JP6822483B2 - Spherical aberration correction calculation device, microscope, spherical aberration correction calculation program and spherical aberration correction calculation method - Google Patents

Description

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

Conventionally, there are known microscope devices that drive a correction ring that adjusts the influence of spherical aberration caused by the optical system of a specimen and a microscope by power from an electric motor or the like (for example, Patent Document 1).

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

With the objective lens having the spherical aberration correction optical system, the stage on which the imaging object is placed, and the spherical aberration correction optical system adjusted by the first aberration correction amount, the relative distance between the objective lens and the stage is determined. The relative distance between the objective lens and the stage in a state where the spherical aberration correction optical system is adjusted by the second aberration correction amount and the first image group obtained by continuously shooting the imaging object while changing. An image group generation unit that generates an image group including a second image group obtained by continuously shooting the imaged object while changing the aberration, and a first of the image groups based on a predetermined reference image. An evaluation value calculation unit that selects three image groups and calculates an evaluation value for each image constituting the third image group, and an optimum value of an aberration correction amount of the objective lens based on the evaluation value. The reference image is imaged in a state where the aberration correction amount of the objective lens is set to an arbitrary aberration correction amount and the image pickup target is in focus of the objective lens. A spherical aberration correction calculation device that is an image of the observed position .

It is a figure which shows an example of the appearance structure of the spherical aberration correction optical system system. It is an enlarged view of a part of the appearance composition of an image pickup optical system. It is a figure which shows an example of the functional structure of the spherical aberration correction calculation apparatus. It is a flow chart which shows an example of the operation of the spherical aberration correction calculation apparatus. It is a figure which shows an example of the captured image. It is a figure which shows an example of quadratic curve fitting of evaluation values. It is a figure which shows an example of the functional structure of the spherical aberration correction calculation apparatus. It is a flow chart which shows an example of the operation of the spherical aberration correction calculation apparatus.

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

The spherical aberration correction unit 20 is a partial configuration of a microscope involved in magnifying a sample and observing and acquiring an observed image by an objective lens provided with a correction ring capable of controlling the degree of correction of spherical aberration by an electric motor. .. 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).
Spherical aberration is an aberration in which light rays emitted from a point light source do not focus on a single 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.

[About the spherical aberration correction unit 20]
Here, the spherical aberration correction unit 20 will be described with reference to FIG. In the following description, the case where the spherical aberration correction unit 20 is partially configured as an inverted microscope will be described, but the present invention is not limited to this. The spherical aberration correction unit 20 may have a partial configuration 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 spherical aberration correction unit 20 which is a part of the microscope. In FIG. 1, the spherical aberration correction unit 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 the revolver (not shown) that holds the objective lens up and down. Can be moved to. In FIG. 2, when the relative position between the objective lens 21 and the imaging target TP changes, the focal position of the objective lens 21 changes. In the following description, the focal position of the objective lens 21 will be referred to as 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 in the optical axis direction of the aberration correction lens arranged on the optical axis of the objective lens 21 in the lens barrel that holds the objective lens 21. 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. 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. The correction amount CL is the degree of spherical aberration corrected by the correction ring 21a. The correction ring 21a having such an annular structure is not indispensable, and a cam structure may be used to move the aberration correction lens in the direction of the optical axis OA. Further, instead of the aberration correction lens, a mirror that corrects 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 as an aberration correction optical system, and by adjusting the transmission of observation light incident on the observation optical system, the spherical aberration is formed. Correct the aberration.

The correction ring driving unit 22 drives the correction position of the correction ring 21a. The correction ring 22a 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 a 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 imaging target TP placed on the stage 23 is imaged on the imaging surface of the imaging unit 25 with an imaging lens (not shown) via the objective lens 21. In this example, the imaging target TP is a biological sample having a thickness of TPD. The image pickup unit 25 captures the 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 imaging target TP is arranged on the cover glass CG. Illumination light is applied to the image target TP. The objective lens 21 receives the light transmitted through the image pickup target TP. Spherical aberration is also caused by the wavelength of light transmitted through the TP to be imaged.

The branching portion 26 branches the light from the imaging target TP imaged by the objective lens 21 into the imaging unit 25 and the 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, so that the fluorescence emitted from the fluorescent reagent added to the cells is imaged as an 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. A treatment for fixing cells is performed using a reagent such as formaldehyde. The biological sample imaged by the imaging unit 25 may be, for example, a fixed sample, a transparent sample, or a living sample.

