the correction amount calculation device 10a includes an evaluation value calculation unit 14a and a degree calculation unit 15a. The evaluation value calculation unit 14a acquires the multi-axis ruler information RPC from the ruler information generation unit 18. The multi-axis ruler information RPC includes a plurality of ruler information RPIs having different types of condition parameters. The evaluation value calculation unit 14a is based on the reference surface image supplied from the reference focus surface information acquisition unit 16 and the plurality of ruler images included in the multi-axis ruler information RPC supplied from the ruler information generation unit 18. The reference plane correlation value is calculated for each parameter type. The evaluation value calculation unit 14a is based on the observation image supplied from the image acquisition unit 13 and the plurality of ruler images included in the multi-axis ruler information RPC supplied from the ruler information generation unit 18, for each type of condition parameter. Calculate the observation surface correlation value. The degree calculation unit 15a acquires a plurality of reference plane correlation values and observation plane correlation values from the evaluation value calculation unit 14a. The degree calculation unit 15a calculates the correction amount of the optical modulation unit 28a based on the plurality of reference surface correlation values and the observation surface correlation values. That is, the degree calculation unit 15a calculates the correction amount based on a plurality of conditions in which the types of condition parameters are different from each other. As a result, the correction amount calculation device 10a can calculate the correction amount in a shorter time than the correction amount calculation device 10 of the first embodiment.

In the above description, the degree calculation unit shown in the first embodiment and the second embodiment has described the case where the correction amount of the correction ring drive unit 22 is calculated, but the control target is not limited to this. .. The degree calculation unit 15 may calculate the correction amount of the control target for correcting the condition indicated by the condition parameter of the ruler information RPI. Specifically, the condition parameters include the positions of the image pickup target TP and the objective lens 21, the degree of spherical aberration correction of the objective lens 21, temperature conditions, the passage of time, and the image pickup target TP and the image pickup unit 25. It is sufficient that at least one of the aberrations on the optical path OP between them is included.

When the correction amount calculation device corrects the positions of the image pickup target TP and the objective lens 21, the ruler information includes the ruler image captured by changing the position of the image pickup target TP and the objective lens 21 with the condition parameter. It suffices if it is included. When this position is the position in the optical axis direction between the image pickup target TP and the objective lens, the correction amount calculation device calculates the correction amount of the objective lens driving unit 24. When this position is orthogonal to the optical axis direction of the image pickup target TP and the objective lens, the correction amount calculation device calculates the correction amount of the stage drive unit 27. As the vertical movement drive mechanism of the stage 23, a stage vertical movement mechanism of a general microscope can be used. For example, a cam mechanism or an engaging member that meshes with a gear can be used. Further, as a mechanism for moving the objective lens side in the optical axis direction, a mechanism used in a general microscope can be used. For example, a gear and an engaging member, a vertical movement mechanism by a link mechanism, a cam mechanism, and the like can be used.

When the correction amount calculation device corrects the temperature condition, the ruler information may include a ruler image captured by changing the temperature as a condition parameter. In this case, the correction amount calculation device may calculate the correction amount of the temperature adjusting device (not shown). For example, the temperature control device is an air conditioner that changes the temperature of the environment in which the microscope 20 is installed.

When the correction amount calculation device corrects the passage of time, the ruler information may include ruler images captured at different times as conditional parameters. In this case, the correction amount calculation device may calculate the correction amount for adjusting the time for capturing the observation image.

When the correction amount calculation device corrects the aberration on the optical path OP between the image pickup target TP and the image pickup unit 25, the ruler information includes a ruler image captured by changing the variables of each term of the Zernike polynomial. It suffices if it is included. In this case, the correction amount calculation device may calculate the correction amount for correcting the degree of light modulation of the optical modulation unit 28a. Further, the correction amount calculation device may calculate the correction amount of the temperature control device. The correction amount calculation device may calculate the correction amount of the correction position CL of the correction ring.

The microscope 20 may be provided with the correction amount calculation device shown in the first embodiment and the second embodiment described above.

Although the embodiment of the present invention has been described in detail with reference to the drawings, the specific configuration is not limited to this embodiment and may be appropriately modified without departing from the spirit of the present invention. can.

The correction amount calculation device 10 and the correction amount calculation device 10a described above have a computer inside. The process of each process of the above-mentioned apparatus is stored in a computer-readable recording medium in the form of a program, and the process is performed by the computer reading and executing this program. Here, the computer-readable recording medium refers to a magnetic disk, a magneto-optical disk, a CD-ROM, a DVD-ROM, a semiconductor memory, or the like. Further, the computer program may be distributed to the computer via a communication line, and the computer receiving the distribution may execute the program.

Further, the above program may be for realizing a part of the above-mentioned functions.
Further, it may be a so-called difference file (difference program) that can realize the above-mentioned function in combination with a program already recorded in the computer system.