Further, the spherical aberration correction unit 20 transmits 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 in the above-mentioned captured image TI. It may be imaged as. As a result, the spherical aberration correction unit 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 acquired. 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 a 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. When the immersion space i is filled with water, the spherical aberration changes depending on the thickness of the cover glass. Further, the distance between the cover glass CG and the focal position OF changes as the observation position changes inside the imaging target TP. When the objective lens 21 is an oil-immersed 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 spherical aberration correction calculation device 10 calculates the correction amount of the spherical aberration of the image.
In the present embodiment, the spherical aberration correction calculation device 10 includes a display unit 11 and a control unit 12. The control unit 12 calculates the degree of correction of spherical aberration of the objective lens 21. The display unit 11 displays the captured image captured by the spherical aberration correction unit 20. The display unit 11 displays the result calculated by the control unit 12. The spherical aberration correction 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 spherical aberration correction calculation device 10]
Next, the functional configuration of the spherical aberration correction calculation device 10 will be described with reference to FIG.
FIG. 3 is a diagram showing an example of the functional configuration of the spherical aberration correction calculation device 10 of the first embodiment.

The control unit 12 includes an image acquisition unit 13, an evaluation value calculation unit 14, a reference focus surface information acquisition unit 16, an operation detection unit 17, and a degree calculation unit 15 for calculating a correction amount. Here, the degree calculation unit 15 is an example of an aberration correction amount calculation unit.

The image acquisition unit 13 acquires an image captured image TI, which is an image signal captured by the image capturing unit 25.
The captured image TI is a captured image TI in which the imaging target TP is captured by a plurality of correction amounts CL that are different from each other at each of a plurality of positions where the absolute positions ZL of the objective lens 21 are different from each other. In other words, the captured image TI is a captured image TI composed of a plurality of images in which the imaging target TP is captured at a plurality of relative positions having different absolute positions ZL for each of a plurality of correction amounts CL different from each other. Here, the absolute position ZL is the position of the objective lens when the relative positions of the imaging target TP and the objective lens 21 are changed. The details of the captured image TI will be described later.
The image acquisition unit 13 outputs the acquired captured image TI to the evaluation value calculation unit 14.

The reference focus plane information acquisition unit 16 acquires the reference focus plane information TFI. In this example, the reference focus plane information TFI is an image in which the focal position OF that the observer wants to observe is captured. The observer is a person who observes the imaging target TP by operating the spherical aberration correction calculation device 10 and the spherical aberration correction unit 20. The reference focus surface information acquisition unit 16 outputs the acquired reference focus surface information TFI 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), a correction ring start position CB, and an imaging start position ZB.

The target area ROI is a part of the imaged pixels of the captured image TI. 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 correction ring start position CB and the image pickup start position ZB are initial positions at which the image pickup of the captured image TI is started. The spherical aberration correction unit 20 aligns the position of the correction ring 21a of the objective lens 21 with the correction ring start position CB and the relative position between the objective lens 21 and the imaging target TP with the imaging start position ZB, and then performs imaging of the imaging target TP. Start.
The operation detection unit 17 outputs the detected operation result to the evaluation value calculation unit 14.

The evaluation value calculation unit 14 acquires the image signal of the image pickup target TP from the image acquisition unit 13.
The evaluation value calculation unit 14 calculates an evaluation value for each image constituting the captured image group. In the present embodiment, the evaluation value calculation unit 14 determines the displacement indicated by the reference focus surface information TFI acquired by the reference focus surface information acquisition unit 16 from among the captured image TIs composed of a plurality of images acquired from the image acquisition unit 13. Select the captured image that has been captured with the corresponding displacement. In other words, the evaluation value calculation unit 14 selects an captured image obtained by capturing an image of the focus plane indicated by the reference focus plane information TFI and a similar focus plane from the captured image TI.
The evaluation value calculation unit 14 determines the evaluation value RV of the correction amount CL from the captured image TIs captured at the absolute position ZL where the selected captured image is captured, based on the captured image TIs having different correction amounts CL. It is calculated for each captured image TI.
The evaluation value calculation unit 14 outputs the calculated evaluation value RV to the degree calculation unit 15.

The degree calculation unit 15 acquires the evaluation value RV from the evaluation value calculation unit 14.
The degree calculation unit 15 obtains the optimum value of the aberration correction amount based on the evaluation value acquired from the evaluation value calculation unit 14. In the present embodiment, the degree calculation unit 15 interpolates the evaluation values RVs calculated by the evaluation value calculation unit 14 to calculate the correction amount CL corresponding to the displacement of the reference focus surface. In other words, the degree calculation unit 15 interpolates the values between the evaluation values RV calculated by the evaluation value calculation unit 14, and estimates the influence of the difference in the correction amount CL on the evaluation value RV. The degree calculation unit 15 assumes that the correction amount CL of the peak position of the estimated evaluation value RV is the correction degree CP that well corrects the spherical aberration. The optimum value of the aberration correction amount described above is the degree of correction CP.
The degree calculation unit 15 outputs a correction degree CP corresponding to the calculated displacement 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 degree CP of the correction corresponding to the displacement of the reference focus surface calculated by the degree calculation unit 15. The correction ring drive unit 22 acquires the degree CP of the correction corresponding to the displacement of the reference focus surface from the degree calculation unit 15. The correction ring driving unit 22 drives the correction ring 21a based on the acquired displacement of the reference focus surface and the degree of correction CP corresponding to it.

[Outline of operation of spherical aberration correction calculation device 10]
Next, an outline of the operation of the spherical aberration correction calculation device 10 will be described with reference to FIG.
FIG. 4 is a flow chart showing an example of the operation of the spherical aberration correction calculation device 10.
The spherical aberration correction calculation device 10 acquires basic parameters by the operation of the observer (step S110).

[About captured image TI]
FIG. 5 is a diagram showing an example of the captured image TI.
In FIG. 5, the horizontal axis indicates the degree CL of each aberration correction in which the amount of aberration correction is changed, and the vertical axis indicates the relative position between the imaging target TP and the objective lens 21 when the absolute position of the objective lens is changed. ..
The captured image TI includes a plurality of images captured at absolute positions ZL in which the positions of the imaging target TP and the objective lens 21 are different for each correction amount CL.
Specifically, the captured image TI1 is an image captured at a plurality of absolute positions ZL in which the correction ring 21a included in the objective lens 21 is adjusted to the correction amount CL1 and the positions of the imaging target TP and the objective lens 21 are different. .. The captured image TI2 to the captured image TI7 are images in which the correction ring 21a included in the objective lens 21 is adjusted from a different correction amount CL2 to a correction amount CL7 and captured at a plurality of absolute positions ZL. The captured images TI1 to the captured images TI7 include seven captured images captured at positions having different absolute positions ZL. In this example, each correction amount CL from the correction amount CL1 to the correction amount CL7 divides the entire range of the settable value of the correction ring 21a of the objective lens 21 into seven equal parts. As the number of divisions of the correction amount CL increases, the accuracy of the correction amount CL corresponding to the displacement of the reference focus surface calculated by the degree calculation unit 15 improves.

The image pickup unit 25 captures an image of an observation object to be a captured image TI. The imaging unit 25 outputs the captured image TI to the image acquisition unit 13. The image acquisition unit 13 acquires a plurality of images (TI1 to TI17) to be captured images TI from the spherical aberration correction unit 20 (step S120). The reference focus plane information acquisition unit 16 acquires the reference focus plane information TFI by the operation of the observer (step S130). Specifically, the observer focuses the objective lens on the part to be observed while looking at the image of the object to be observed (the image visually confirmed or the image captured). At this time, it is not necessary to correct the aberration. Either the process of step S120 or the process of step S130 may be performed first. The evaluation value calculation unit 14 calculates the correlation with the reference focus plane information TFI for each captured image TI acquired by the image acquisition unit 13. Details will be described below.

[How to select the absolute position ZL close to the reference focus plane information TFI]
A method in which the evaluation value calculation unit 14 selects an image whose focus position is close to the reference focus plane information TFI from the captured image TI will be described. As a method of selecting an image having a focus position close to the reference focus plane information TFI from the captured image TI, there is a method of selecting an image having a spatial distribution of luminance close to each other. In order to select an image having a similar spatial distribution of brightness, in addition to calculating the correlation between the reference focus plane information TFI and the captured image TI, a method of calculating the difference between the reference focus plane information TFI and the captured image TI, etc. is there.

Specifically, the image obtained by capturing the same focal position OF as the reference focus plane information TFI 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 reference focus plane information TFI and the captured image TI, respectively. The brightness correlation is calculated by normalization cross-correlation or the like.
The evaluation value calculation unit 14 may calculate the correlation between the reference focus plane information TFI and the captured image TI, and compare the calculated correlations with each other. In this case, the evaluation value calculation unit 14 determines that the captured image showing a higher correlation has captured the focus plane close to the focus position OF captured by the reference focus plane information TFI.