10, 10a ... Correction amount calculation device, 11 ... Display unit, 12, 12a ... Control unit, 13 ... Image acquisition unit, 14 ... Evaluation value calculation unit, 15 ... Degree calculation unit, 16 ... Reference focus plane information acquisition unit, 17 ... Operation detection unit, 18 ... Ruler information generation unit, 20, 20a ... Microscope, 21 ... Objective lens, 21a ... Correction ring, 22 ... Correction ring drive unit, 23 ... Stage, 24 ... Objective lens drive unit, 25 ... Imaging unit , 26 ... Wavelength branch, 27 ... Stage drive, 28a ... Optical modulator, 100 ... Microscope system

Claims (8)

A correction amount calculation device for calculating an aberration correction amount for an image pickup device including an image pickup unit that captures an image pickup target.
The pre SL correction amount a calculated possible conditions the device itself, the positions of the imaging target and the objective lens, the degree of spherical aberration correction of the objective lens, and temperature conditions, and time elapsed, the imaging target A first image acquisition unit that acquires a plurality of images captured by the same imaging target by varying the degree of imaging conditions including at least one of aberrations on the optical path between the image and the imaging unit.
A second image acquisition unit that acquires a second image which the imaging target is imaged,
A third image acquisition unit that acquires a third image captured by the image pickup target at the focal position that the observer wants to observe, and a third image acquisition unit.
A first evaluation value calculation unit that calculates a first evaluation value that is an evaluation value based on a plurality of the images acquired by the first image acquisition unit and the second image acquired by the second image acquisition unit. When,
A second evaluation value calculation unit that calculates a second evaluation value that is an evaluation value based on the plurality of images acquired by the first image acquisition unit and the third image acquired by the third image acquisition unit. When,
A correction amount calculation unit that calculates the correction amount based on the first evaluation value calculated by the first evaluation value calculation unit and the second evaluation value calculated by the second evaluation value calculation unit. A correction amount calculation device including. The correction amount calculation unit is
The correction amount calculation device according to claim 1, wherein the correction amount is calculated based on a plurality of conditions in which the conditions are different from each other. The first evaluation value calculation unit is
The evaluation value with the second image is calculated for each of the plurality of images, and the first evaluation value is calculated by complementing the calculated plurality of evaluation values.
The second evaluation value calculation unit is
The second evaluation value is calculated by calculating an evaluation value with the third image for each of the plurality of the images and complementing the calculated evaluation values with each other. The second evaluation value is calculated according to claim 1 or 2. Correction amount calculation device. It said objective lens having a spherical surface aberration correction optical system,
A drive unit that adjusts the aberration correction amount of the spherical aberration correction optical system of the objective lens based on the correction amount calculated by the correction amount calculation unit according to any one of claims 1 to 3. microscope. An imaging unit that captures the image to be imaged, and an imaging unit that captures the image.
An aberration correction unit that corrects aberrations on the optical path between the image pickup target and the image pickup unit,
Equipped with
The photoaberration correction unit is
A microscope that adjusts the aberration correction amount of the optical aberration correction unit based on the correction amount calculated by the correction amount calculation unit according to any one of claims 1 to 3. A microscope provided with a drive unit that moves the relative position between the objective lens and the stage on which the image pickup target is placed based on the correction amount calculated by the correction amount calculation unit according to any one of claims 1 to 3. .. A computer equipped with a correction amount calculation device for calculating an aberration correction amount related to an image pickup device including an image pickup unit that captures an image pickup target.
The conditions under which the correction amount can be calculated by the own device are the positions of the image pickup target and the objective lens, the degree of spherical aberration correction of the objective lens, the temperature condition, the passage of time, and the image pickup target. Acquiring a plurality of images in which the same image pickup target is captured by different degrees of imaging conditions including at least one of aberrations on the optical path between the image pickup unit and the image pickup unit.
Acquiring the second image captured by the imaging target, and
Acquiring a third image in which the image pickup target is captured at the focal position that the observer wants to observe.
And calculating a plurality of the images acquired, the first evaluation value is an evaluation value based on the acquired said second image,
And calculating a plurality of the images acquired, the second evaluation value is obtained evaluation values based on said third image,
To calculate the correction amount based on the calculated first evaluation value and the calculated second evaluation value.
Correction amount calculation program for executing. Among the imaging conditions, the condition is such that the own device can calculate the amount of aberration correction for the imaging device including the imaging unit that captures the imaging target, and the positions of the imaging target and the objective lens and the spherical surface of the objective lens. The same imaging object with different degrees of aberration correction, temperature conditions, time lapse, and imaging conditions including at least one of aberrations on the optical path between the imaging object and the imaging unit. The first image acquisition step to acquire multiple images captured by
A second image acquisition step of acquiring a second image of the imaging target is imaged,
The third image acquisition step of acquiring the third image captured by the image pickup target at the focal position desired to be observed by the observer, and the third image acquisition step.
A first evaluation value for calculating a first evaluation value, which is an evaluation value based on a plurality of the images acquired from the first image acquisition step and the second image acquired from the second image acquisition step. Calculation steps and
A second evaluation value for calculating a second evaluation value, which is an evaluation value based on the plurality of images acquired from the first image acquisition step and the third image acquired from the third image acquisition step. Calculation steps and
Correction amount calculation to calculate the correction amount based on the first evaluation value calculated from the first evaluation value calculation step and the second evaluation value calculated from the second evaluation value calculation step. A correction amount calculation method having steps and.

Images