The evaluation value calculation unit 14 selects an captured image having a high correlation with the reference focus plane information TFI from a plurality of images forming the captured image TI. The evaluation value calculation unit 14 calculates the evaluation value of the captured image TI captured at the relative position ZL where the image having a high correlation with the reference focus plane information TFI was captured (step S140).

[Calculation method of correction amount CL corresponding to displacement of reference focus surface]
The captured image TI 8 shown in FIG. 5 is an image selected from each captured image TI based on the result calculated by the evaluation calculation unit 14. This is an image captured in each captured image TI, which is the same surface as the captured image obtained by capturing the focus surface close to the focus position OF captured by the reference focus surface information TFI. In other words, the captured image TIs are images obtained by capturing the same focus plane. However, the degree of aberration correction of each captured image TI is different.
The captured image TI8-4 is a reference image that is the first captured image and is a reference focus plane image. That is, the spherical aberration correction unit 20 captures the captured image TI by changing the absolute position ZL and the correction amount CL after imaging the absolute position ZL4 and the correction amount CL4. The absolute position ZL and the correction amount CL for starting the imaging of the captured image TI may be the first time to take an image from any absolute position ZL and the correction amount CL, and are not limited to the above-mentioned absolute position ZL and the correction amount CL. Further, the captured image TI8-4 may be the target region ROI of the captured image.
The evaluation value calculation unit 14 calculates the evaluation value of each of the captured images included in the captured image TI8. Specifically, the evaluation value calculation unit 14 calculates the evaluation value based on 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 like. calculate.

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

When the evaluation value calculation unit 14 calculates the evaluation value based on the frequency component of the spatial frequency of the image of the captured image TI8, the value of the frequency component obtained by Fourier transform or the like is used as the evaluation 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 evaluation value based on the point image distribution function, the evaluation value distribution function is used as the evaluation value. The better the spherical aberration is corrected, the smaller the point spread function.

The evaluation value calculation unit 14 outputs the calculated evaluation value RV to the degree calculation unit 15. The degree calculation unit 15 acquires the evaluation value RV from the evaluation value calculation unit 14. The degree calculation unit 15 interpolates the acquired evaluation values RVs. The degree calculation unit 15 calculates the degree CP of the correction corresponding to the displacement of the reference focus plane based on the interpolation result (step S150). The degree calculation unit 15 outputs a correction degree CP corresponding to the calculated displacement of the reference focus surface to the correction ring drive unit 22.

The spherical aberration correction calculation device 10 does not have to calculate the absolute position ZL of the objective lens 21 when a thin sample is imaged. In this case, the spherical aberration correction calculation device 10 may perform the calculation from the evaluation value without calculating the absolute position ZL.

The degree calculation unit 15 interpolates the evaluation value RV calculated by the evaluation value calculation unit 14. In this example, the degree calculation unit 15 fits the evaluation values RVs to each other by a quadratic curve. Specifically, the degree calculation unit 15 fits the evaluation values RVs to each other by a quadratic curve fitting method by the least squares method. The degree calculation unit 15 may fit the evaluation values RV to each other by the evaluation value RV by the linear function fitting or the exponential function fitting.
FIG. 6 is a diagram showing an example in which evaluation values RVs are fitted to each other by a quadratic curve.
The graph shown in FIG. 6 is a graph having a correction amount CL on the horizontal axis and an evaluation value RV on the vertical axis.
The evaluation value RV1 to the evaluation value RV7 are the evaluation value RV calculated by the evaluation value calculation unit 14, respectively. The curve L1 is the result of fitting a quadratic curve. The evaluation value of the peak portion of the curve L1 is the evaluation value RVP. The degree of correction corresponding to the evaluation value RVP CP is the degree of correction corresponding to the displacement of the reference focus plane.

[Summary of the first embodiment]
As described above, the spherical aberration correction calculation device 10 includes an evaluation value calculation unit 14 and a degree calculation unit 15. The evaluation value calculation unit 14 selects an captured image having a high correlation with the reference focus plane information TFI from the captured image TI. The evaluation value calculation unit 14 calculates the evaluation value RV for each captured image TI captured at the absolute position ZL where the captured image having a high correlation with the reference focus plane information TFI was captured. The degree calculation unit 15 interpolates the evaluation value RV calculated by the evaluation value calculation unit 14. The degree calculation unit 15 calculates the degree CP of the correction corresponding to the displacement of the reference focus plane based on the interpolated evaluation value. The spherical aberration correction calculation device 10 can calculate the degree of correction of spherical aberration in which the focal position is aligned with the reference focus plane information TFI. That is, the spherical aberration correction calculation device 10 can correct the spherical aberration according to the focal position desired by the observer.

In other words, in the spherical aberration correction calculation device 10, the degree of correction of the spherical aberration of the spherical aberration correction unit including the objective lens corresponds to the displacement of the focus surface of the spherical aberration correction unit in the optical axis direction. Image acquisition that acquires a plurality of captured images in which the imaging target is captured by a plurality of correction degrees that are different from each other for each of the plurality of relative positions in which the relative positions of the imaging target and the objective lens are different from each other via the aberration correction optical system. Of the plurality of captured image TIs acquired by the unit 13 and the image acquisition unit 13, correction is performed based on a plurality of captured image TIs having different correction amounts CL in which the image corresponding to the image of the reference focus plane is captured. The degree to which the optimum correction amount is calculated by obtaining a curve from the evaluation value calculation unit 14 that calculates the evaluation value RV of the degree for each of the plurality of captured image TIs and the plurality of evaluation value RVs calculated by the evaluation value calculation unit 14. It is an apparatus including a calculation unit 15.

In the above description, the case where the imaging target TP is a thick sample has been described. In this case, the reference focus plane information TFI is the focus plane captured in the reference image. However, when the imaging target TP observes an imaging target TP having a thin thickness such as beads, the reference focus plane information TFI does not have to be an captured image. In this case, the spherical aberration correction unit 20 may fix the focal position OF on the surface where the cover glass CG and the image pickup target TP come into contact with each other. The evaluation value calculation unit 14 may calculate the evaluation value RV of the captured image TI imaged by fixing the absolute position ZL between the objective lens 21 and the image pickup target TP.

In the above description, the case where the objective lens 21 is an oil immersion objective lens or an immersion objective lens such as a water immersion 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 evaluation value RV at high speed.

In the above description, the evaluation value calculation unit 14 is based on the brightness 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 evaluation value was described.
When the evaluation value calculation unit 14 calculates the evaluation value based on the spatial frequency component, the spatial frequency indicated by the reference focus plane information TFI and the captured image TI for each captured image TI having a different correction amount CL. The evaluation value for a certain captured image is calculated based on the spatial frequency indicated by a certain captured image.

Specifically, the evaluation value calculation unit 14 calculates the evaluation value based on the spatial frequency of a predetermined band among the spatial frequencies. In this example, the evaluation value calculation unit 14 calculates the evaluation value based on the ratio of the values of the low frequency component and the high frequency component.

[Second Embodiment]
Up to this point, the configuration in which the spherical aberration correction calculation device 10 calculates the degree of correction CP corresponding to the displacement of the reference focus plane has been described. Next, the spherical aberration correction calculation device of the second embodiment fixes the degree of correction CP corresponding to the displacement of the reference focus surface and calculates the relative position corresponding to the displacement of the reference focus surface of the objective lens 21. The configuration 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 spherical aberration correction calculation device 10a will be described with reference to FIG. 7.
FIG. 7 is a diagram showing an example of the functional configuration of the spherical aberration correction calculation device 10a of the second embodiment.
The spherical aberration correction calculation device 10a includes a control unit 12a. The control unit 12a includes a second image acquisition unit 130, an imaging state evaluation value calculation unit 131, and a relative position calculation unit 132.

The second image acquisition unit 130 acquires the second captured image ZI from the image pickup unit 25.
The second captured image ZI is a correction corresponding to the displacement of the reference focus surface calculated by the degree calculation unit 15 for each of a plurality of second relative positions in which the relative positions of the image target TP and the objective lens 21 are different from each other. It is a plurality of captured images captured by the degree CP.
The second image acquisition unit 130 outputs the second image ZI acquired from the image pickup unit 25 to the image pickup state evaluation value calculation unit 131.

The image pickup state evaluation value calculation unit 131 acquires the second image capture image ZI from the second image acquisition unit 130.
The imaging state evaluation value calculation unit 131 calculates the second evaluation value ZV based on the second image ZI acquired from the second image acquisition unit 130. The second evaluation value ZV is an evaluation value indicating the degree of the imaging state of the imaging target TP. The evaluation values indicating the degree of the imaging state of the imaging target TP are the brightness value of the captured image of the second captured image ZI, the brightness distribution of the captured image, the contrast of the captured image, the frequency component of the captured image, and the point image of the captured image. It is an evaluation value such as a distribution function.
The imaging state evaluation value calculation unit 131 outputs the calculated second evaluation value ZV to the relative position calculation unit 132.

The relative position calculation unit 132 acquires the second evaluation value ZV from the imaging state evaluation value calculation unit 131. The relative position calculation unit 132 interpolates the second evaluation values ZV acquired from the imaging state evaluation value calculation unit 131. The relative position calculation unit 132 calculates the absolute position ZLP corresponding to the displacement of the reference focus plane based on the interpolated second evaluation value ZV. The relative position calculation unit 132 outputs the absolute position ZLP corresponding to the calculated displacement of the reference focus surface to the objective lens driving unit 24 and the display unit 11.

The objective lens driving unit 24 acquires the absolute position ZLP corresponding to the displacement of the reference focus surface from the relative position calculation unit 132. The objective lens driving unit 24 adjusts the relative position between the objective lens 21 and the imaging target TP to the absolute position ZLP. The spherical aberration correction unit 20 images the image target TP in a state where the objective lens 21 has an absolute position ZLP corresponding to the displacement of the reference focus surface and a degree of correction CP corresponding to the displacement of the reference focus surface.

The display unit 11 acquires the absolute position ZLP corresponding to the displacement of the reference focus surface from the relative position calculation unit 132. The display unit 11 displays the absolute position ZLP corresponding to the displacement of the reference focus plane acquired from the relative position calculation unit 132.

[Outline of operation of spherical aberration correction calculation device 10a]
Next, an outline of the operation of the spherical aberration correction calculation device 10a will be described with reference to FIG.
FIG. 8 is a flow chart showing an example of the operation of the spherical aberration correction calculation device 10a.

The second image acquisition unit 130 acquires the second image ZI from the image pickup unit 25 (step S210). The second image acquisition unit 130 outputs the second image ZI acquired from the image pickup unit 25 to the image pickup state evaluation value calculation unit 131.

The image pickup state evaluation value calculation unit 131 acquires the second image capture image ZI from the second image acquisition unit 130. The imaging state evaluation value calculation unit 131 calculates the evaluation value of the second captured image ZI acquired from the second image acquisition unit 130 as the second evaluation value ZV (step S220).
The imaging state evaluation value calculation unit 131 calculates the second evaluation value ZV for each second captured image ZI in the same manner as the evaluation value calculation unit 14 described above. The imaging state evaluation value calculation unit 131 outputs the calculated second evaluation value ZV to the relative position calculation unit 132.

The relative position calculation unit 132 acquires the second evaluation value ZV from the imaging state evaluation value calculation unit 131. The relative position calculation unit 132 interpolates the acquired second evaluation value ZV by two-dimensional fitting. The relative position calculation unit 132 interpolates the second evaluation value ZV in the same manner as the degree calculation unit 15 described above. The relative position calculation unit 132 calculates the absolute position ZLP corresponding to the displacement of the reference focus plane from the interpolated second evaluation value ZV (step S230). The relative position calculation unit 132 calculates that the relative position corresponding to the peak of the interpolated second evaluation value ZV is the absolute position ZLP corresponding to the displacement of the reference focus plane.

[Summary of the second embodiment]
As described above, the spherical aberration correction calculation device 10a includes an imaging state evaluation value calculation unit 131 and a relative position calculation unit 132. The image pickup state evaluation value calculation unit 131 fixes the degree of correction CP corresponding to the displacement of the reference focus surface, and calculates the second evaluation value ZV of the second captured image ZI having different absolute positions ZL. The relative position calculation unit 132 interpolates the second evaluation values ZVs and calculates the absolute position ZLP corresponding to the displacement of the reference focus plane. The spherical aberration correction unit 20 adjusts the objective lens 21 to the degree of correction CP corresponding to the displacement of the reference focus surface calculated by the spherical aberration correction calculation device 10a and the absolute position ZLP corresponding to the displacement of the reference focus surface. The spherical aberration correction unit 20 can adjust the focal position of the objective lens 21 to a surface closer to the reference focus surface information TFI.

[About the calculation order of evaluation values]
In the above description, the evaluation value calculation unit 14 calculates the evaluation value RV in order from the correction ring start position CB and the imaging start position ZB input by the observer.

Instead of the correction ring start position CB, the evaluation value calculation unit 14 may calculate the evaluation value RV in order from the captured image captured by the degree of correction near the median in the range where the correction ring 21a can be corrected. Specifically, the evaluation value calculation unit 14 calculates the evaluation value RV from the correction amount CL4 shown in FIG. 5, and calculates the evaluation value RV in the order of the correction amount CL4 from the correction amount CL7 and the correction amount CL4 to the correction amount CL1. ..

Further, the evaluation value calculation unit 14 may calculate the evaluation value RV from the degree of correction in the vicinity of the correction amount CL4. Specifically, the evaluation value calculation unit 14 calculates the evaluation value RV in the order of CL3 and the correction amount CL5, the correction amount CL2 and the correction amount CL6, the correction amount CL1 and the correction amount CL7.

The evaluation value calculation unit 14 may determine the order in which the evaluation value RV is calculated from the captured image TI based on the optical characteristics of the objective lens 21. Based on the optical characteristics of the objective lens 21, the evaluation value calculation unit 14 associates the displacement of the focal position OF with the adjustment of the correction amount CL with the change of the absolute position ZL. The evaluation value calculation unit 14 can determine the order in which the evaluation value RV is calculated based on the displacement of the focal position OF. The evaluation value calculation unit 14 can reduce the number of captured images TI required for calculating the peak of the evaluation value RV. The spherical aberration correction unit 20 can reduce the number of times the captured image TI is captured. That is, in the case of a sample in which the TP to be imaged is stained, the degree of fading of the sample can be suppressed by imaging for calculating the degree of correction CP corresponding to the displacement of the reference focus plane, and clearer imaging can be performed. Images can be captured.

[About the calculation of the degree of correction CP corresponding to the displacement of the reference focus plane]
The degree calculation unit 15 may be able to sequentially interpolate the evaluation value RVs calculated by the evaluation value calculation unit 14 and calculate the peak of the complemented evaluation value RVs. The degree calculation unit 15 may end the process when the peak of the evaluation value RV can be detected, and calculate the degree CP of the correction corresponding to the displacement of the reference focus surface. Since the degree calculation unit 15 ends the process when the peak can be detected, the time required for calculating the degree CP of the correction corresponding to the displacement of the reference focus plane can be shortened.

[Adjustment of absolute position ZL]
In the above description, the absolute position ZL between the objective lens 21 and the captured image TI has been adjusted by driving the objective lens 21 by the objective lens driving unit 24, but the stage 23 is moved in the optical axis OA direction. You may adjust it. When adjusting the stage 23, the spherical aberration correction unit 20 includes a stage drive unit (not shown). In this case, the stage drive unit drives the stage 23 when it acquires the absolute position ZLP corresponding to the displacement of the reference focus surface. When the stage 23 is driven, the absolute position ZL between the objective lens 21 and the imaging target TP is adjusted. As the vertical movement drive mechanism of the stage, a general vertical stage vertical movement mechanism of a 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.

Next, a microscope including the spherical aberration correction unit 20 will be described.
The microscope includes an objective lens having a spherical aberration correction optical system, a stage on which an imaging object is placed, and an image group generating unit. Here, the microscope is an example of an image group generating device.

The image group generation unit generates an image group including the first image group and the second image group. The image group generating unit is a device provided in, for example, the image acquisition unit 13 in FIG. 7. The image generation device is a device having an image acquisition unit.

The first image group is obtained by continuously shooting an image-imaging object while changing the relative distance between the objective lens and the stage in a state where the spherical aberration correction optical system is adjusted by the first aberration correction amount. The relative distance is the distance between the objective lens and the stage, and is synonymous with the relative position when the positions of the objective lens and the stage are targeted.

The second image group can be obtained by continuously shooting an imaging object while changing the relative distance between the objective lens and the stage in a state where the spherical aberration correction optical system is adjusted by the second aberration correction amount. In FIG. 5, the first image group is, for example, a captured image TI1, and the second image group is, for example, a captured image TI2.

Continuous shooting means that images are taken with the degree of correction of the correction amount CL1 in FIG. 5 described above, the position ZL of each objective lens is stopped at each position, and images of different objective lens positions ZL are continuously imaged. is there.

The image group generating unit is a device provided in, for example, the image acquisition unit 13 of FIG. 7 described above. The image generation device is a device having an image acquisition unit.

Although the embodiments of the present invention have 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. it can.

The spherical aberration correction calculation device 10 and the spherical aberration correction calculation device 10a described above have a computer inside. The process of each process of the device described above 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, this computer program may be distributed to a 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, a so-called difference file (difference program) may be used, which can realize the above-mentioned functions in combination with a program already recorded in the computer system.

10 ... Spherical aberration correction 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, 20 ... Spherical aberration correction unit, 21 ... Objective lens, 21a ... Correction ring, 22 ... Correction ring drive unit, 23 ... Stage, 24 ... Objective lens drive unit, 25 ... Imaging unit, 100 ... Spherical aberration correction optics System system, 130 ... second image acquisition unit, 131 ... imaging state evaluation value calculation unit, 132 ... relative position calculation unit

Claims (8)

An objective lens with a spherical aberration correction optical system and
The stage on which the object to be imaged is placed and
With the first image group obtained by continuously shooting the image pickup object while changing the relative distance between the objective lens and the stage in a state where the spherical aberration correction optical system is adjusted by the first aberration correction amount. ,
With the second image group obtained by continuously shooting the image pickup object while changing the relative distance between the objective lens and the stage in a state where the spherical aberration correction optical system is adjusted by the second aberration correction amount. With the image group generation unit that generates the image group including
An evaluation value calculation unit that selects a third image group from the image groups based on a predetermined reference image and calculates an evaluation value for each image constituting the third image group.
With the aberration correction amount calculation unit that obtains the optimum value of the aberration correction amount of the objective lens based on the evaluation value.
With
The reference image is an image of an observation position imaged in a state where the aberration correction amount of the objective lens is set to an arbitrary aberration correction amount and the objective lens is in focus on the imaging object.
Spherical aberration correction calculation device. The aberration correction amount calculation unit is
The spherical aberration correction calculation device according to claim 1 , wherein a plurality of the evaluation values calculated by the evaluation value calculation unit are interpolated to calculate the aberration correction amount. The evaluation value calculation unit
For each of the plurality of images having different aberration correction amounts,
The spherical aberration correction calculation device according to claim 1 or 2 , wherein an evaluation value is calculated based on the spatial frequency indicated by the reference image and the spatial frequency indicated by a certain image among the plurality of the images. The evaluation value calculation unit
The evaluation value is calculated based on the spatial frequency of a predetermined band among the spatial frequencies.
The spherical aberration correction calculation device according to claim 3 . Based claims 1 to evaluation value calculated by any of the spherical aberration correction calculation device according to claim 4, the driving unit of the aberration correction optical system moves in the optical axis direction the spherical aberration correcting optical system of the objective lens Equipped with a microscope. The microscope is a microscope according to claim 5, comprising a drive unit that moves in the optical axis direction relative position of the objective lens and the stage after moving the aberration correction optical system by the driving unit. In computer
With the spherical aberration correction optical system adjusted by the first aberration correction amount, the image pickup object is continuously photographed while changing the relative distance between the objective lens having the spherical aberration correction optical system and the stage on which the image pickup object is placed. The first image group obtained by doing
With the second image group obtained by continuously shooting the image pickup object while changing the relative distance between the objective lens and the stage in a state where the spherical aberration correction optical system is adjusted by the second aberration correction amount. an image group generating step of generating an image group including,
An evaluation value calculation step of selecting a third image group from the image groups based on a predetermined reference image and calculating an evaluation value for each of the images constituting the third image group.
An aberration correction amount calculation step for obtaining an optimum value of the aberration correction amount of the objective lens based on the evaluation value, and
A spherical aberration correction calculation program for execution,
The reference image is an image of an observation position imaged in a state where the aberration correction amount of the objective lens is set to an arbitrary aberration correction amount and the objective lens is in focus on the imaging object.
Spherical aberration correction calculation program . With the spherical aberration correction optical system adjusted by the first aberration correction amount, the image pickup object is continuously photographed while changing the relative distance between the objective lens having the spherical aberration correction optical system and the stage on which the image pickup object is placed. The first image group obtained by doing
With the second image group obtained by continuously shooting the image pickup object while changing the relative distance between the objective lens and the stage in a state where the spherical aberration correction optical system is adjusted by the second aberration correction amount.
Generates a group of images containing
A third image group is selected from the image group based on a predetermined reference image, and an evaluation value for each image constituting the third image group is calculated.
Based on the evaluation value, the optimum value of the aberration correction amount of the objective lens is obtained.
Spherical aberration correction calculation method
The reference image is an image of an observation position imaged in a state where the aberration correction amount of the objective lens is set to an arbitrary aberration correction amount and the objective lens is in focus on the imaging object.
Spherical aberration correction calculation method.